CHONDROID BONE, SECONDARY CARTILAGE, AND METAPLASIA

William A Beresford MA DPhil © West Virginia University, Morgantown, USA

This book was published in 1981 by Urban & Schwarzenberg with the identifications
ISBN 0-8067-0261-3 Baltimore & ISBN 3-541-70261-3 Munich
The publishers, now Waverly, have kindly returned the copyright to me, so that this online version is to be downloaded and printed out solely for personal use, and not for profit.
In-text commentary with citations updating it to 1999 will be italicised (new preface).

CONTENTS IN BRIEF
1981 Preface
Chapter 1 . Chondroid Bone
Chapter 2 . Nomenclature of Chondroid Bone
Chapter 3 . Secondary Cartilages I
Chapter 4 . Secondary Cartilages II
Chapter 5 . Metaplasia I
Chapter 6 . Metaplasia II
Chapter 7 . Undifferentiated Mesenchymal Cell
Chapter 8 . Modulation
Chapter 9 . Long-Bone Periosteal and Insertion-Structure Chondrifications
Chapter 10 Chondroid Bone II
Chapter 11 Mammalian Skull
Chapter 12 Avian Skull
Chapter 13 Antlers and Horns
Chapter 14 Phallic Bones
Chapter 15 Clavicle
Chapter 16 Fracture Callus
Chapter 17 Transplantations of Skeletal Tissues
Chapter 18 Bone and Cartilaginous Tumors
Chapter 19 Piscine Chondroid Bone
Chapter 20 Cavian Dental Chondroid Bone
Chapter 21 Osteocytic Osteolysis: Mistaken Chondroid Bone?
Chapter 22 Temporal Bone
Chapter 23 Rickets
Chapter 24 Conclusions
References
New Preface


CONTENTS IN FULL

Chapter I       Chondroid Bone
  Introduction
  Chondroid bone, type II
  Chondroid bone, type I
  Relations between CB I and II
  Chondroid bone, type III: miscellaneous kinds
  Secondary cartilages and chondroid bone
  Conclusion

Chapter 2   Nomenclature of Chondroid Bone
  Names for chondroid bone
  "Spurious" chondroid bones
  Knorpelknochen
  Knochenknorpel (Osteoidknorpel)
  Chondroider Knochen
  Chondroidknochen
  Chondroidal osteogenesis
  Chondroid membrane bone
  Osteoider Knorpel
  Chondroid bone
  Chosen nomenclature

Chapter 3    Secondary Cartilages I
  Introduction and overview
  Secondary cartilage: definitions and listings
  Schaffer's category of secondary cartilage
  Secondary cartilage as lately defined
  Compilation of secondary cartilages on Schaffer's criterion
  Anomalous secondary cartilages
  Fuch's refutation of secondary cartilages
  Secondary cartilages: comparisons and special studies
  Explicit comparisons
    Between secondary cartilages
    Between secondary and primary cartilages
  Some individual histochemical and ultrastructural studies
  General remarks

Chapter 4    Secondary Cartilages II
  Mechanical evocation of secondary cartilage Schaffer
  Pressure-elicited chondrogenesis on endochondral bones
  Pressure-elicited cartilage related to membrane bones
  Experimental evocation of avian secondary cartilage: Murray & Hall
  Altmann's arguments against extrinsic mechanical stimulation of
   Chondrogenesis
  Unstressed sites of secondary chondrogenesis
  Secondary chondrogenesis, at but unrelated to, mechanical disturbance
  Altmann and Pauwel's unifying chondrogenic hypothesis
  Mechanical determinants of osteogenesis
  Determinant stimuli arising from differential growth

Chapter 5   Metaplasia I
  Introduction and context
  Virchow's metaplasia
  Explanations of metaplasia
   Direct metaplasia
   Embryonic rests
   Displacement or migration of differentiated cells
   The undifferentiated mesenchymal cells
   Indirect metaplasia
   Defining metaplasia out of existence
   Modulation as a substitute for metaplasia
  Experimental narrowing of the accounts of metaplasia

Chapter 6     Metaplasia II
  Examples of metaplasia
  Novel stem cell differentiations ("tissue" metaplasia)
  The differentiated cell as stem cell
  The completeness of tissue metaplasia
  Lens regeneration in newts
  Lens formation by transdifferentiation in vitro: Urodela, Aves,
    Mammalia
  Transformations in chromatophores
  Glandular metaplasia of renal smooth muscle
  Connective tissue metaplasias
    Fibrous connective tissue to bone
    Fibrous connective tissue to cartilage
    Cartilage to fibrous connective tissue
    Hyaline cartilage to fibrocartilage
  Rapid transformations in connective tissues
  The role of stem cells
  Regeneration as the context of indirect metaplasia
  Terminology of differentiation and metaplasia
    Dedifferentiation
    Redifferentiation
    Terminal differentiation
  Slow transformations of connective tissue: issues arising
  Terminal differentiation
  The tissue legacy
  Myoid transformations
  Biochemical and morphological indices of differentiation
  Degrees of metaplasia
  Physiological and pathological metaplasia
  "Immaturity" of cartilage
  The matrix in metaplasia
  Transdetermination in imaginal disks
  Nuclear transplantation & cell hybridization: nuclear metaplasia

Chapter 7   Undifferentiated Mesenchymal Cell
  Introduction
  Maximow's concept of UMCs
  Maximow and the fibroblast
  Maximow and Bloom's textbook
  Doubts regarding UMC and its role in ectopic ossification

Chapter 8     Modulation
  Introduction and summary
  Modulation: Weiss's concepts and modern usage
  Schemes of modulation for bone
    McLean and Bloom
    Bloom, Domm, Nalbandov, and Bloom
    Willis
    Young
    Hall
  Schemes of modulation for connective tissues
    Schaffer
    Pritchard
    Johnson
    Hall
    Willmer
    Willis, Moss, Klaatsch, etc
  Variations between schemes in the cells participating
  Formative-destructive schism
  Osteoblast-to-osteocyte modulation
  Reticular cells in bone modulations
  Osteogenic cells of marrow: experiments
  Consequences of omitting a cell type
  Average cellular condition
  Stability of the modulated state
  Diagrams of modulation: implicit simplifications
  Modulation 2 by chondrocytes and skeletal muscle
  The connective tissues: one tissue?
  Modulation and metaplasia

Chapter 9 Long-Bone Periosteal and Insertion-Structure Chondrifications
  Introduction
  Endochondral bone's subperiosteal chondrogenesis: Zawisch-Ossenitz
  Other instances of subperiosteal chondroid bone
  Reports with long-bone chondroid bone absent
  Circumscript periosteal attachments
  Tibio-fibular fusion in rodents
  Schaffer's chondroid tissues
  Fibrocartilage-vesicular tissue distinction
  Cartilage in tendons and ligaments
  Cartilaginous metaplasia in tendons
  Other peri-skeletal metaplasias

Chapter 10   Chondroid Bone II
  Mineralization versus ossification
  Mineralized fibrocartilage at insertions
  Other mineralized fibrocartilages
  Pathological chondroid bone II (fibro)
  Articular chondroid bone II
  Chondroid bone II (hyaline)
  Chondroid bone II (fibro)
  The Nature of chondroid bone II (hyaline)
  Chondroid bone II (hyaline) in epiphyseal plates
  Chondroid bone II (hyaline) in other permanent cartilages
  Cellular viability and chondroid bone
  Occurrence of bone
  Stimuli for degeneration, calcification, and ossification
  Chondroid bone II (elastic)
  Metaplastic interpretations of CB II
  Non-mammalian chondroid bone II
  The patella and cartilage canals
  Tracheal cartilages
  Sites of insertion
  Conclusions
  Confusions between chondroid bone I and II
  The cells and mineralization
  Functions of CB II
    Growth
    Mechanical roles
    Non-mechanical roles
  Secondary cartilages and CB II

Chapter 11      Mammalian Skull
  Introduction
  The mandible
    The number of chondrogenic sites
    Angular and coronoid cartilages
    Anterior mandibular cartilages
    Cartilage on alveolar processes
    Mandibular condyle
  Introduction
  Uebergangsgewebe: Chondroid Bone I?
  Condylar cartilaginous tissues: a stable basis for comparisons?
  Persisting cartilage cells
  Periosteal bone: living cartilage boundary
  The disk of the mandibular joint
  Squamosal fossa and tubercle
  Fuchs' comparative studies
  Human articular tubercular covering
  Chondroid bone in rodent fossa
  Temporal chondroid bone: reactions and remodeling
  Maxilla and pterygoid
  Pterygoid
  Maxilla and palatine bone
  Cranial vault sutures: two kinds of cartilage?

Chapter 12      Avian Skull
  Sites of secondary cartilage
  Avian cranial chondroid bone I
  Reptilian secondary cartilage
  Markers of chondroblastic differentiation

Chapter 13      Antlers and Horns
  Introduction
  Postulated tissues and mechanisms of antlerogenesis
  Cartilage, bone or chondroid bone?
  Zonation and the position of chondroid bone
  Chondroid bone and chondrobsteoid
  Chondroid bone and metaplasia
  Cornugenesis

Chapter 14      Phallic Bones
  Occurrence
  Secondary cartilages
  Development of the penile skeleton in the rat
  The Anterior fibrocartilaginous process
  The Penile bone
  Chondroid bone
  Penile skeletogenesis in other species

Chapter 15      Clavicle
  Introduction
  Initial clavicular chondroid bone
  Hyalinzelliges chondroides Gewebe (Pseudoknorpel: early clavicular cartilage
  Mischgewebe: Chondroid bone I
  Conclusions

Chapter 16      Fracture Callus
  Callus chondrogeneses
  The chondrogenic stimulus
  Callus chondroid bone I: by metaplasia or blastema
  Metaplastic interpretations
  Blastemal interpretations
  More evidence and an evaluation
  Chondroid bone on healing membrane bones
  Bony stumps

Chapter 17   Transplantations of Skeletal Tissues
  Introduction
  Chondrogenesis by periosteal grafts in vivo
  In vivo grafts of bone and periosteum
  Secondary cartilage from avian periosteum in vitro or on the CAM
  Perichondral osteogenesis in transplant and explant
  Stimuli for periosteal and perichondral switching
  Osseous transformation in transplanted primary cartilage
  Osseous transformation in transplanted secondary cartilage
  "Metaplastic" changes in the matrix or cells of transplanted cartilage
  Chondrocytic escape and metaplasia in the intact animal
  Cartilage formed by grafted periosteum and regenerating tissue: signs of its metaplasia
  Conclusions   

Chapter 18     Bone and Cartilaginous Tumors
  Introduction
  Secondary cartilages
  Instances of neoplastic secondary cartilage
  Sources of cartilaginous tumors
  Virchow's hypotheses
  Tumors on membrane bones: role of previous secondary cartilage
  Experimentally evoked secondary cartilage
  Chondroid bone in tumors
  Nomenclature
  Chondroid bone II
  Chondroid bone I
    In malignant tumors (skeletal)
    In malignant tumors (extraskeletal)
    In benign tumors
    With experimentally induced ectopic skeletal tissues
    Metaplasia in tumors?
    Creeping substitution

Chapter 19      Piscine Chondroid Bone
  Introduction
  Selachian calcified cartilage (CB II)
  More truly intermediate chondroid bone
  Bony fishes and chondroidal ossification

Chapter 20     Cavian Dental Chondroid Bone    335

Chapter 21      Osteocytic Osteolysis: Mistaken Chondroid Bone?
  Introduction
  Osteocytic osteolysis: Belanger and colleagues' work
  Evidence against osteocytic osteolysis
  Avian medullary bone
  "Chondroid bone" from excess PTH and vitamin A deficiency
  Chondroid bone in osteogenesis imperfecta congenital
  Osteopetrosis
  Bone-to-cartilage metaplasias?
  Conclusion

Chapter 22      Temporal Bone
  Introduction
  Globulus osseous (Knochenkugeln)
  Interglobularraum
  Residual cartilage and chondroid bone
  Cartilaginous interglobular spaces
  Otosclerotic bone - how chondroid?
  Metaplasia in interglobular spaces?
  Secondary cartilages
  Gussen's observations, nomenclature and conclusions: a critique

Chapter 23      Rickets
  Introduction to rachitic "chondroid bone" and the metaplastic question
  Metaplastic interpretations: Virchow's
  Non-metaplastic assessments: Muller's
  Nature and origin of the fibrous cartilage
  Similar "chondroid bone" in congenital syphilis
  Rachitic cartilage as secondary?

Chapter 24      Conclusions
  Secondary cartilage
  Metaplasia
  Chondroid bone
  Some questions

     References

     New Preface

1981 PREFACE

This book examines three skeletal phenomena whose perplexing nature is made both harder and easier to penetrate by their being closely intertwined. They are chondroid bone, secondary cartilage, and metaplasia. In the briefest of terms, chondroid bone is any tissue intermediate in nature between bone and cartilage; secondary cartilages are ones forming after the primary cartilaginous skeleton; and metaplasia and modulation involve the transformations of differentiated cells - skeletal and non-skeletal - to other cell kinds.

The impulse to write came because: there is neither book nor review article on chondroid bone; secondary cartilages have received only fragmentary attention since 1930; and metaplasia is a perennial challenge to the concept of differentiation which is seldom tackled in the round.

Although chondroid bone and secondary cartilage clearly belong in the province of the skeletal biologist, bone pathologist and orthopedic surgeon, the implications of their genesis and transformations extend to the very general and current concerns of: What is differentiation? What is a tissue? What are the relations of a tissue with its cells? How well can tissues maintain their identity? If they fail, what is the nature of their transformation? And, what factors influence such transformations?

Analysis and synthesis are involved. The topics of the title will first be analyzed to establish what is meant by, say, indirect metaplasia, or accessory secondary cartilages. With this as foundation, in the second part I shall take sites and situations one by one, looking critically at the evidence for chondroid bone, secondary cartilage and metaplasia.
Synthesis enters in the sense that the very many examples of intermediates between bone and cartilage may be summed to the conclusion that there is a family of tissues for which the name chondroid bone is apt.

Because of its intimate connection with metaplasia and for other reasons rooted in the founding of histology, chondroid bone has never received general recognition as a class of tissue. This neglect has made difficulties for many students of the skeleton who must often meet chondroid bone, the tissue being present at many boundaries of bone. Thus, the tissue is often not recognized for what it is, and when so acknowledged, its observer has not even a brief review in a journal to turn to for information. Therefore, as its main task, this book seeks to establish the identity of chondroid bone and explore its subtypes.

A murky crust of varied and sometimes conflicting meanings overlies concepts such as modulation that had their origin well into this century. The opacity of nuances has grown even thicker around the metaplasia and secondary cartilage of nineteenth-century histology and histopathology.
The only path to understanding was to trace each concept from its inception, where I could find it, up to the present day. For me, none of three principal ideas was easy to dissect and I claim neither to have gone about the tasks the best way, nor always to have reached scientifically convincing conclusions.
Nevertheless, the same difficulties face anyone interested in the concepts, and I hope that, to the extent that it is systematic, my attempt to confront the trio will help others, despite its lacunae and naivete.

Where so much confusion exists on what a tissue or process really is, I chose as the safest course the device of extensive quotation, so that the reader can judge for himself what the cited authors had in mind.

A related problem arose with the figures. Most instances of chondroid bone have been noted, in passing, in materials prepared for routine histology, and therefore have the blandness of detail associated with such stains as hematoxylin and eosin. Within the subclasses of chondroid bone I and II, most illustrations published depict very similar tissues. Therein lies the strength of the evidence that chondroid bone exists. To draw on this strength, but avoid pointless and expensive repetition in the figures here, I have made frequent reference to the figures of others, with their numbers, depicting chondroid bone in its various forms, and confine my own to those needed for an introduction to the tissue. For clarity, the latter are often identified in the text as "my" Figure (with its number).
Another minor departure from convention is not to indicate that a quotation has been translated, since this information can readily be inferred from the foreign title of the article in the bibliography.

Many acknowledgments are due. I owe my start in histology to Professor Paul Glees, Oxford and Goettingen, and my interest in bone to Professor Norman Hancox, Liverpool. This book has benefited from the encouragement and critical comments of Professors Brian Hall, Dalhousie University, and Randall Reyer of West Virginia University. Its faults are, of course, all mine.
I am especially grateful to my wife, Margaret, for turning scribble into typescript, the metaplasia of which into manuscript was achieved by Mss. April Blosser, Joann Cox, Angie Police and Mildred Wilson. For the metamorphosis into a book, it is my pleasure to thank Mr. Braxton Dallam Mitchell, president of Urban & Schwarzenberg Inc., and his associates, Ms. Carola Sautter and Mr. Detlev Moos.
I also appreciate the generous help of the librarians of West Virginia University Medical Center, the technical work of Ms. Artemis Khatcherian, Mrs. Elsie Syner and Ms. Kathy Ellifritz, the loan of slides by Professor T. Walley Williams, Mss. Teresa Elmore, Jeanne Henninger and Anne Stockhausen's checking of the references, and the willingness of many experimenters to send offprints. For their permission to reproduce figures, I thank Professors M. Listgarten and I. Shapiro and the Pergamon Press (Figure 37); N. Kemp and S. Westrin and the Wistar Press (Figures 9, 10, 35, 36); K. H. Knese and the SpringerVerlag (Figures 4, 8, 12, 13); D. Dahlin and Charles C Thomas, Publisher, (Figures 33 and 34); and S. Clayton (Figures 28, 29 and 30). Autumn, 1980 - WA BERESFORD


Chapter 1 CHONDROID BONE

Introduction
Chondroid bone, type II
Chondroid bone, type I
Relations between CB I and II
Chondroid bone, type III: miscellaneous kinds
Secondary cartilages and chondroid bone
Conclusion

INTRODUCTION

This book came about because of an observation that I made in the course of a histological study of the developing mandibular joint and penile bone of the rat. The growing mandibular condyle is noted as the site of a so-called secondary cartilage. It is less widely known that the rat's penile bone has very similar cartilage (Ruth, 1934; Beresford, 1975a; Vilmann and Vilmann, 1978), and as shown in Figure 1.

Separate from these secondary cartilages, the young penile and temporal bones each bear a small region of tissue sharing bony and cartilaginous characteristics - the matrix appears mineralized, collagenous and very like that of adjacent bone, but the cells are large and spheroid or ovoid, and react with alcian blue as shown in Fig. 2.

I had occasionally seen such tissue referred to in papers by the name "chondroid bone," (CB) but a search for a general article on chondroid bone was fruitless. What I found were many isolated reports of sightings of chondroid bone that had been made, like mine, by happenstance, while studying some other aspect of bone or cartilage. The purpose of this book is to collect and make sense of these many observations.

The handicap is that there is no agreed terminology for the tissues occupying the spectrum between cartilage and bone. For example, one person may name a tissue "chondroid bone," or some synonym thereof, but actually mean typical bone. Another may describe a tissue as intermediate between bone and cartilage, but may either fail to name it or may call it something other than chondroid bone.
How can one know that the individual has chondroid bone in mind? By noting a writer's descriptions, illustrations, and remarks in the paper in relation to comparisons with the CB of other workers can one ascertain whether or not the researcher is referring to CB. Thus, one chapter in this text deals exclusively with the problems of nomenclature, because these problems arise again and again.

Once identified, the literature on chondroid bone began to display patterns that reflect the organization of the material here. Thus, there is not one chondroid bone, but a family of tissues differing in their structure, histogenesis, chemistry, fate, etc. These "chondroid bones" have relations not only with tissues - bone, cartilage, chondroid (Schaffer, 1930), and one another - but with various processes - metaplasia, secondary chondrogenesis, osteocytic osteolysis, to name a few.

The unravelling of chondroid bone's relationships with such tissues and events requires that one understand the nature of, say, chondroid. This requirement created a second quest, namely an inquiry into such phenomena as metaplasia and secondary cartilages. This, in turn, generated tertiary attempts to resolve such problems as the nature of the undifferentiated mesenchymal cell and modulation, and the use of a mechanical etiology as a criterion for distinguishing cartilages as secondary.

Some examples of the relation of chondroid bone with other entities are given below, because they may provide a means of separating the mass of reported chondroid bones into smaller and somewhat more homogeneous classes.
Metaplasia - the apparent transformation of an established mature tissue into another - is skeletal biology's own skeleton in the closet. For many years most bones were thought to form by a direct metamorphosis from cartilage. Sharpey (1849) and H. Muller (1858) are credited with convincingly establishing that the major process of endochondral osteogenesis is not such a metaplasia, but rather the supplanting of cartilage by bone. Muller also made two often neglected observations.

CHONDROID BONE, TYPE II

First Muller observed that calcified cartilage is not all destroyed; some persists into adulthood at boundaries where endochondral ossification has ceased, e.g., subjacent to articular cartilages, the synchondroses of the vertebrae and pelvis, where the bony and cartilaginous portions of the ribs adjoin, and in the auditory ossicles (Figure 3).

Meyer (1849a) had seen many examples of this mineralized cartilage, but because he took them to be metaplastic bone, his "ossified cartilage" might refer to either bone or to calcified cartilage.
Only for the subarticular cartilage did he draw a clear distinction between eigentlicher Knochen (true bone) and verknocherter Gelenkknorpel (ossified joint cartilage).

The other names that mineralized cartilage has acquired over the years are "superficial osseous crust" (Pittard, 1852); "calcified cartilage" (Muller, 1858, and many others); "incompletely developed bone" (Kolliker, 1889); "eigenthumliche verknocherter Zwischenschicht" (peculiarly ossified intermediate layer) (Gebhardt, 1903; "Knorpelknochen" and "ossified vesicular fibrous tissue" (Weidenreich, 1923c); "parallelfaseriger chondroider Einstrahlungsknochen" (Knese and Biermann, 1958); and "metaplastic tissue" and "metaplastic bone" (Haines and Mohuiddin, 1968).

The names express two controversies: one on the tissue's nature and the other on its possible metaplastic significance. Regarding its nature, the list of nomenclature above has precedents for naming the material "chondroid bone," but with what justification? Muller (1858) stressed that such enduring calcified cartilage is not true bone. As Haines and Mohuiddin (1968) emphasized, neither is it what is generally conceived of as calcified cartilage - namely, the kind of hypertrophic cartilage about to be eroded in endochondral ossification.

Although a failure to resorb typical epiphyseal growth cartilage may in some species leave noticeable amounts of persistent calcified cartilage, other examples of this latter tissue form away from sites of endochondral ossification. Also, in the longstanding calcified cartilages the cells are not usually hypertrophied, and the matrix appears more collagenous.
In all locations, doubts have been expressed about the viability of the tissue's cells, some researchers believing calcification of the matrix brings death to the cells, others thinking to the contrary.

The intermittent attention paid to the tissue since Muller's time has furnished it with several names, revealed more sites of its occurrence, e.g., the permanent cartilages of the airway and external ear, and established that the tissue has some living cells. Functioning chondrocytes in a collagenous, but well mineralized matrix, place the tissue somewhere between cartilage and bone, making it a kind of chondroid bone.

Calcification of enduring cartilage can be experienced by all three classic types of cartilage, so that the particular mineralized variety can be indicated by a suffix, e.g. chondroid bone II (fibro). This name skirts around the other controversy concerning the tissue; namely, whether it can eventually undergo metaplasia to true bone, while it does convey the known; that it is only after a typical soft hyaline, elastic or fibrocartilage has developed that mineralization comes about.

CHONDROID BONE TYPE I

Chondroid bone I is connected with the second of Muller's minor observations. He saw that at the onset of endochondral ossification bone is also laid down on living undecalcified cartilage. In other words, the germinal tissue of the perichondrium rapidly switches to osteogenesis, leaving a very narrow zone of a rather ambiguous tissue along the interface between living cartilage and bone.

On the early long-bone's shaft the amount of such tissue is almost negligible. However, in many other situations where a switching periosteum (or perichondrium) is nearby, enough of a tissue intermediate between cartilage and bone is present to have attracted regular comment. This intermediate tissue most often has chondrocyte-like cells in a bony matrix, and is set amidst new bone and cartilage with which it merges.
Chondroid bone I is the term that will be used to designate the tissue closest in appearance to the midpoint between bone and cartilage.

Many examples of CB I first came to light around the mid-nineteenth century, when the metaplastic theory of osteogenesis still had many adherents. The CB I generally accompanied new bone and cartilage, so that its linking the latter two tissues physically and its intermediate nature were promptly taken as proof that cartilage was turning directly into bone. If cartilage can turn into bone directly, or bone into cartilage, chondroid bone as a form intermediate between bone and cartilage should occur, unless the metaplasia takes place very rapidly, which is unlikely, considering how much the matrices differ and the high matrix-to-cell ratio.
But, chondroid bone I is not of itself proof that a metaplasia of mature tissues is happening. Chondroid bone could come about by the differentiation directly to that intermediate tissue of indifferent, precursor cells of the periosteum or other soft tissue, an interpretation favored by Schaffer (1888), among others.

The ability of seemingly germinal tissues to alter their destination of differentiation, e.g., periosteum to produce callus cartilage, has led to the view that certain mesenchymal cells become specialized to a degree at which they can then change into chondroblasts, osteoblasts or other cells - the partly specialized but not finally committed cells to be called "osteogenic cells" (Ham, 1930); "skeletogenous cells" (Fell, 1933); "Skelettzellen" (Knese and Biermann, 1958); "scleroblasts" (Moss, 1964a); or "myxoblasts" (Willmer, 1960).

The factors that finally direct such "osteogenic" or "sclerogenic" cells into a particular specialization are believed to include extrinsic chemical and mechanical stimuli. In such a cellularly responsive situation, one anticipates that a determining factor, e.g., oxygen tension (Ham, 1930; Bassett and Herrmann, 1961), would sometimes stand at a halfway level (or several factors could balance out) to give the scleroblast a message that it might follow by forming an intermediate tissue - chondroid bone, as Jolly (1961) predicted.

If the determination of the scleroblast to form bone and not cartilage is specified by the measure of simple factors such as gas tension or mechanical pressure, one may wonder why the intermediate tissue - CB, as specified by halfway values of the factors - is not seen and reported more often. One answer might be that halfway values are not enough of a stimulus for the cells.
However, it will emerge that CB I is, in small amounts, quite cornmon, but occurs mostly in sites subject to rapid reshaping, so that it is short-lived and hence may escape notice.

The tissue appears to be underreported for another reason. Mindful that CB has been used as evidence for metaplasia, doubters of this phenomenon have seen CB but often have called it something else thus expressing their reservations about its nature origin, or fate, or have left it unnamed altogether..

RELATIONS BETWEEN CB I AND II

Since the most frequently met expressions of CB I and II are very similiar - cells of mostly chondrocytic form and dimensions in a mineralized collagenous matrix - is not the distinction between the two artificial? It is not when the probable origins and the speed of transformation of the tissues are heeded.
In CB II the cells are already differentiated as chondrocytes, so that the key processes are 1) its formation by mineralization, with whatever changes in the cells that calcification requires, and 2) whatever assures its endurance, i.e., its resistance to erosion.

The essence of CB I, on the other hand, is that it could have arisen de novo from a blastema by a particular mode of differentiation of skeletal precursor cells. These cells, starting in a primordial situation, presumably have to elaborate and control more products and processes to deliver CB I than are needed for making CB II from existing cartilage. Also, for CB II the cartilage formation and calcification take longer than the few days needed for subperiosteal chondroid bone I to form in a fracture callus or tumor, or on young cranial bone.

If some distinction between CB I and CB II is not made, the danger exists that one may seek to explain, say, the origin of CB I with evidence acquired from sites of CB II where differentiation runs a different course. One can easily be led to this step in certain locations where chondroid bones I and II both appear concurrently or successively.
Reports by two groups serve as illustrations. The development of the mandibular symphysis of the hamster involves CB I, while in the adult animals the symphyseal fibrocartilage experiences mineralization to take on the form of CB II, but the authors (Trevisan and Scapino, 1976b) do not distinguish the processes involved at the two different times.
Next, Haines and Mohuiddin (1968), in a paper that deals mostly with CB II, refer to the secondary cartilage and CB I of the young alveolar crest as if they were CB II forming by the same kind of mineralization as produces CB II in subarticular cartilage and ligamentous insertions.

The only anatomists to specifically separate two kinds of chondroid bone were Knese and Biermann (1958) who, in describing events at the human trochanter minor, identified "parallel-faseriger chondroider Einstrahlungsknochen" CB II (fibro), and "metaplastisch gebildete chondroide Knochen" (probably CB I).

While chondroid bone I often gives the appearance of having formed directly from germinal cells, nevertheless, it could have arisen in the course of a metaplasia of cartilage to bone. Despite the general skepticism, it has not been possible to disprove such a metaplasia. Indeed, almost every instance of CB I and 11 has at one time or another been taken as evidence of metaplasia, and one of the later tasks will be to assess this interpretation, site by site.

Metaplasia is not only too controversial to be made the basis for classifying chondroid bone, but it also sometimes appears to intervene in several ways around the one skeletal site. For example, Knese and Biermann (1958) referred to "metaplastically formed chondroid bone" by the minor trochanter, and presumably meant the bone they described that forms directly by metaplasia from the cartilage-like tissue - chondroid.
This would follow the practice of referring to some teleost bone as "chondroid bone" to indicate its origin, rather than to identify the preceding intermediate material - the actual CB acting as evidence for the transition.
(Knese and Biermann also applied nearby at the trochanter the name "chondroid bone" to an intermediate tissue forming directly from periosteal cells.)

At and by the joints of long bones, the following metaplasias and chondroid bones have been reported:
calcified subarticular cartilage or CB II (hyaline), and its metaplasia to bone;
a metaplasia of chondroid to bone, implying CB I as an intermediate;
subperiosteal CB I, with a subsequent metaplasia to bone;
a metaplasia of tendon and ligament to "fibrocartilage";
calcification of this "fibrocartilage" to CB II (fibro), followed by its metaplasia to bone; and
a metaplasia of fibroblasts to osteoblasts and bone, but not involving chondroid bone.

Hence, the classification into CB II and I seeks to distinguish between two types of intermediate tissues: those apparently formed by a mineralization of cartilage (CB II), and those where available germinal cells may have formed a tissue falling squarely between bone and cartilage (CB II). This classification avoids commitment to any metaplastic interpretation

CHONDROID BONE TYPE III: MISCELLANEOUS KINDS

The above division brings an initial working order to many examples of chondroid bone. However, there are several other instances of cartilage more bony than usual and atypically cartilaginous bone that lie outside the categories of CB I and II. Some examples are the cementum of guinea pig's molar teeth, the medullary bone of birds, the bone of individuals afflicted with osteogenesis imperfecta fetalis, bone of the otic capsule, and bone seeming to undergo cartilaginous metaplasia. These and other instances are so heterogeneous that any attempt to fit them into anything other than a third, miscellaneous, class of chondroid bones would be unjustified. Their only similarity is that in one way or another they are unlike CB I and II in their appearance and the circumstances of their formation. Some occur deep within the bone, whereas CB I and II are usually at the boundaries of a bone. This position gives CB I and 11 a potentially major role in the skeleton's biomechanical relations with other tissues; this role has been discussed mainly in connection with the concept of secondary cartilages.

SECONDARY CARTILAGES AND CHONDROID BONE

Schaffer's (1888) critique of metaplasia, which still illuminates many current problems in skeletal differentiation, coincidentally gave the first review of chondroid bone and an early listing of secondary cartilages. He argued to such good effect on direct metaplasia that the concept was and has remained discredited outside the histopathological laboratory, and, as a tissue, chondroid bone has languished because of its entanglement with metaplasia. (Something similar befell the hormones, which did not emerge until this century from the oblivion to which the humors of early physiology were cast.)
When the microscope came into wide use, several men rejected the notion that in intracartilaginous ossification cartilage becomes bone. Muller (1858) explicitly held that cartilage is destroyed, and then bone is laid down by a process distinct from the destruction, in holes in the remains of the cartilaginous matrix. His theory of osseous histogenesis (the neoplastic as distinct from the metaplastic) soon gained general, but not universal, acceptance. The few steadfast adherents of the earlier school such as Lieberkiihn (1863) pointed to several instances of an apparent cartilage-to-bone metaplasia as proof of a general, metaplastic, theory of bone formation. In his rebuttal, Schaffer (I 888) made a catalog of these special cases: some tumors of bone, fracture callus, the pubic symphysis, sites of ossification in reptiles, developing horns and antlers, avian tracheal cartilages, the spine of the scapula, the mandible, ossifying avian tendons, rachitic cartilage, the clavicle, the tuberosity of the radius, the bone of teleost fishes, and places at the periphery of long bones by the joints.

This list is most pertinent. It was the presence of tissues intermediate between bone and cartilage at all these sites that led to their use as confiriiiation of an osseous metaplasia of cartilage. The "metaplasists," desperate for support, had thrown into the argument everything chondroid-bony that they could lay eyes on. Thus, the list embraces sites of CB 1, e.g., tumors and callus, of CB II, e.g., ossifying avian tendons and tracheal cartilage, and the very diverse members of CB III, e.g., piscine chondroid bone and rachitic cartilage. In the first place, the list was one of sites of purported metaplasia, the evidence for which was chondroid bone in one form or another. At the same time, many of the locations were also ones that Schaffer (1903) was later to identify explicitly as harboring secondary cartilage, that is, cartilage developing after the formation of the primordial cartilaginous skeleton. The secondary cartilage was in some skeletal tumors, fracture callus, developing horns and antlers, the clavicle, the tuberosity of the radius and other points of attachment to longbones, the mandible's protuberances, and margins of bones of the cranial vault. That these instances comprise a majority of Schaffer's compilation of chondroid bones makes one ask whether chondroid bone, or one of its particular forms, and secondary cartilage are identical; or, if not identical, related in some way.

Early in my search, I found two recent papers which, in asides, equated chondroid bone with an ossified kind of secondary cartilage (Moss, 1958; Gussen, 1968b). What was meant in these papers by chondroid bone was bone formed by a nietaplasia of cartilage that earlier had been present in a large enough amount to be recognized as cartilage. Except for the smallest examples on some of the cranial bones, secondary cartilages develop as tissues that can be assigned unequivocally to the category of non-bony connective tissues. However, while they exhibit no histological affinity with bone, they are not identical with other hyaline or fibrocartilages. Some are partly related to the chondroid tissue of lower vertebrate classes. Schaffer (1930) in particular was reluctant to classify the more fibrous secondary cartilages as "fibrocartilage," preferring instead to group them with fibrous vesicular or chondroid tissue.

Although secondary cartilage cannot be equated with any form of chondroid bone, the two tissues do have intimate relations.

  1. When a secondary cartilage forms, it is often from an existing periosteuni. At the junction between the forming cartilage and old or concurrently forming bone, an intermediate tissue may appear. This CB I could be the secondary cartilage experiencing metaplasia, or be a de novo product of the same germinal population as gave rise to the cartilage.
  2. The histogeneses of chondroid bone and secondary cartilage lend themselves to the same two alternative explanations. Since a secondary cartilage, by definition, forms relatively late, this means the cartilage forms after other tissues have differentiated. The appearance of cartilage in established tissues requires either that a differentiated tissue has transformed -- a metaplasia-or some population of stem cells (i.e., cells with more than one potential) persisted in the maturing tissues.
  3. The mineralization that affects certain long-standing primary cartilages to make them CB II can also establish itself in such secondary cartilages as that of the mandibular condyle and chondrified tendon insertions, to convert part of them to CB II.
  4. Many of the secondary cartilages have been described as undergoing a metaplasia to bone, principally on the basis of their being accompanied by CB I or II. This later metaplasia of which they are suspected is distinct from any metaplasia of a soft connective tissue that might have earlier brought the secondary cartilage into being.
  5. If, as rarely happens, an area of chondroid bone should develop without any clearcut cartilage accompanying it, to the extent that the chondroid bone itself is cartilaginous it might be viewed as a secondary chondrification.

CONCLUSION

I can liken this review of chondroid bone to the fabrication of a category or net and its comparison with other nets that have long trawled the same waters-metaplasia and secondary cartilage, To understand the catch one must know the net. For example, the general order of mesh for these categories has been light microscopic, bringing in cells and tissues, but only recently some ultrastructural and macromolecular phenomena. The net for chondroid bone spans the full distance between bone and cartilage in their common forms, and is aimed at catching for examination any deviation by each in the chondroid or bony direction.

How a tissue may stray from bone or cartilage into the domain of chondroid bone is indicated in a crude way by Table I, which sets some characters of typical bone and cartilage in a matrix, and then shows how mineralized fibrocartilage CB II (fibro), chondroid bone I, and avian medullary bone to some degree straddle the two halves of the matrix. The extent of mineralization in much CB I is unknown, but some studies have shown that it does mineralize (see the chondrodsteoid problem, Chapter 13).

Table 1. Bone-Cartilage Overlapping: Three Examples

TISSUE mineral collagen proteoglycans cell size cell processes BONE **** **** * * **** Hyl CARTILAGE - ** **** *** - CB II fibro **** *** ** *** - CB I *** *** ** *** - Avian medullary **** **** ** ** **** bone

Some entities that do sit astride the bone and cartilage line, as defined by the criteria listed, enter the discussion, but are not to be considered as chondroid bone beyond this point:
calcified hyaline cartilage of endochondral ossification that is resorbed;
certain hyaline cartilages and chondroid of lower vertebrates that have stellate cells (Schaffer, 1930);
most early woven bone, despite its quite large cells with their polysaccharide capsules;
osteoid, which is not simply bone without mineral (Thomas, 1961);
fibrocartilage, even that sharing type-I collagen with bone; and
some other subtle chemical crossings-over, such as the deposition of mineral crystals pathologically in articular cartilage in the condition of "chondrocalcinosis" (McCarty, 1977).

Other criteria distinguishing bone from cartilage are omitted from the table, because they concern only a few instances of chondroid bone. For example, certain cartilages are brought nearer to bone by their trabeculae and vascularity.

After discussing the nomenclature of CB, I shall look at the other nets for their initial construction, their tears and patches, and present state. Thus, metaplasia is much mended but scientifically serviceable, though its offshoot, modulation, is rent. Secondary cartilage started small, was greatly enlarged, then left in pieces, only one of which is in current use.


Chapter 2 NOMENCLATURE OF CHONDROID BONE

Names for chondroid bone
"Spurious" chondroid bones
Knorpelknochen
Knochenknorpel (Osteoidknorpel)
Chondroider Knochen
Chondroidknochen
Chondroidal osteogenesis
Chondroid membrane bone
Osteoider Knorpel
Chondroid bone
Chosen nomenclature

NAMES FOR CHONDROID BONE

Tissues intermediate between bone and cartilage have acquired various names, including "chondroid bone." Chondroid bone or its equivalent foreign term has also been used for tissues other'than those combining characteristics both of bone and of cartilage. If one wishes to restrict chondroid bone to the intermediate tissue, such other applications of the term are spurious, even though they make sense in another way. Those cases where chondroid bone does not mean a bony-cum-cartilaginous material will be reviewed, but first I list almost all the terms that have been used for "true" chondroid bone: Uebergangsgewebe (Brock, 1876); chondroid Modification des osteoiden Gewebes (Kassowitz, 1881); chondroid Cewebe (Grohe, 1899; Axhausen, 1909; Asada, 1927); Knorpelcement (Brunn, 1891); Knochenknorpel (Funkenstein, 1903); chondroider Knochen (Schaffer, 1933); tessuto di transizione (Momigliano-Levi, 1930c); Knorpelknochen, verknocherter Knorpel, mineralisierte vesiculose Bindegewebe (Weidenreich, 1923c); sclerotischer Knorpel (Eggeling, 1911; Heidsieck, 1928); tissu preosseux (Robin and Herrmann, 1882); preosseous tissue (Modell and Noback, 1931); fibrovesicular tissue (Wislocki, Weatherford and Singer, 1947); Chondroidknochen, Chondroid (ZawischOssenitz, 1929a,b); chondroitic tissue (Plasmans, Kuypers and Slooff, 1978); chondro-osseous tissue (Leriche and Policard, 1928); cbondro- osteoid (Urist and Johnson, 1943; Zadek and Robinson, 1967; Hancox, 1972; Buring, 1975); osteochondroid (Aegerter and Kirkpatrick, 1975); osteoidchondroid (Bassett, 1959); chondro- osseoid (Frost, 1973); Mischgewebe (Zawisch, 1953; Altmann, 1964); intermediate secondary cartilage, (Moss, 1958); cartilage (Hunt, 1959); metaplastic bone (Haines and Mohuiddin, 1968); metaplastic tissue (Sprinz, 1970); tissu de transition (Pensa, 1913); knorpeighnliches Mischgewebe (Lehner and Plenk, 1936); osteoid-like material (Melcher, 1971); Knorpel-Knochen and parallel-faseriger chondroider Einstrahlungsknochen (Knese and Biermann, 1958); grone I and II (Beresford).

The diversity of names reflects the various anatomists' wishes to stress the tissue's transitional nature, its possible metaplastic origin, or a kinship of the tissue with bone, osteoid or cartilage. The frequency with which an affinity to osteoid is expressed may have something to do with a common histological appearance of the tissue - bone-like, but not quite the same as proper bone. Another factor could be that the choice of "osteoid" expresses the authors' suspicion that the tissue seen is mineral-poor, although most sightings have been chance ones in routinely decalcified material, where only special stains allow a distinction of osteoid from bone, and even then the mineral content is only inferred.

The names devised are many, suggesting a tissue often met, but puzzling to the observers. However, with only one exception, (Knese and Biermann, 1958), the terms do not differentiate between chondroid bone I and II. My reasons for adding a new name - grone - to so long a list are as follows: First, to provide chondroid bone, the tissue, with a name designating only those tissues intermediate between bone and cartilage. As will be seen shortly, chondroid bone has had many other connotations. Second, to give the tissue a terse, distinctive name having no other associations. Grone, an obsolete form of groin (Oxford English Dictionary), can serve, expressing roots in gristle and bone, but favoring neither tissue. Chondroid bone conveys a bias towards bone. Third, by employing a monosyllable, one avoids the use of cumbersome expressions for the two principal classes of chondroid bone: grone I and grone II; blastemal grone and cartilaginous grone. Fourth, grone avoids the imprecise term "chondroid" discussed below. With time, grone may commend itself to others interested in the tissue.

A few of the terms listed above that have been applied to true chondroid bone not only give no hint of the nature of the tissue, but in several instances "chondroid bone" has referred to materials at some remove from chondroid bone or grone, or quite unrelated to it. Examples of these spurious chondroid bones are given below with some of their historical context.

"SPURIOUS" CHONDROID BONES

The circumstances of the period 1830-1860 in which the nomenclature developed were complex. Microscopists who differed sharply in their belief in metaplasia were attempting to discern orderly patterns among the tissues of many classes, including mammalian and reptilian bone, the special calcified cartilage, acellular bone and osteodentin of fishes, and the varied tissues of rachitic and healing bones, skeletal and extraskeletal tumors. The metaplastic theory of general osteogenesis gave rise to terms expressing the relationships believed to hold between the transforming and transformed tissues. When metaplasia was dislodged as the dominant conception of osteogenesis, the terms lingered on, never to be clearly and completely adapted to the new interpretations of the relations between bone and cartilage. This terminological lag was to be the fate of osteoid, and such hybrids of bone and cartilage as Knochenknorpel, which appear to refer to some kind of bony cartilage or chondroid bone, but in fact merely express an hypothesis on the origin of the bone so named.

Microscopy of sites of normal ossification at first seemed to confirm the prevalent view that in the vertebrae and long bones the cartilage transformed directly into bone and marrow. The osseous transformation involved primarily a gain of calcium salts, to be followed by a secondary metamorphosis to give it a lamellar construction and smaller cells. The bone that was modified cartilage was therefore to be called Knorpel-knochen, and, when its mineral was removed by acid decalcification, it reverted to soft Knochenknorpel.

Knorpelknochen
Knorpelknochen was, therefore, bone formed from cartilage of the primordial cartilaginous skeleton. The term continued in use after the overthrow of the metaplastic interpretation to specify the general embryological history of that skeletal element. Kolliker (1850) classified what we now distinguish as endochondral and membranous bones as primary and secondary ossifications, but exceptions to the temporal sequence and the collapse of the superstructure of hypotheses based on the distinction brought the primary-secondary dichotomy into disfavor. However, we still speak loosely of the "endochondral" bones of the body to call attention to the nature of the tissue that preceded them. The many attempts at improving on the membranous-endochondral terminology have not been accepted, mostly because of their clumsiness, as admitted by Felts (1961) in making his offerings: "cartilage-preceded and non-cartilage-preceded skeletal organs."

Thus, names such as Knorpelknochen (Virchow, 1853, 1884) and chondriform bone (Adami, 1908) mean only bones of endochondral origin, ignore the precise relation, if any, of particular regions of the bone's bony tissue to the original cartilaginous primordium, and do not imply that the bone present is now at all cartilaginous. But, Knese (1978b) uses Knorpelknochen in the legend to Figure 178 in the sense of true CB for a tissue "with cells very like chondrocytes in a matrix like bone's" (Chapter 9).

Knochenknorpel
Knochenknorpel (Osteoidknorpel), the pliable material resulting from the experimental demineralization of bone, could nowadays be known as osteoid, but an abnormal osteoid produced retrogressively, as opposed to osteoid that arises by the production of an organic bone matrix whose calcification lags or fails to occur. The early ideas of osteoid and Knochenknorpel were concerned with the tissues of four situations: the formation of the cranial vault; the calcification of fibrous connective tissues such as tendons; the histogenesis of skeletal tumors; and the changes in endochondral bones in rickets. Virchow in particular dealt with the last three pathological circumstances, and his influence and belief in metaplasia were to prolong the pathologists' misconception of osteoid as a cartilaginous kind of tissue.

The linking of the two tissues came about primarily because of the strength of the conviction of Reichert (1849) and others that even the flat cranial bones were transformed cartilage. Meyer (1849a) cited as part of the evidence for this event what were probably the small regions of secondary cartilage and chondroid bone often present at cranial bones' margins, his sogenannte Nahtknorpel, best seen on the parietal bone of the 11/2-2-year-old child. Reichert, on the other hand, was more definite in stating that the tissue preceding the bones was not hyaline cartilage. He suggested instead that it was faserig or hautig knorpelig (fibrously or desmally cartilaginous). Kolliker (1850b) disputed the cartilaginous nature of the tissue, because it contained neither chondrocytes nor yielded chondrin, but only held collagen and indifferente Bildungszellen. Nevertheless, the name Hautknorpel (desmal cartilage) stuck as one term for the tissue giving rise to the flat and facial skull bones, an acceptance perhaps reinforced by a reluctance of some, to recognize two different routes in bone formation. The basic mechanism of initial osteogenesis was viewed as the same everywhere, namely, the deposition of mineral, and this undoubtedly occurred in the soft precursory tissue of the skull bones.

Virchow (1853, 1863) examined all kinds of pathological specimens, and "normal" bones of all ages, the latter probably including some affected to a degree by the very prevalent rickets. Because of the widespread occurrence of regions of transitional tissues in such material, he proposed that bone, cartilage, connective tissues, and marrow could experience metaplasia into one another. A potential distinction was between hyaline cartilage, which gave rise to Knorpelknochen, and other tissues that apparently could turn into bone, such as fibrocartilage, and another material - osteoides Bindegewebe, osteoide Bindesubstanz or Osteoid. The last was to be found under the periosteum of long bones, turning into the bones of the face and cranial vault, and comprising many skeletal neoplasms, and a good part of the rachitic skeleton.

The variety of the situations in which the osteoid material was held to occur, especially by embracing tumors and calcified tendons, meant that different tissues were subsumed under one name. Already in 1858, Muller warned of the imprecision of osteoide Substanz, and was careful to call the material that he saw deposited first by osteoblasts by a different name - osteogene Substanz, which matches exactly what is now known as osteoid: poorly mineralized bone with nothing especially in common with cartilage save a negative -- they both are mineral-poor.

Virchow (1863) was in a position to emphasize the difference between osteoid tissue and cartilage, because he stated that microscopy revealed Osteoid to be quite different from the usual kinds of cartilage, including fibrocartilage. But since Osteoid mineralized and appeared to participate in intramembranous ossification in a way similar to his conception of the metaplastic role of cartilage in long bones, he could not forbear to endorse the use of the term Hautknorpel as a synonym for Osteoid.

An immediate consequence was that he included tumors composed of osteoid among the chondromas as the osteoiden Chondrome. Before long, tumors with various combinations of osteoid, bone, cartilage, the intermediary chondroid bone, and sarcomatous tissue, were categorized by this name, leaving osteoid with no agreed morphological meaning in pathology. Even now, Jackson, Reckling and Mantz (1977) recommend a clinical distinction between "osteoid osteoma" and "osteoblastoma," although "significant histological differences could not be demonstrated among our cases."

From what is now known of the extremely narrow width of the osteoid seam in the bones of well nourished humans and animals (for example, Kassowitz, 1911; McLean and Bloom, 1940, Bonucci, 1977; Fornasier, 1977), it is likely that the "normal" bones where Virchow saw his Osteoid were somewhat rachitic. Although many of the circumstances in which osteoid was seen were abnormal, and the idea that it underwent a metaplasia to bone was ill-conceived, the tissue undoubtedly exists and needs a name: (Knese and Knoop (1961) suggested praossale Gewebe). For example, the bone of many tumors is poorly mineralized (Sela and Boyde, 1977) and is truly osteoid, and normal osteoblasts generally deposit collagen and other macromolecules before mineral crystals precipitate. Maturation of the collagen and mineralization are not instantaneous, so that osteoid is not one homogeneous entity, but itself is divisible into zones (Knese, 1963; Fornasier, 1977).

The history of the term osteoid has been such that when dealing with neoplasms and rickets where osteoid, bone, cartilage, and true chondroid bone may be present, one has to be very wary of names suggesting the presence of a chondroid bone. For example, Osteoidknorpel may turn out to be mere osteoid (Funkenstein, 1903), whereas Virchow's (1853) rachitic knorpelig-osteoides Gewebe had more morphological claim on an intermediate status, but was probably cartilage (Chapter 23).

Much chondroid bone I probably is poorly mineralized and should therefore be called "chondroid osteoid," but osteoid is so enveloped in equivocation and chondroid is even more imprecise (see Chondroidal Osteogenesis below) that to couple osteoid with chondroid must spawn misconceptions. Except in the instance of CB 11, chondroid bone as used hereafter accordingly carries no implication as to the tissue's state of mineralization.

Chondroider Knochen
Chondroider Knochen was Schaffer's (1888) infelicitous choice to describe young forming bone in the mandible. The trabeculae within the condyle comprised a bone with large, closely spaced cells, between which was a matrix not staining as strongly as more mature bone, but reacting more like cartilage with the limited methods available to him - Busch's hematoxylin and eosin, picric acid, and safranin. For its cartilaginous features, Schaffer called the tissue grosszelliger chondroider Knochen, but he was in no doubt that it was bone. (In the condyle, he also observed a transitional tissue where the bone's periosteum was about to form cartilage. This Uebergangsgewebe is a chondroid bone.)

In many forms of "true" chondroid bone, the cells are large because the are more like chondrocytes than osteocytes, but the mere plumpness of the osteocytes in immature bone does not thereby make young, fast-growing bone cartilaginous enough to be chondroid bone.

Chondroidknochen
Chondroidknochen was kept alive by Schaffer's colleague, Zawisch-Ossenitz (1927, 1929a,b, 1953), but she robbed it of meaning by putting it to not one but several uses. First, she applied the term, as Schaffer did, to young bone, but then subdivided chondroid bone into fiber-less or fine-fibered versus coarse-fibered. Coarse-fibered chondroid bone was simply bone, but some subperiosteal instances of fiber-less chondroid bone almost certainly were true chondroid bone, others were just large-celled bone. At other times, Zawisch (1953) referred to true chondroid bone as a Mischgewebe. Finally, she believed the eosinophilic matrix around the canals in hyaline cartilage to be Chondroidknochen. Thus, her Chondroidknochen sometimes was not true chondroid bone.
Chondroidal Osteogenesis
Chondroidal osteogenesis (Moss, 1961) one might expect to lead to chondroid bone, but the matter is far from that simple, for there is more than one kind of chondroid. In German, Chondroid found a use as a noun (Schaffer, 1930) to designate tissues intermediate in nature between the chordoid or notochordal kind and hyaline cartilage. It is more common in lower animals than in mammals, where it is restricted to sesamoids, ligaments and extraskeletal sites such as the penis, heart and tongue of particular species. Chondroid also goes under the name "vesicular tissue" because of its large pale cells.

In German, chondroid is the adjective signifying a relation with the tissue Chondrold, e.g., chondroid Gewebe, but also has the usual sense of cartilaginous, as it has in English. Occasionally, chondroid is used as a noun in English, but this is rare; far more often chondroid means cartilage-like and carries no implication of the tissue Chondroid, as it occurs, say, in fish. Unfortunately, since there are exceptions to this general practice, chondroid cannot be used without qualification. The following instances will illustrate the difficulties the word can introduce.

Clinicians dealing only with the skeleton of mammals generally apply chondroid not to identify the kind of tissue in question as Chondroid, described by Schaffer and his predecessors and still being studied as a tissue in its own right with its own subtypes (Cowden, 1967; Curtis and Cowden, 1977), but to mean that it has a resemblance to hyaline cartilage. For example, Urist and Johnson (1943) wrote of chondroidal osteogenesis for what seemed to be a direct transformation of cartilage to bone in fracture callus; Yamagishi and Yoshimura (1955) called the cartilaginous tissue at a fracture site chondroid, and Cotchin (1958) employed the same name for the cartilage in mixed tumors such as osteochondromas.

Last, Meachim and Osborne (1970) and Meachim (1972) defined the Chondroid developing in osteoarthritic femoral cartilage thus: "this tissue contains rounded cells resembling chondrocytes, but has an intercellular matrix that shows little or no basophilia with haematoxylin and remains uncoloured after the section has been stained with toluidine blue."

In distinct contrast, Moss's (1961) chondroidal osteogenesis described the process in teleost fishes whereby Schaffer's Chondroid is transformed into some of the animal's bones. Chondroidal osteogenesis in fishes thus involves a direct metaplasia of chondroid to cellular bone (Moss, 1961, 1962), the cells of which may then disappear, leaving an acellular bone. The bone forming in this way could be called chondroid bone, but since it no longer has anything about it of chondroid or cartilage, the name is inappropriate in the present context. On the other hand, in the course of such an osteogenesis, while the somewhat cartilage-like tissue is changing into bone, a tissue should be present with cartilaginous and osseous characteristics, and would be a kind of true chondroid bone.

One more use of chondroid should be mentioned. Zawisch-Ossenitz (1929a) had Chondroid as an abbreviation for chondroid bone, which is doubly unhelpful since the German name by custom refers to an altogether separate entity, and her use of the full term was itself ambiguous (see Chondroidknochen earlier and Chapter 9).

The multifarious meanings attached to chondroid make one reluctant to apply it to bone in the chondroid-bone sense used throughout this review. It won selection only by being the least ill-suited to the task of introducing and defining an elusive entity with a long history of periphrasis and mistaken identity.

Chondroid Membrane Bone
Chondroid membrane bone was a name introduced by Gussen (1968) while reporting her study of the labyrinthine capsule. Her papers (1968a,b) are accessible and can mislead the unwary reader on the nature of chondroid bone. She failed to appreciate the difference between chondroid as a tissue and secondary cartilage. Thus, she wrote, "Moss (1961) describes secondary cartilage in fish bone, but refers to it as chondroid," and "Enlow (1962a) refers to secondary cartilage as chondroid."

Her substitution of the name chondroid cartilage for secondary cartilage trapped her into this definition: "Chondroid bone is, therefore, membrane bone which, instead of being formed directly has gone through a stage of secondary cartilage formation and has continued directly through this secondary cartilage stage to bone."
Thus, her chondroid membrane bone is, like the final tissue of Moss's (1961) chondroidal ostedgenesis, real bone, although its claimed path of development is out of the ordinary. The development may indeed have involved true chondroid bone while cartilage was becoming bone, if that direct transformation actually took place. Gussen's (1968b) use of secondary cartilage, chondroid and chondroid cartilage as synonyms was unhelpful.

Osteoider Knorpel
Osteoider Knorpel was Schaffer's (1888) fortunately short-lived name for the calcified hypertrophic cartilage on which bone (his chondroider Knochen) was laid down in the young mandibular condyle.
Chondroid Bone
Chondroid bone could be applied to true bone formed by osteoblasts derived from chondrocytes freed, alive, from their lacunae by resorption, if such an event really happens. To the same degree, the name would also fit bone formed by a conversion of the matrix of cartilage after the death of the chondrocytes. Both eventualities have had their spokesmen in the not too distant past. As a potential example of the former, Lufti (1971) wrote of "cartilage bone" lining the spaces resorbed in the chick's tibial cartilage. Whether the term referred to bone formed by the many chondrocytes he believed to have dedifferentiated after surviving erosion, or to the "areas where the calcified cartilage is apparently directly transformed into 'bone' remains unclear.
Chosen Nomenclature
I shall employ chondroid tissue or Chondroid as Schaffer defined it and, except for calling cartilage-like bone chondroid bone, elsewhere use only the term cartilaginous when cartilage-like properties are involved so as to avoid the use of chondroid as an adjective synonymous with cartilaginous. The name chondroid bone is too well embedded in the literature as the name of some intermediates between bone and cartilage for it not to be the choice to start the discussion. However, a new name - grone - is called for, unencumbered by the assorted associations of a century.

In conclusion, this survey has to consider: 1) whether or not any tissue called by a name implying that it falls between bone and cartilage proves to be deserving of the name; and 2) all intermediates of cartilage and bone, even though they may never have been called chondroid bone.


Chapter 3 SECONDARY CARTILAGES I

. Introduction and overview
. Secondary cartilage: definitions and listings
. Schaffer's category of secondary cartilage
. Secondary cartilage as lately defined
. Compilation of secondary cartilages on Schaffer's criterion
. Anomalous secondary cartilages
. Fuch's refutation of secondary cartilages
. Secondary cartilages: comparisons and special studies
. Explicit comparisons
. Between secondary cartilages
. Between secondary and primary cartilages
. Some individual histochemical and ultrastructural studies
. General remarks

INTRODUCTION AND OVERVIEW

To the query, what is secondary cartilage? there have been two answers, each by itself quite straightforward. One is that it is the cartilage forming on bones of membranous origin; the other, obviously far broader in scope, is that it is any cartilage that is not primary. The existence of these two different but related answers is one source of difficulty in defining the concept. Another difficulty is that, as usual, various other terms have been proposed in place of secondary, and applied haphazardly for one or another of its principal meanings.

Aside from differing times of first formation, can one otherwise justify a separation of secondary chondrogenesis from primary? Part of the answer has been thought to lie in the possible extrinsic mechanical etiology of secondary cartilage, a hypothesis resting on many disputed observations and experiments. The biomechanical arguments are an impediment to following the development of the concept of secondary cartilage chronologically.
Another handicap to a speedy progression of ideas is my choice to precis the reasoning of Fuchs and Altmann in more space than it has had in the English literature, because respectively they offer the most detailed counterarguments to the notion of secondary cartilage and the role of extrinsic forces in secondary chondrogenesis. For an overview, here in outline are the main topics and historical landmarks in the story of secondary cartilage.

  1. For most of the skeleton, cartilage precedes bone in development, but some cranial bones form without cartilage (Nesbitt, 1736), and on long bones bone is added subperiosteally when cartilage is not longer present (Duhamet, 1743).
  2. Cartilage can form on such bone as has had no previous direct cartilaginous associations: When these chondrifications came to light, they were more than just intriguing illustrations of the skeleton's potential for differentiation. Since the common theory of the 18th and early 19th centuries was that cartilage preceded and turned into bone, they represented a startling turnaround in the conceived usual order of events. To-this day, they have retained their fascination for skeletal biologists.
  3. The cartilages appearing on the forming cranial bones, especially the mandible, came to be called accessory (Stieda, 1875). Also, they differed subtly in their histology and manner of ossification from primary cartilages (Schaffer, 1888).
  4. The cartilage forming on long bones because of rubbing or fracture obviously came after the establishment of the primary cartilaginou skeleton, but so did fetal cartilaginous accessory formations on membrane bone, such as the mandibular condyle, and by a much closer margin.
  5. With nineteenth-century biologists applying microscopy wholesale to the structures of vertebrates, many instances emerged of chondrogenesis that occurred later and was separate from the primordial cartilage, either as normal "extraskeletal formations," e.g., in tendons and antlers, or pathologically ectopic in soft tissues. (Many of these cartilages experienced ossification.)
  6. The early idea of Duhamel, Hueter, Virchow and others that mechanical stimulation caused by movement could elicit chondrogenesis was adopted by Kassowitz (1881) and Schaffer (1897) as a general explanation for the formation of any cartilage appearing after the construction of the primary skeleton. Kassowitz had particularly in mind the cartilage of fracture callus and at what he called apophyses - in essence any cartilaginous protuberance on bone - and Schaffer the transformation at tendinous insertions. Although the two men dissented over whether such cartilages could experience a direct metaplasia to bone, their thinking coincided on the probable role of external mechanics and in viewing all the diverse late cartilages as one class: for which the name secondary came into style (Schaffer, 1930).
  7. The work of Roux (1895, 1897), who tried to put the mechanics and mechanisms of developmental processes on a more rigorous and experimental basis, gave impetus to a biomechanical interpretation of secondary cartilage. For example, Rathcke (1898) believed muscularly induced stresses to have caused the chondrogenesis in a case of myositis ossificans,
  8. Kassowitz (1881), however, had held that some cartilage, especially of primary structures, appeared to form where no obvious mechanical action existed. Roux, too, envisaged a period when the primordial cartilage underwent an intrinsically determined differentiation, before coming under the influence of outside stresses.
  9. Experimental evidence started to accumulate that implied that mechanical factors such as pressure did not always precede secondary chondrogenesis. For example, Kapsammer (1898) found cartilage in the medullary canal of broken bones and under infected periosteum, and Solger (1892) questioned the validity of Wolff's and Roux's hypotheses for certain extraskeletal ossifications.
    Thus, the pattern of future developments was already established by the turn of the century.
In tracing the history and nature of secondary cartilage in more detail, some confusion can be dispelled if "secondary" be kept for all late, nonprimary cartilages, "accessory" for cartilages on membranous bones, and the existence of secondary cartilages be treated apart from hypotheses on what caused the cartilage to form.

Table II

The concept of secondary cartilage in its broad sense is of value for three reasons. It calls attention to the great extent of chondrogenesis within the tissues (see Table II) and over the individual's lifetime: a potential for differentiation to be considered in any scheme seeking to explain how cartilage arises and the significance of the tissue. Second, by contrasting secondary with primary cartilage, one is obliged to seek answers about their differences, not only in what elicits chondrogenesis and its part in the different timing, but in their morphology and functions. Comparisons between some secondaries and primaries and among secondaries are dealt with briefly on pp. 41-43. link Third, any secondary cartilage may involve chondroid bone or metaplasia in its formation or fate, so that the broad category provides an inventory of structures to be checked for these phenomena.

SECONDARY CARTILAGE: DEFINITIONS AND LISTINGS

Instances of secondary cartilage were noticed long before their histogenetic significance could be appreciated. Thus, in the eighteenth century cartilage was known to occur in healing fractures, and Duhamel (1742) observed cartilage where a rachitic deformity had caused the ulna to rub against the radius. The prevailing conception of osteogenesis then was that all bone was cartilage transformed by mineralization, so that any cartilage appearing later on a bone could be ascribed to the same mechanism that had established the earlier cartilaginous primordium, or to a metaplastic reversion of bone to cartilage. The concept of a cartilage's being secondary came only after a revolutionary idea had taken hold: that most bone was not transformed cartilage, but formed by an independent process.

The groundwork for this neoplastic hypothesis of osteogenesis, as opposed to the metaplastic, had been laid by Nesbitt (1736), who saw that the bone of the skull's vault was not preceded by cartilage, and, according to Rollett (1870), by Grew (1681) and Havers (1691) whose suggestion that bone forms on long bones under the periosteum where cartilage is no longer present was confirmed experimentally by Duhamel (1743). The observation that most cranial and facial bones develop without a cartilaginous precursor was to be the key to establishing the idea of secondary cartilages. This does not mean that secondary cartilage can only appear in relation to craniofacial bones, only that for bones arising endochondrally there was always the suspicion that cartilage developing under the periosteum was somehow related to the primordial cartilage. However, if, as in the mandible, there was no cartilaginous precursor, the cartilage arising after the onset of osteogenesis was clearly a formation temporally, spatially and histogenetically distinct from the primary cartilages.

An accurate description of the tissues and events involved in the formation of the skull had to await the microscopy of Reichert, Kolliker, Virchow and others outlined in Chapter 11. Together they showed that many cranial and facial bones formed directly from a soft tissue that was not the hyaline cartilage of long-bone development, but then truly cartilaginous additions appeared at certain protuberances, most noticeably at the mandibular condyle where an articulating surface was needed.

The principal problem of nomenclature was to distinguish this new mode of ossification from that based upon preformed cartilage. The wrangle over Kolliker's secondary-primary terminology and its phylogenetic significance overshadowed the issue of a collective name for the small cartilages appearing on the secondary or membranous bones. It appears to be Stieda (1875) who systematically listed the cartilages of the mandible under the designation accessory cartilage nuclei. These were on the condyle and angular process, and anteriorly at the symphysis. Accessory conveyed the thought that the cartilage was not critical to the mandible's origin.

The accessory cartilages differed from other hyaline cartilage in their appearance and relations with adjacent bone, and they mostly disappeared later in development. These factors led to the cartilages joining the variegated company of sites of purported chondro-osseous metaplasia. Anatomists of the metaplastic school fell back to the argument that, "The primordial cartilages may ossify by destruction and replacement, but many other cartilages ossify directly by metamorphosis and at these sites there is the chondroid bone to prove it." The most persistent proponent of this dual hypothesis was Kassowitz (1877, 1881).

Kassowitz believed that a metaplasia of cartilage to bone occurs in two kinds of situation: fracture callus, and at what he called sites of apophyseal growth. His apophyses were the coronoid, angular and coronoid processes of the mandible, the ends of the clavicle, antlers, the scapular spine, and certain tuberosities on long bones, e.g., of the radius. Thus, the restricted concept of cartilages accessory to membrane bones was broadened by situation and significance: from the craniofacial bones to the clavicle; from bones formed in membrane to bones principally of endochondral origin, such as the scapula and the radius; and from normal development to sites of fracture.
Kassowitz seems to have used no term to embrace both his apophyseal cartilages and that of fracture callus, but all of them, with the possible exception of the scapula's spine, are examples of chondrogenesis independent of the primary cartilages.

Schaffer (1888) used his painstaking histological analysis of the forming mandibular coronoid and condylar cartilages to argue against the occurrence of skeletal metaplasia at any of the sites proposed by Kassowitz and others. As noted earlier, the sites of postulated metaplasia rejected by Schaffer included a number of cartilages formed separately from the primaries: in some tumors, fracture callus, horns and antlers, the spine of the scapula, mandible, the clavicle, the cranial vault, and the periphery of long bones near the joints. What these cartilages had in common did not cause Schaffer to name them collectively at the time, but germinated in his mind as he embarked on his extensive studies of cartilage in all its forms.

For the mandibular cartilages, Schaffer used Stieda's term "accessory cartilage nuclei." He emphasized that the cartilage was hyaline and played a provisional role for the growing bony mandible, but noted that the scant matrix stained weakly with hematoxylin and thus was immature (unreif). This constituted an early argument that secondary cartilages, at least those accessory to cranial bones, were distinctive for their histology, as well as by the timing and circumstances of their development.

The cartilages forming on established bones appeared to come from periosteum. This was unexpected activity for a periosteum and called for an explanation. One was forthcoming from some earlier observations that had led to the suggestion that mechanical disturbances can stimulate a periosteum to chondrogenesis. By the turn of the century, Schaffer had seen or learned of many examples of late chondrogenesis, had noted that their cartilage's histology was sometimes distinctive, and he was able to link some of them with a prior mechanical disruption. His category of secondary cartilages had taken shape, in the form in which it was to attract the criticisms of Fuchs (1906, 1909b, see p. 38link).

Once the concept of accessory cartilage nuclei had been broadened by Kassowitz and Schaffer to encompass late-forming cartilages, it could not be kept from expanding until it took in all non-primary cartilages. That became Schaffer's explicit position when he offered his major compilation in 1930, based upon his extensive experience with all kinds of chondrifications and the voluminous literature then accumulated.

SCHAFFER'S CATEGORY OF SECONDARY CARTILAGE

Schaffer defined as secondary or accessory any cartilage forming after and separate from the primary cartilaginous skeleton. His sites for secondary cartilage seen during development were: the mandibular symphysis, condyle, coronoid process, angular process, and alveolar processes; the ends of the clavicle; on several bones of the cranial vault; on the maxilla and its alveolar processes; and on the forming penile bone of many mammals. In addition, the cartilage participating temporarily in fracture-callus was secondary.
Secondary cartilages that developed and persisted were in: the sesamoids of interphalangeal joints; the muscle fascia of the mole's back; the hearts of some animals and the diaphragms of others; the sclera and nictitating membranes of many species; the lyssa of the tongue; the penis, and more rarely, the clitoris of some mammals. Finally, all elastic cartilages were secondary.

Schaffer drew a distinction between the foregoing secondary cartilages that arise by normal development in the appropriate species, and, heterotopic cartilages that are occasional abnormal formations not typical of the site in that species, e.g. cartilage in human arterial walls. Schaffer viewed heterotopic cartilages as a subclass of secondary ones, since they too arise after and independently of the primary skeletal cartilages, but result from abnormal stimuli.

Schaffer's list obscured the older distinction between accessory and other late-forming or secondary cartilages, embraced the unusual in extraskeletal cartilages, and the pathological with fracture callus and heterotopic cartilages, but omitted the antler, cartilaginous tumors, and the scapula's spine which had been brought into consideration by Kassowitz (1877).

These subdivisions will be taken up in due course, but first there is a fundamental problem. The secondary cartilages in Schaffer's (1930) compilation are so heterogeneous that the classification appears at first sight to lack what gives any system of sets meaning: that each set comprises only members having something significant in common, say, a developmental process or a function.

The first group of secondary cartilages that Schaffer specified form in a close relation with the periosteum of pre-existing bone. He expressly attributed most of these formations to mechanical influences arising from use, to pressure, in particular, citing Strasser (1905), who had postulated pressure as the likely stimulus for the cartilage found at the articulating end of the pterygoid bone in the dove.

There is reason to believe that Schaffer conceived of all the diverse cartilages in his secondary category as owing their origin to some mechanical stimulus, thereby justifying their inclusion in one group. Another secondary periosteal cartilage is that of healing fractures, and Schaffer (1930) mentioned authors of the last century who believed that mechanical factors such as pressure, pull, and shear to be the stimuli for the periosteal chondrogenesis of broken bones. These authors included Kapsammer (1898), who showed that stabilization of the fracture went along with an absence of chondrogenesis. Regarding the remainder of his secondary cartilages, Schaffer did not consider mechanical stress in the histogenesis of elastic cartilage, but for some of the other hyaline ones, for example, scleral cartilages, muscle pull was discussed as playing a role in their development. Schaffer (1916) also attributed the development of the symphyseal cartilage to the mobility of the two mandibular halves.

Omitted from Schaffer's list (1930) of secondary cartilages was the first firm tissue in the growing antler. Schaffer may have left it out because it did not accord with his attempt to unify the diverse secondary cartilages by one main mechanical cause, or, as Hall (1978) points out, because it was not universally accepted as cartilaginous (Chapter 13).

SECONDARY CARTILAGE AS LATELY DEFINED

By excluding from Schaffer's secondary cartilage 1) the elastic cartilages; 2) cartilages in sites atypical for most mammals - what may be termed extrakeletal cartilages, e.g., cardiac, diaphragmatic and lyssal; and 3) the abnormal heterotopic cartilages, one approaches the current idea of seeondary cartilage. They are now those formed by the periosteum of an existing bone, with the implication, or the explicit stipulation (e.g., Patterson, 1977), that the bone is of dermal origin.
The narrowing of the once broad concept of secondary cartilages did not take place in an openly agreed way. It happened more by the focusing of interest on the cranial bones and by change and restitution in the nomenclature of secondary cartilages, for which de Beer and Murray were principally responsible.
De Beer (1937) in his book on the development of the vertebrate skull reviewed the early theories of ossification and endorsed Schaffer's (1930) classification of secondary cartilages. Schaffer's concepts were thus broadcast to the English-speaking world in the context of the cartilages on cranial bones whose histology, form, etc. de Beer discussed.
Murray was the next to grapple seriously with secondary cartilages, coining a new name and invigorating the topic by controlled experiment. Although he himself did not exclude pathological sites or chondrogenesis on endochondrally formed bones, his experimental work on avian cranial bones furthered the practice of restricting the accepted sense of secondary cartilage.

Murray (1954) divided mammalian cartilages into three categories: 1) those of the primordial cartilaginous skeleton; 2) those formed by normally nonchondrogenic skeletal tissues, and probably in response to mechanical stimuli, e.g., fracture callus cartilage, and that formed by tibial and fibular periostea as a prelude to fusion in young rats and guinea pigs; and 3) cartilages induced ectopically by experiment, or appearing ectopically as pathological formations. Of these, categories 2 and 3 were secondary. In a short paper (Murray, 1957), he drew attention to the many sites of secondary cartilage on membranous bones of the chick's skull, and added to his list of secondary cartilages those developing at pseudarthroses. As Schaffer had, Murray called the cartilages secondary "because of their late development."

In introducing his major work on chondrifications at joints in the chick skull, Murray (1963) gave thought to a term which would express the situation more satisfactorily than did the term secondary for cartilages that develop on membrane bones, e.g., on the mammalian mandible. He chose the term adventitious, used by botanists for structures arising in unexpected places, but he granted that there is nothing accidental or casual about these cartilages.
Right away his neologism stumbled, first over the ectopic cartilages to which, as he wrote, "adventitious" applies even better; and second over the separate meniscal cartilages within the chick's cranial articulations which owed their development to the same masses of germinal cells as gave rise to the cartilages upon the membrane bones. He excluded the chondrified articular membranes, because they differed histologically from the cartilages on the bones, and because of the practical consideration of the need in his further discussion for a term to encompass only the latter cartilages.
Murray did not mention the term "accessory," which has nearly the meaning he desired, although the status of meniscal cartilages would still have needed special clarification.

His last words on secondary cartilages in general clarify his position vis-a-vis Schaffer's: "Thus the main distinction lies between the cartilages which are components of the chondrocranium or of other parts of the embryonal cartilaginous skeleton, and those, the adventitious cartilages, which develop in separation from it, and apparently independently of it. This seems also to be the criterion used by Schaffer (1930) in distinguishing primary from secondary cartilage. Among the adventitious cartilages are those which develop in all normal individuals of a species, including the examples described in this paper and those occurring in the skull bones of many mammals, and those which are occasional occurrences, and may often be associated with pathological processes (like cartilages in the walls of blood vessels, or in repairing fractures), or may be induced experimentally."

After 1963, Murray's term, adventitious, was favored for a while by him and his colleagues. Durkin (1972), from extensive histological work on the mandibular condyle (Durkin, Heeley and Irving, 1972), then proposed embryonic cartilages instead, writing that his intent was to draw attention to the special character of such cartilages rather than to alter the terminology.

Trevisan and Scapino (1976a) take Murray and Durkin to task for tampering with the nomenclature, and claim "the term 'secondary cartilage' has historical priority and its general meaning is well known." To the contrary, "accessory" seems to have priority, and the general meaning is uncertain.
Durkin's term brought home the points he intended-that mandibular condylar cartilage is, as Schaffer (1888) noted, unreif, and in its endochondral growth it differs from epiphyseal plates in many ways.

Trevisan and Scapino (1976a) themselves offer a narrow and clumsy definition: "Secondary cartilages appear in association with membrane bones and are temporally and physically distinct from primary cartilages." The definition beggs the question of what to do with cartilages that are distinct from primary ones but unrelated to membranous bone. Also, the same authors view the mechanical evocation of secondary cartilages as settled.
A mechanical stimulation of chondrogenesis has been shown for very few secondary cartilages, and Murray (1957) admitted in his first experiment that the mechanical hypothesis snagged on one avian secondary cartilage that had behaved anomalously.
Although, since then, movement, or factors related to it, has been proved necessary for several secondary catilages to form on avian cranial bones (Murray and Smiles, 1965; Hall, 1968d), and normal use is needed to maintain the recruitment of chondroblasts (Hall, 1978, 1979), the mandibular condylar cartilage develops from its blastema in vitro in the absence of movement (Glasstone, 1968, 1971); and it is hard, though not impossible, to see movement as evoking the growth cartilages of the antler (Goss, 1970), or penile bone (Ruth, 1934), particularly since the latter forms without a direct connection with muscles and in infant rats. For now, there is insufficient experimental evidence to base the category of secondary cartilages on the mechanical stresses associated with movement as the stimulus for chondrogenesis.

COMPILATION OF SECONDARY CARTILAGES ON SCHAFFER'S CRITERION

Over some forty years Schaffer developed the concept of secondary cartilages as ones forming after the primary cartilaginous skeleton. Holding to that one clear criterion, but noting the anomalous timing of clavicular chondrogenesis, his category can be modified somewhat in its subdivisions and slightly extended to give the following situations (for a listing in extenso see Table II, p. 26).
I. Cartilage formed by periosteum or endosteum
    a. on membrane bones during 1) normal development, 2) healing, or 3) the formation of tumors
    b. on endochondral bones under the same three circumstances
    c. in cultures or grafts
2. Cartilage formed ectopically and abnormally in soft connective tissues
3. Extraskeletal cartilages formed as normal developments in uncommon sites
4. Elastic cartilages
Schaffer kept aloof from the histogenesis of cartilaginous tumors, and also did not include I.c., although he had discussed this earlier (Schaffer, 1903). Categories I.a. and b. are separated not because the evidence favors a real difference between the periostea (it does not), but because the distinction has been proposed and tested, starting with the periosteal grafts of Buchholz (1863).

The current idea of secondary cartilages is confined to I.a. I) - only one expression of a far wider phenomenon of late-forming cartilages. There is clearly some justification in considering membrane-bone chondrification separately, but at the risk of overlooking the extent of secondary chondrogenesis and the possible existence of common mechanisms, for example, the role of induction in the formation of pathological ectopic cartilage and the natural avian scleral cartilage (Toro, 1935).

The setting aside of elastic, ectopic and extraskeletal cartilages is better made as a conscious and explicit act than an unwitting one. Indeed, Schaffer himself provided some basis for not grouping elastic and extraskeletal cartilages too closely with skeletal secondary chondrogenesis.

Schaffer (1930) labelled the major elastic cartilages as secondary for three reasons: 1) their relatively late development; 2) because in the same organ in different species other tissues, such as hyaline cartilage or adipose tissue, took the place of elastic cartilage, showing that elastic cartilage was not the obligatory skeletal tissue for that organ; and 3) elastic cartilage could merge into other tissues and structures, such as the vocal cords. Here, in 3), what Schaffer was trying to convey is hard to divine. This may be an allusion to the usual existence of primordial cartilages as discrete pieces enclosed in perichondrium, in contrast with the development of secondary cartilages within and merged with connective tissue.

The inclusion in the class of some of the extraskeletal cartilages could be queried on the grounds that the tissue is more chondroid than cartilaginous, but this distinction (discussed in Chapter 9) may have little to it.

Thus, the secondary cartilages have no manifest unity, being in the first place merely non-primary cartilages. Even those of I.a. 1) cannot be unified by a known mechanical etiology, and they differ from one another in some aspects of their morphology and behavior. This last is a clue to one use for the broad classification of "secondary cartilage": to encourage observation, experiment, and thought on how to break it into entities more meaningful for the nature of cartilage and the underlying mechanisms of chondrogenesis.

The remarkable extent of the late chondrifications is indicated in Table II, where Schaffer's (1930) examples are on the left. On the right I have added others that are eligible by the criterion of appearing late. By holding strictly to the criterion of timing, certain cartilages formed in close association with primary cartilages are included in the secondary category. These anomalies are starred in the table and discussed below.

ANOMALOUS SECONDARY CARTILAGES

The structures starred as questionable members of the class of secondary cartilages fall into three categories: 1) those that share other characteristics with the conventionally recognized secondary cartilages; 2) others whose histogenesis is too obscure for one to know whether they are secondary or primary, and 3) some that undoubtedly develop from primary cartilages, but do so late.
Category 2
Category 2) is exemplified by the bar of cartilage, observed (Figure 5, Yagiela and Woodbury, 1977)) under the anterior lambdoidal suture and lying between the falx and the endocranial periosteum in perinatal rats, but disappearing soon after birth. Pritchard, Scott and Girgis (1956), who had earlier described this cartilage, credited the tectal region of the chondrocranium as its source, but until this is shown to be so, the possibility remains that the cartilage is secondary, its typically hyaline appearance notwithstanding.
Category 1
The hyaline appearance of the avian pterygoid pad likewise raises doubts concerning how secondary it is, but it is an example of 1) above. The avian pterygoid bone, developing intramembranously, acquires a cartilaginous articular pad at its joint with the parasphenoid (Hall, 1968b). Because of its origin on a membrane bone, Hall classified it as an adventitious (i.e., secondary) cartilage, but with some hesitation, for these reasons. First, unlike most avian cranial secondary cartilages, that of the pterygoid develops where the fibrous capsules of the two bones meet, and not from germinal cells of the erstwhile osteogenic Iayer of the bone.
Second, in its histology and histochemistry, the pterygoid articular cartilage closely resembles small-celled primary cartilages such as Meckel's and the quadrate's, with the distinction of having less glycogen but a greater endowment of collagen. Indeed, in the adult bird, the cartilage has become fibrocartilage. Of itself, this fibrous richness is immaterial to the cartilage's being secondary. The secondary cartilages of the mammalian clavicle and mandibular condyle tend towards the fibrous, while avian primary articular cartilages in general were noted long ago by van der Stricht (1890) and others for their perceptibly collagenous texture.
Third, the lack of alkaline phosphatase activity and the failure of the pterygoid cartilage to mineralize are not bars to its admission to the secondary class, when membership is based upon the circumstances of its development, not its fate. Many of Schaffer's (1930) secondaries do not experience ossification. Moreover, Hall's (1968b) supposition that alkaline phosphatase is limited to hypertrophic calcifying cartilages was disproved by the findings of Badi (1972b) in the small-celled cartilage at ligamentous insertions.
As he did for other avian adventitious cartilages, Hall (1968b) demonstrated that, if kept in vitro free from movement for its developmental period, the structure's progenitor cells would turn to osteogenesis instead of chondrogenesis. Thus, however different in its morphology, its behavior in this respect was typical of avian cranial secondary cartilage. In sum, the pterygoid pad is an avian secondary cartilage.

Schaffer (1930) mentioned all manner of cartilages in his section on secondary cartilage formations, including the squamosal and mandibular cartilages and various sesamoid structures, but he deferred detailed discussion of the meniscus of the mammalian temporomandibular joint (TMJ) to his section on fibrocartilage. There, he considered the menisci in general without any discussion of whether the meniscus of the TMJ is a secondary cartilage.
The omission, if actual, would be surprising for two reasons. Schaffer concluded from his own observation of cartilage in the human mandibular meniscus (Figure 286, Schaffer, 1930)) that this appeared to be another example of mechanical function bringing about chondrogenesis in an erstwhile fibrous connective tissue.
Second, in reviewing the forms of cartilage reported in the mandibular menisci of a variety of mammals, he remarked in two on the presence of some elastic fibers; and he unquestionably regarded elastic cartilages as secondary. In fact, Schaffer had earlier stated in his separate consideration of chondroid tissue that the meniscus of the human jaw was part chondroid fibrous tissue, and part fibrocartilage and hyaline cartilage, which were to be considered as secondary.

Category 3
Cartilage can arise anew from primary cartilage. Thus cartilage forms within cartilage canals (Hintzsche, 1931), and regenerates from perichondral grafts (Wasteson and Ohlsen, 1977). In their study of the development, maturation and senescence of hyaline cartilage, Amprino and Bairati (1933a) came across tissues that they chose to call secondary cartilage, despite their presence on and in a primary cartilage. After the age of about 40, a layer of relatively recently formed cartilage was commonly present between the perichondrium and the original cartilaginous mass of tracheal cartilages. Another frequent finding was new cartilage growing within the cavities that developed in the zones of asbestotic transformation in costal cartilage.

The scapula, long recognized as having an origin in primordial cartilage, also entered into the early discussions of secondary cartilage, but dropped out without an explicit reason having been given. Why was the scapula considered in the secondary context? Zawisch's (1954) account of the formation of the human scapula makes it easier to understand why Kassowitz (1881) listed the spine of the scapula among his apophyseal sites of accessory cartilage with supposed osseous metaplasia.

A cartilaginous primordium forms and the experiences a perichondral (subperiosteal) ossification akin to that on the shafts of long bones, to produce the main body of the scapula. For an embryo of 44 mm crownrump length, while the bone still lies upon the primary cartilage, Zawisch's text at first implied that secondary cartilage develops outside on the bone, "On it the main mass of the spine is built of layers of bone. In the medial part a new, essentially sharper ridge of cartilage has developed, that I wish to name medial spinal margin." In the next paragraph, "However, a new cartilage growth has been deposited in an apophyseal way from the medial angle directed towards the coracoid to the original upper margin. This growth apparently originates in the small accessory cartilage focus depicted in black in Figure model I . . . ." But in her later interpretation and summary she indicates that the cartilage in question is an outgrowth of the original primary cartilage: "Medially from which the cartilage of the scapular body, still able to grow, builds a dorsally directed ridge - the medial secondary spinal margin. This then ossifies endochondrally from the scapular body . . . ."
Thus, the building of the spine is complex enough to involve perichondral and endochondral ossification, and some of the cartilage thereof grows in a sufficiently roundabout way as to attract the terms secondary and accessory; as in her English summary, "The spine and the supraspinal plate do not exist in the cartilaginous model. They are secondary formations composed each of several parts which appear in the course of the ossification."
It comes as little surprise that Kassowitz earlier had believed the spine to hold a secondary cartilage, but despite its late arrival the cartilage seems to stem from a primary cartilage.

As a final example of the problems of fitting the observations to the primary-secondary classification, one may note certain behaviors of the nonsclerogenic lateral somitic mesoderm: In his review of somitic chondrogenesis, Hall (1977) referred to experiments where the somites' lateral region, normally destined to become dermomyotome, could be persuaded by proximity to various tissues to form cartilage. Other inductions leading to secondary cartilage typically occur in established soft connective tissues well after the somites have formed their primary cartilages. What is involved in the experiments analyzed by Hall is a similar kind of ectopic chondrogenesis, but one effected before or concurrently with primary chondrogenesis and having as the responding population cells closely related to those of the somitic sclerotome in their origin, position, and ability to react to neural tissues.

Fuchs many years ago covered most of the points arising in the preceding discussion of the difficulties to be met with in seeking to erect a category of secondary cartilage based upon a late time of appearance, or special histology.

FUCHS'S REFUTATION OF SECONDARY CARTILAGE

Schaffer broadened the earlier concept of accessory cartilages to encompass such entities as elastic cartilage and fracture callus in the more general category of secondary cartilages. His clearest enunciation of the category was in a late publication (Schaffer, 1930), but he had developed the idea much earlier (Schaffer, 1897, 1907).
It was then that Fuchs (1906, 1909a) made the only serious attempt to question the notion of secondary cartilages. Schaffer (1930) brushed aside Fuch's objections, but these are of more than just historical interest.

Fuchs (1906) noted cartilage in the glenoid region of the squamosal bone of several species: bone of certain intramembranous origin. His first question was directed at the prevailing general hypothesis that such secondary cartilages arose by the action of mechanical factors, such as pressure. He argued that the relation between secondary cartilage and pressure was inconsistent: cartilage occurred on membrane bones where there was no pressure from neighboring skeletal structures; and elsewhere, sites of definite pressure on such bones had no cartilage. He offered no specific examples of either circumstance at that point in the text. Further on, he noted that the temporomandibular menisci of one grown hedgehog contained cartilage; those of another hedgehog did not. He was unable to conceive how, if this cartilage developed because of a mechanical stimulus, presumably identical physical influences in the two animals had led to the formation of different tissues.
The disparity in outcome, of course, poses a problem for any theory of development. Fuchs' refuge in construing the meniscal cartilage as a residue of a phylogenetically reducing primordial skeletal component still did not explain the difference that he observed.
In the newborn badger and the fetal cat the wide-ranging glenoid cartilage extended beyond the region on which the mandibular condyle could press, further convincing Fuchs that the mechanical action of the condyle played no role in the formation of the cartilage.
He proposed that the zygomatic bone in mammals had a dual phylogenetic origin, in a membrane bone, and an endochondral component that earlier in its phylogeny had been more developed. Thus, the temporal cartilage was a primary entity.

At this time Fuchs did not question that the cartilage formed later than visceral cartilages such as those of the first bronchial arch. The kernel of his argument was not that secondary cartilages were not temporally second, but that the stimulus to their formation - pressure - had no reliable relation with the presence of a secondary cartilage. The unfounded step in his reasoning is clear. He jumped from the justified conclusion that the hypothesis of pressure causing chondrogenesis on membrane bone was insecurely based, to the assumption that membrane bones are incapable of forming cartilage and, hence, any cartilage seen must be a derivative of separate chondroblasts, to be attributed somehow to primordial cartilaginous skeletal elements.

Fuchs (1909a) developed more systematically his refutation of secondary cartilages. He started by listing the characteristics by which others had distinguished secondary cartilages from the primary cartilages, namely: 1) the establishment of the cartilage later in development; 2) the cartilage is histologically different in that a) it often has large vesicular cells, and b) not uncommonly there are fibers (often elastic) between the cells; and 3) cartilage-specific stains frequently react less strongly with secondary than with primary cartilages.
(The mention of elastic fibers came about because Schaffer (1907) had recently denied the epiglottal cartilage an origin in the visceral arches and, instead, included it among the secondary cartilages as a new formation.)
Fuchs sought to undermine Schaffer's category of secondary cartilages by showing that there were exceptions in each of the aspects that apparently made secondary cartilages distinctive.

Among the elements of the accepted primordial cartilaginous skeleton, a rare few form in cartilage much later than the rest. Fuchs cited Graefenberg's (1905) observation of this nature of the lunate bone of the human carpus. Regarding the special histological characteristics of secondary cartilages, Fuchs suggested that since primordial hyaline cartilages also had hypertrophied cells just prior to the encroachment of ossification, the one thing peculiar to secondary cartilages was the early onset of cellular hypertrophy, which did not constitute a fundamental difference.
Nor was the frequent occurrence of fibers in the matrix of secondary cartilage anything special. Undoubtedly primordial cartilages could be fibrocartilaginous, as in the articular cartilages of birds, or elastic, as in the nasal cartilages of bats.
Fuchs conceded that secondary cartilages often are stained more feebly than primary ones, although he noted a good reaction in the rabbit's mandibular condylar cartilage with thionin. He viewed the usually weaker staining as another, chemical, expression of the reduction during phylogeny that he believed these cartilages experienced. Some primordial cartilages also did not stain intensely.
Fuchs did not therefore accept that the differences in staining intensity provided any basis for considering the secondary cartilages other than tardily expressed primary cartilages.

He then returned to the hypothesis of secondaries forming in response to mechanical stimuli. He noted that the mandibular condylar cartilage is well developed when the zygomatic part of the temporal bone is little advanced and, he speculated from his histological observations, lies too far from the condyle for the latter to exert pressure upon it. Thus, the argument that secondary cartilages can be distinguished by their being the consequence of mechanical stimuli was unfounded.
Another attempt to separate secondary cartilages on the basis of special etiological circumstances concerns the supposed rapidity of skeletal growth at sites where such cartilage appears. Noting the occurrence of secondary cartilage at regions of rapid skeletal growth, some authors have taken the chondrogenesis to be the expression of high Wachstumsenergie (growth energy), without offering any exact definition of what this is, or what mechanism might relate it to the formation of cartilage. Fuchs (1909a) discounted Wachstumsenergie on the grounds that in carnivores, such as the cat, the clavicle is rudimentary and does not grow particularly fast, but still has a sizeable secondary cartilage.
Moreover, the premise that cartilage grows faster than bone, whatever that may mean exactly, is not beyond doubt (Murray 1963). How one could even fairly compare the growth of cartilage and bone is a major hurdle, since one is solid, and the other usually trabecular in its fast growing regions; cartilage can proceed by interstitial growth, bone cannot. Usually the presence of cartilage or a chondroid kind of bone serves in a circular fashion both as evidence for rapid growth and as the result of rapid growth.

Fuchs' attack on secondary cartilages had two prongs. The first was to minimize the importance of the differences between secondary and primary cartilages, and to demonstrate that no criterion - whether of timing, histology or etiology - precisely separated the two categories. That he was able to accomplish this is not surprising, given the heterogeneity and abundance of the cartilages classified by Schaffer as secondary.
The second line of attack was to trace the secondary cartilage back to a primordium (Chondroblastem), not only separate from membrane bone but recognizeably cartilaginous in the period when the other, and acknowledged, primary cartilages were forming. In this way Fuchs contended that he had positive evidence that secondary cartilages were in fact primary, to complement the negative argument that other explanations of the chondrogenesis - pressure or growth-energy - were inadequate.
Fuchs's claim to be able to recognize a Chondroblastem at sites of future secondary cartilage met strong criticism from Gaupp (1906), who demanded a strict histological definition, which Fuchs (1909a) was unable to give. Schaffer (1930) also criticized Fuchs for calling mesenchymal condensations bone or cartilage before there was the slightest indication which tissue would emerge.

Fuchs's contention that all secondary cartilages represent elements of the primordial cartilage skeletons of ancestral reptiles is not justified by the developmental history and the histology of the secondary cartilages in existing mammals and birds. Nevertheless, some of the details marshalled by him are worthy of note.

If the boundaries of Schaffer's broad class of secondary cartilages are so vulnerable, has the concept any value? Its practical worth lies in bringing attention to all examples of non-primary chondrogenesis, so that the erection of subclasses, such as accessory cartilage, for purposes of comparison and hypothesis is not founded on a narrow range of instances, which would neglect examples that might modify or negate particular hypotheses.

Such difficulties, arising from a concept of secondary cartilage that is too restricted, are foreshadowed in Patterson's (1977) discussion of the phylogenetic relations of membrane, dermal, and cartilage bones. He defines a dermal bone in endothermous tetrapods as one not preformed in cartilage, and homologous with the bones of fishes that are tied to the ectodermal basement membrane by a surface coat of dentin and/or enameloid. Examples are the mammalian parietal and clavicle and the many avian cranial bones on which Murray (1957) and Hall (1968b) found secondary (adventitious) cartilage.

Patterson then defines adventitious cartilage as arising "at the periphery of a dermal bone," and speaks "of the phenomenon of adventitious cartilage in birds mammals. In that case 'the factors influencing and chondrogenesis and osteogenesis are thoroughly investigated, and do not seem to be applicable elsewhere in vertebrates." Patterson overlooked the extensive evidence (see Chapter 4, Pressure-elicited Chondrogenesis on Endochondral Bones) connecting movement and late chondrogenesis in mammalian "cartilage" bones that had encouraged Schaffer to group his many secondary cartilages as one.

Patterson's category of "membrane bones" includes neoformations such as the mammalian baculum and patella. Although his distinction between dermal and membrane bones serves his phylogenetic analysis well, it has the effect of defining away the secondary cartilage on penile bones, if only dermal bones have adventitious cartilage and the baculum is not dermal. The very close resemblance between the secondary cartilages on the "membrane" penile bone and the "dermal" mandible suggests a common histogenesis and function, that can be recognized by placing them together in the accessory division of secondary cartilages. Other comparisons have been made within this secondary sub-class and between its members and the primary cartilages.

SECONDARY CARTILAGES: COMPARISONS AND SPECIAL STUDIES

Among problems of the skull worth further study, de Beer (1937) listed that regarding the nature of secondary cartilage. The use of modern methods has yielded a wealth of information on the mandibular condylar cartilage, but far less on other secondary cartilages, and none on most of them when they are considered in their entirety. In the mandibular condyle the following is a far from complete list of the aspects so far investigated: the light (Carlson, McNamara and Jaul, 1978) and electron microscopy of its development; its contribution to the growth of the whole bone (e.g., Koski, 1968; Meikle, 1973b); the kinetics of its proliferative cells (e.g., Kanouse, Ramfjord and Nasjleti, 1969); its histochemistry; and its reaction to metabolic stresses, e.g., to papain (Irving and Rdnning, 1962), and altered mechanical functioning, (e.g., Lindsay, 1978).

While the secondary cartilages deserve a wide-ranging review, space and emphasis here do not allow more than the elaboration of a few themes close to the principal thread. First, it can be demonstrated that the mandibular condyle, while generally representative of accessory secondary cartilages, differs in particular respects from, say, those of the antler, clavicle and penile bone.
Thus one can look at the comparative histology within the class of secondary cartilages. Next, many studies have set the secondary cartilages alongside those primary ones undergoing endochondral growth for an assessment of the differences and resemblances between the structures and the ossification processes.
The techniques used most often for the above comparisons have been histochemical, and these same methods are applied to another problem arising especially for the clavicle and antler, but of concern to all secondary cartilages. The difficulty lies with the earliest matrix-rich tissue to form. If the tissue is bone, any cartilage later appearing upon it is secondary; but should the tissue be cartilage, any more cartilage and bone forming on and in it are no more than typical participants in the endochondral sequence of a primordial cartilaginous element.

Returning to the comparisons of the secondary with primary cartilages and among the secondaries, some of the studies, primarily histochemical, are outlined below. This list will serve as a peg upon which to hang some general remarks on secondary cartilages.

Explicit Comparisons
Between Secondary Cartilages Hall (1968a, 1968c) looked at the mouse's mandibular condyle and the chick's cranial adventitious cartilages for glycogen, collagen, calcification, lipids, alkaline phosphatase, and acid mucopolysaccharides. These materials are typical of those examined in secondary cartilages in general. Koch (1960) stained for collagen with azan, and for cartilage matrix with thi onin and the PAS method in the clavicle and developing human mandible (bone only).
While his histology did not use alcian blue in a precise histochemical way, Beresford compared the cartilages of the rat's penile bone and the mandibular condyle for their general structure (1975b), their pattern of zonation (1975a), and their reaction to excess vitamin A (1971). Vilmann and Vilmann (1978) have embarked on a histochemical comparison of the same two cartilages. Their finding is that the distributions of acid phosphatase are identical, but the penile cartilage is exceptional in the greater intensity of reaction for alkaline phosphatasc and Its presence in chondrocytes of all layers.

Between Secondary and Primary Cartilages The several early German anatomists who contrasted cartilages and ossification in the mandible and long bones are mentioned elsewhere (see Chapter 11). Weinmann and Sicher (1964) and Symons (1965) returned to the comparison. Symon's analysis in the rat used histochemical methods for glycogen, acid mucopolysaccharides, nucleic-acids, and alkaline phosphatase.
Ronning, Paunio and Koski (1967) examined the rat's mandibular condyle, several long bone epiphyses, and two cranial synchondroses for their biochemistry and reactions with Sudan black, alcian blue, toluidine blue, and PAS with and without a prior digestion by diastase. Durkin, Heeley, and Irving (1973) reviewed their extensive work on the normal mandibular condyle and the two cartilages of the tibial epiphysis in guinea pig and rat. They emphasized that the epiphyseal plate is a specialized structure differing from both the nearby epiphyseal articular cartilage and the condylar cartilage, both of which serve growth and articulation.
Their studies embraced the responses and recovery of all three structures in scurvy, and to a deprivation of vitamin D. Baume (1970) also disrupted the three kinds of cartilage with a vitamin, namely, vitamin A.
The growth of the rat's clavicle, mandibular condyle, and several endochondral bones was compared in organ culture (Petrovic, 1972). The clavicular cartilage was surprising because it behaved like epiphyseal cartilage rather than the mandibular condyle's, in that its growth depended on the division of differentiated chondroblasts.

The disposition of acid and alkaline phosphatases was looked at by Kenrad and Vilmann (1977) in the mandibular condylar, synchondroseal, epiphyseal, and growth-plate cartilages of suckling rats. In his examination of the cranial adventitious cartilage of the embryonic chick, Hall (1968c) compared it with two primary cartilages, the quadrate bone's and Meckel's, and also noted changes in the histochemistry of its development when the parent bone was immobilized (Hall, 1972b). Last, there is a long tradition of contrasting the growing antler with ossifying long bones, e.g., by Landois (1865a) and Banks (1971).

Some Individual Histochemical and Ultrastructural Studies
Hall and Storey (1968) worked on the fine structure of the cartilage and bone in the chick's quadratojugal.
The histochemistry of the developing antler drew the attention of Wislocki, Weatherford, and Singer (1947), Mollelo, Epling, and Davis (1963), and Frazier, Banks, and Newbrey (1975) in a study complementing Newbrey and Bank's (1975) electron microscopy.
Andersen (1963) investigated the forming human clavicle for its polysaccharides and alkaline phosphatase, but the fine structure of the clavicle appears not to have been reported anywhere.
The phallic secondary cartilages (see Chapter 14) were the interest of Clayton (1977). He applied the electron microscope and histochemical techniques for proteoglycans and polysaccharides to the proximal growth cartilage of the mouse's penile bone, a region of chondroid bone at the bone's distal tip, and the separate fibrocartilaginous anterior process.

The following is a sample of the investigations of the various components of the secondary cartilage of fracture callus. Tonna (1964) used polarized light after metachromatic staining to observe the collagen. Many enzymes have been examined histochemically (Raekallio and Makinen 1968; Timmer, Hadders, Hardonk, and Koudstaal, 1968; Balogh and Hagek, 1965; Severson and Tonna, 1970) and biochemically (Semb, Gudmundson, Westlin, and Hallander, 1971; Lenart, Szell, and Csorba, 1971; Kahn, Jafri, Lewis, and Arsenis, 1978).
Lindholm, Lindholm, Rosengard, and Hackman (1971) cited some of the numerous works on the histochemistry and radioautograpby of callus proteoglycans, while Aho and Isomako (1962) and Aho (1966) have performed fine-structural examinations.

The ultrastructure of another secondary cartilage, that of certainly chondrogenic tumors, is the subject of some of the papers cited by Halliwell and Kinden (1977). Experimentally induced cartilage has had its fine structure noted in vitro (Anderson and Griner, 1977) and in vivo (Anderson, 1967).

Greenspan and Blackwood (1966) made a thorough histochemical analysis of the mandibular condyle in perinatal and suckling rats, looking for several enzymes, carbohydrates, lipids, and protein. The chondrocytes were richly endowed with enzymes. The meaning of the acid phosphatase and plentiful glycogen in hypertrophic chondrocytes particularly engaged these writers.

Silbermann and Frommer's series of reports involved reactions in the mouse's mandibular condyle for succinic dehydrogenase (1972), acid phosphatase (1973a; 1973b), and other hydrolytic enzymes (1974b), and for acid mucopolysaccharides (1973a, 1974a). Two of the reactions - for acid phosphatase (1973b) and glycosaminoglycans (1974a) - were examined in the electron microscope. Meikle (1975a) studied at the ultrastructural level the distribution of acid phosphatase and aryl sulphatase in the rat's mandibular condyle. Papers on the general ultrastructure of condylar cartilage are cited by Silbermann and Lewinson (1978).

General Remarks

Process of Ossification The fate of many secondary cartilages is to be totally or largely replaced by bone, so that a principal interest lies in the process of their ossification. Recent work has confirmed the basic morphology established in the previous century, for example, the scarcity of matrix, the disorder of the chondrocytes, and the obvious participation by chondroclasts in the erosion (Schaffer, 1888; Durkin, Heeley, and Irving, 1972).
The latter authors noted that the distribution of calcium takes on a distinctive pattern related to the disposition of the chondrocytes and obviously different from that in epiphyseal plates.

General Similarity Among Secondary Cartilages When the presence or absence of palisading is taken into account, in terms of the overall chemistry of matrix materials, most workers (Symons (1965) and Hall (1968a,c) inter alios) have been struck more by the general similarity among secondary cartilages and between these and the primary cartilages involved in endochondral growth than by the small differences, for instance, in the content and distribution of glycogen. Close attention has been repaid by the discovery of other subtle differences between the condylar cartilage and other sites, for example, in the distribution of alkaline phosphatase (Kenrad and Vilmann, 1977; Vilmann, 1977).

Value of Age-Specific Comparison There is a growing doubt concerning the validity of comparing several cartilages in animals of just one age. Not only has each cartilage been growing for its own specific period, thus putting it at a particular stage in its development, but Kenrad and Vilmann (1977) intimate that inequalities in the cellular kinetics over the entire period of growth may invalidate comparisons.

Aspects of Secondary Cartilages for Comparison The scope of the comparisons has included only a few of the secondary cartilages. For example, Kenrad and Vilmann (1977) used for a secondary only the mandibular condyle's, but had three examples of primary cartilages. When using one or two cartilages as representative of the secondary class, there are some aspects to be kept in mind.

  1. Most of the work has been done on a very limited range of species, namely, some rodents and man, thus possibly further limiting the worth of the analysis.
  2. Some secondary cartilages are restricted to particular species or classes, e.g., those of antlers and penile bones, but these cannot be excluded from any general comparisons simply because, unlike the mandibular cartilages, they are not present in all mammals.
    Working from the standpoint of only the condylar cartilage, one may be led to imply properties for secondary cartilages as a whole which are unlikely and certainly unproven. Ronning, Paunio, and Koski (1967) linked the secondary nature of the principal mandibular cartilage to its mesectodermal origin, but while it is an intriguing idea worth pursuing that, say, the penile bone's growth cartilage may also be an ectodermal derivative of the neural crest, for sesamoids and fracture callus cartilage such a genesis must be questionable.
  3. Another notion, this one based on the phylogeny of the mammalian mandible, was Koski's (1968) approval of the name secondary, because it reflects not the nature of the cartilage, but the late appearance in evolution of its parent bone. The concept of secondary cartilages is in difficulty enough, without trying to give it an evolutionary significance.
    Certain invertebrates can convince one of how troublesome this mixture of ideas can be. Schaffer (1930) included among the secondary cartilages such extraskeletal cartilages and chondroid tissues as are seen in the heart and eye of birds and reptiles. Now, since invertebrates have no endoskeleton, their ocular and other cartilages (Person and Philpott, 1967) are "extraskeletal" and hence secondary. If the comparable invertebrate ancestors of the vertebrates had such cartilages, "secondary cartilage" preceded primary in phylogeny. But, since there is no primary cartilage in an invertebrate, the secondary or extraskeletal cartilage develops as the only firm structure and therefore is a primary or first, which has no meaning without a second.
    For a sensible discussion of the role of cartilage, chondroid bone (Orvig, 1951), and bone in evolution, the reader has Hall (1975a, 1978), Schaeffer (1977), and Kemp and Westrin (1979). One notes the general failure of primary-secondary dichotomies to make a helpful contribution to the understanding of skeletal evolution.

    One invertebrate cartilage, that supporting each cluster of tentacles in the feather-duster marine worm, is remarkable for its component of chondroid bone (Person and Philpott, 1967), although this was not the chosen name.
    They wrote: "The endoskeletal complex is comprised of several differing cell and tissue types, including one which in section bears a very close resemblance to osteoid precursor of bone." In the legend to their Figure 18, they referred to the tissue's marked vascularity, "giving the tissue section an appearance remarkably similar to that of bone sections. However, it is stressed that the Eudistylia tissue does not mineralize."

  4. Struck by the resemblance of the mandibular condyle to the articular cartilage of long bones, and the contrast between both these structures and the epiphyseal growth plates, Durkin, Heeley, and Irving (1972) grouped the condylar, articular, and initial central region of a long bone's primordium as "embryonic" cartilages, distinct from the specialized cartilage of the epiphyseal disk. However, Durkin's (1973) advocacy of the use of "embryonic" as virtually a synonym for secondary then flies in the face of Schaffer's definition of secondary - distinct from primordial cartilaginous skeletal elements - and can only stir more mud into the water. Moreover, the parallel between mandibular condylar and long bone articular cartilages can be taken only so far, since Kenrad and Vilmann (1977) have called attention to differences in the clustering of the cells and the amount of matrix.
Histochemistry The enzyme histochemistry can bring the basic morphological pictures to life, in the sense that it can reveal the ability of the cells to participate in the changes happening in the tissues. The histochemistry of the mandibular condyle has provided evidence of the continued enzymatic activity of the hypertrophic chondrocytes after calcification of their matrix (Greenspan and Blackwood, 1966; Silbermann and Frommer, 1972, 1974b).
This information does not, however, tell of the eventual fate of those cells. For example, Meikle (1975a) believed that the acquisition of lysosomal enzymes was to bring about autolysis of the chondrocytes, and hence signified imminent death, rather than the vitality assumed by other observers. Others have interpreted the accumulation of lipid to indicate degeneration, a conclusion challenged by Silbermann and Lewinson (1978).

Conclusions A survey of the information on secondary cartilages gained by histology, histochemistry, electron microscopy, radioautography, etc., brings one to the following conclusions:

  1. Several cartilages resemble each other in many respects; for example the clavicle, penile bone, and mandibular condyle have a similar layering and disorder in their growth cartilages and, at an early stage in their morphogenesis, they all have long bony sleeves encasing carrot-shaped plugs of cartilage.
  2. Secondary cartilages alike in many ways differ in others; unlike the condyle's, the penile cartilage does not have a free articular surface, and it contributes much to the longitudinal growth of the bone, a property shared with the antler. However, the antler is special in the pattern of erosion of its cartilage, its late histogenesis and its renewal.
    Thus, no one secondary cartilage can represent all the properties of the class. This is particularly so when the category is construed in Schaffer's wide sense, embracing extraskeletal and ectopic cartilages, fracture callus, sesamoids, tendinous insertions, in vitro growths, tumors, elastic cartilages, etc. Each instance is special, but used cautiously can throw light on skeletal behavior and the properties of cartilage.
  3. Likewise, while secondary cartilages have properties in common with the primary ones, they also differ; and the primaries are themselves heterogeneous, the synchondroses of the cranial base being distinct from long-bone epiphyses (Ronning, Paunio, and Koski, 1967; Kenrad and Vilmann, 1977), and articular cartilages and growth plates differing within the epiphyses.
The argument that secondary cartilage can validly be distinguished from primary ones has hinged not only on morphology and histochemistry, but on whether a mechanical factor underlies secondary chondrogenesis. First to be considered are the data favoring the hypothesis; then will follow an overview of Altmann's arguments against it.

Chapter 4 SECONDARY CARTILAGES II

. Mechanical evocation of secondary cartilage Schaffer
. Pressure-elicited chondrogenesis on endochondral bones
. Pressure-elicited cartilage related to membrane bones
. Experimental evocation of avian secondary cartilage: Murray & Hall
. Altmann's arguments against extrinsic mechanical stimulation of chondrogenesis
. Unstressed sites of secondary chondrogenesis
. Secondary chondrogenesis, at but unrelated to, mechanical disturbance
. Altmann and Pauwel's unifying chondrogenic hypothesis
. Mechanical determinants of osteogenesis
. Determinant stimuli arising from differential growth
MECHANICAL EVOCATION OF SECONDARY CARTILAGE: SCHAFFER
The stimulation of periosteum to chondrogenesis by a mechanical disturbance has a long history, which provided Schaffer with support for his hypothesis. Virchow (1853) cited Duhamel's (1742) observation on rachitic bones so deformed that one bone rubs against another, for example, the ulna and radius. Where the bones had rubbed together, a thick articular-like cartilage replaced the periosteum.
Hueter (1863) was able to confirm microscopically the occurrence of cartilage at both pathological and normal periosteal sites where bones were in moving contact. Cartilage formed on the surfaces between the Malleolus externus and the Processus anterior calcanei in cases of marked Pes valgus, and on the anterior surface of the talus when a contracture held the foot in abduction. At the elbow of normal newborn infants, parts of the coronoid process of the ulna and the head of the radius are covered by a thick periosteum, but in adults these sites, where the bones touch the humerus when the limit to motion of the joint is reached, are sometimes clad in a fibrocartilage. Hence Hueter concluded that rubbing can elicit new cartilage from periosteum. (Scapinelli and Little (1970) offer similar observations.)

In the same year as Hueter, Buchholz (1863) reported his repetition of Ollier's experiment of transplanting flaps of periosteum under the skin or between muscles. Buchholz found periosteum from the tibia formed bone and sometimes cartilage, whereas that from the roof of the skull formed no cartilage, and bone only once. This appears to be the first experimental test of whether the periosteum of a bone formed in membrane differs in its reactions from that of an "endochondral" bone.
This possibility excited the interest of Koller (1896), who studied the tissues in the callus of healing cranial and facial membranous bones in rabbits. Callus in the zygomatic arch always held cartilage, but that of the supra-orbital margin seldom, and of the cranial vault never. Koller attributed the difference in the amount of cartilage to the differing extents of displacement and the rubbing of the bony pieces resulting from their various relations with the facial musculature. (Incidentally, his conclusion that membranous bones can have cartilage in their fracture callus has been disputed on occasion, but confirmed experimentally many times, for example, by Hall and Jacobson (1975), who refer to earlier work.)

Koller's paper elicited a prompt response from Schaffer (1897) to the effect that there was already ample evidence that the periosteum of membrane bones could form cartilage. Schaffer offered an instance with a probable mechanical cause in the hyaline and fibrocartilage forming on the adult terminal phalanx by the tendon's insertion. The bone here is membranous only in the subperiosteal sense, because the phalanx develops in cartilage (Schuscik, 1918).
Schuscik saw two interesting peculiarities in the early ossification of this element. First was the relatively long persistence of a large island of cellular calcified cartilage separating two marrow space (Figure 3). Second, the unusually large cells and basophilia of the Tuberositas unguicularis (Figure 2) prompted her to describe the tissue as chondroid bone. This separate bony structure is not preformed in cartilage.

Kassowitz (1881) in his monograph on ossification proposed changing pressure and rubbing as the stimuli for periosteal cbondrogenesis, but Schaffer (1930) took the idea further with his attempt to account mechanically for all secondary cartilages. Schaffer's hypothesis, however, was founded on observations where the pressure or movement to which the chondrogenesis was supposedly a response was more a deduction than a property actually measured and shown to increase.
No such measurements have been made, but experimentalists have contrived situations where there can be little doubt that movement and pressure change. In addition, observations continue of cartilage in non-experimental circumstances in which an increase of pressure is a reasonable surmise. Experimental and observational evidence favoring the notion that movement evokes cartilage comes from both membrane bones and their attachments such as menisci, and those bones formed in cartilage and their attachments.

PRESSURE-ELICITED CHONDROGENESIS ON ENDOCHONDRAL BONES
Among the straightforward anatomical observations are those of Stillwell and Gray (1954). They found that one-third of the sites on bone underlying, and in contact with, tendons comprise fibrocartilage, although not a very cellular variety. In one instance, over the facet of the cuboid bone's tuberosity, hyaline cartilage was present. The deeper, bony tissue did not appear to have been modified. The common finding was for the deeper periosteum to have formed fibrocartilage, i.e., a secondary chondrification, while the superficial periosteal layer was fibrous.
Balogh and Foldes (1955) found cartilage in many tendon grooves on bones, with hyaline cartilage at points of considerable pressure, as on the Os cuboideum where the tendon of the peronaeus runs.

Although the mechanical circumstances and the likelihood of movement are insufficiently established, the chondrogenesis at the sites of fusion of the tibia and fibula in rodents (Murray, 1954; Moss, 1977a) and the more restrained periosteal chondrogenesis described by Zawisch-Ossenitz (1927, 1929a,b) (see Chapter 9) may prove to be examples of a natural movement-evoked differentiation. In an experiment, Storey (1972) reported cartilage forming where exostoses of the ulna and radius rub together in rabbits suffering from osteofluorosis.

Momigliano-Levi (1930a) was the first to deliberately evoke secondary cartilage. His brief report described it on the hind-limb phalanges of young guinea pigs and on the lower third of the newly-hatched chick's metatarsus, after a month of bearing what may have been a loose collar, "con adatte morsette", to cause an intermittent compression. Most of the cartilage had randomly ordered cells in sparse matrix, and merged by a transitional zone with the bone. Both the periosteum and the surrounding connective tissue seemed to contribute to the secondary cartilage.

Krompecher (1937a) cut the sartorius muscle free at its caudal insertion and re-routed it so that it ran through the Foramen ischiadicum major, bearing upon the pelvic bone as it did so. The periosteum under the moving muscle formed a fibrocartitage. He concluded that pressure caused the cartilage to form, but in 1955 broadened his idea of the evocative stimulus for the fibrocartilage to include tension, sideways pressure, and movement.
The reference to the last is ambiguous, "but in which, in practice, an essential promoting role is to be accorded the movement stimulus," in that it is unclear whether movement is essential to the differentiation of the cartilage or just increases the growth of differentiated cells. And then, what the movement stimulus comprises in mechanical terms is specified in his next sentence as an intermittent pressure component and a sideways displacement component.
But these components are arrived at solely by induction, and their relation - identity, overlap, or whatever - to the pressure already designated as a chondrogenic stimulus is not elucidated. The omission is typical of those emphasized by Altmann as defects in the mechanical hypothesis (see Altmann's arguments against extrinsic mechanical stimulation of chondrogenesis,link p. 53).

Ploetz (1937a,b) displaced the tendon of M. flexor digitorum in the rabbit's hind-leg so that it ran in front of rather than behind the tibial malleolus, which normally served as a hypomochlion on which the tendon rubbed. After dislocation, the pad of cartilage disappeared from the tendon's surface at this site, and the chondrocyte-like vesicular cells within the tendon appeared to become fibroblasts, although Ploetz could not be sure that such a metaplasia really had taken place.

Where the tendon normally ran under the sole, it appeared grossly also to be a Gleitsehne (gliding tendon), but microscopically there was no pad and the tendon cells were not especially chondrocytic. However, after the operation, the tendon here experienced more loading and abnormal movement, and a pad of hyaline and fibrocartilage formed from the epitendineum, while the cells of the subjacent tendon became larger and enclosed by basophilic matrix.
Thus, what was in effect a Zugsehne (pulling tendon) region turned into a Gleitsehne, and at the malleolus the reverse happened: in response to alterations in both the loading and the movement. Ploetz specified changes in pressure as playing the key role, following Roux's thinking.

Ploetz's (1937) experiment is illuminated by a recent biochemical and TEM analysis of various regions of the unoperated rabbit's Flexor digitorum profundus tendon by Gillard et al. (1977). The sesamoidal region subjected to pressure had a "two- to three-fold increase in the proportion of chondroitin sulphate and a concomitant decrease in proportion of dermatan sulphate," a proteoglycan profile "similar to, but not identical with, cartilage"; for instance, the tendon has dermatan sulphate and hyaluronate, the latter perhaps being involved in the gliding action, as is likely in paratenon. Also, the axial periodicity of the tendon's collagen fell 13-15% in the sesamoid zone.
The authors offered the hypothesis that mechanical factors, "friction, pressure and some tensional forces," influence the matrix which, by virtue of the piezoelectric responses of collagen, might inform the cells to synthesize and maintain mechanically appropriate glycosaminoglycans. Gillard et al. (1979) have now experimented with this tendon.

Barfred (1971) saw a cartilaginous metaplasia, sometimes with calcification, in the free region of the Achilles tendon in nearly half the normal rats he examined. He referred to Ploetz's work and attributed the tendon's cartilaginous zone to pressure from the crossing plantaris tendon. When he tested the Achilles tendon for strength, the cartilaginous region was not the part to rupture.

Before the above findings on tendons, clinicians suspected that rubbing promotes the histogenesis and growth of the patella. An analysis of cases where the patella was absent or rudimentary, but sometimes grew when the child's knee came closer to normal functioning, led Brunner (1891) to conclude "that the patella, which has no genetic relation with the tibia, develops after this as a sesamoid that owes its development to the rubbing of the well functioning quadriceps tendon on the already established femoral epiphyseal cartilage." Walmsley (1940), however, doubted the intratendinous origin of the patella.

PRESSURE-ELICITED CARTILAGE RELATED TO MEMBRANE BONES

Findings that suggest comparison with those just mentioned are by Kopp (1976), who looked at the distribution of proteoglycans in four regions of the menisci of human temporomandibular joints. The central zone, thought to experience most loading, scored highest for metachromasia with toluidine blue and staining with alcian blue. Use of critical electrolyte concentrations and different pHs for staining suggested that the proteoglycans were chondroitin and/or dermatan sulfate.
By personal contact, Kopp learned of Majersjo's biochemical assay of rabbits' pooled TMJ disks, which showed the presence of hyaluronate, dermatan and chondroitin sulfates. Kopp's Figures 8 and 9 show the large, encapsulated chondroid cells believed to make the proteoglycans. Centrally, the cells were "mostly of chondroid type but fibrocytes were also seen." Such chondroid cells and a metachromatic zone were also prominent as an incomplete margin to the central surface ot the disk.
Thus, in the rabbit's tendon and mandibular meniscus the region of most chondrogenicity matches that probably experiencing more pressure than tension.

EXPERIMENTAL EVOCATION OF AVIAN SECONDARY CARTILAGE: MURRAY AND HALL

Murray and his co-workers sought by analysis of the comparative anatomy and histology of the avian cranial joints, and by experimental manipulations to establish two truths for the secondary cartilages:
first, that the cartilage was not derived by a displacement of cells from the blastemata of the chondrocranium - Fuchs's (1906) notion, designated by Murray as the "transference" hypothesis;
second, that, no presumptive chondrogenic cells from any source were present at the sites of secondary cartilage, but there were cells which could respond to the compressive component of use by producing cartilage.
Since primordial cartilage participates in, or close by, all the sites of secondary cartilage seen in the chick, Murray (1963) could not eliminate the transference hypothesis, although the difference in histological nature between secondary and chondrocranial cartilages argued against it. The clinching refutation was Hall's (1967b) finding adventitious cartilage at the frontal-maxillary and jugal-maxillary articulations of the eastern rosella, which involve solely membranous bones.

The second property of avian secondary cartilages demonstrated by Murray (1957) was that their chondrogenic cells were not intrinsically resolute in their determination in the manner characteristic of, say, the precursor cells of Meckel's cartilage, but required some extrinsic stimulus to hold them to chondrogenesis. The quadratojugal bone and quadrate of nine-day embryonic chicks were transplanted together to the chorio-allantoic membrane. The chondrogenic cells on the quadratojugal ceased forming secondary cartilage and commenced osteogenesis, so covering over any existing secondary cartilage.
Suspecting that the condition of the graft inhibiting chondrification might be an absence of movement, Murray injected other eggs with a variety of curare to paralyze the embryos, the immobility to be observed through a window in the shell. While there was some variety in the results, the overall effect was a reduction, total or partial, in the secondary cartilages, with the surangular's as susceptible as the quadratojugal's; this puzzled Murray, because the surangular is, from the lack of an articular cavity, apparently immobile with respect to Meckel's cartilage. These findings made him hesitate to accept a mechanical hypothesis for the evocation of cranial secondary chondrogenesis, but caused him to wonder whether there may, in fact, be slight movement between Meckel's and the surangular.

Murray tested his mechanical hypothesis further in the chick embryo in a well-developed series of experiments (Murray and Smiles, 1965). They eliminated the action of muscles on avian cranial joints by transplanting the nine-day quadratojugal-quadrate and surangular-Meckel's cartilage articulations to the chorio-allantoic membrane (CAM). Joints formed and the tissues were healthy, but secondary cartilage failed to appear. Grafts of 11- and 14-day joints, where the cartilage had already formed, ceased chondrogenesis, and the superficial cells laid down bone upon the secondary cartilage.
This osteogenesis proved that the lack of cartilage in nine-day grafts was not caused by a general inability of the germinal cells to differentiate and synthesize matrix on the CAM, but resulted from the lack of some stimulus to chondrogenesis.

In a second series of experiments, Murray and Smiles injected decamethonium iodide into chick embryos to paralyze them at various ages and for different periods. The amount of movement was observed through a window, and at the end of the experimental period, for comparison with the histologically assessed state of development of secondary cartilage at the four cranial sites illustrated in their Figure 1. They concluded that reduced movement, in general, prevented secondary chondrogenesis or brought established secondary chondrogenesis to a halt, but permitted osteogenesis by the germinal cells that would in vivo have become cartilaginous.
If the chick partially recovered from the paralysis, the sequence of events appeared to have been chondrogenesis, osteogenesis, and a reversion to chondrogenesis, evidenced by a triple layering of the appropriate tissues at the site of secondary cartilage. How well modulation fits what occurred in Murray and Smiles's experiments is taken up elsewhere (seeLINK p. 144).

The outcome of their work was that some mechanical factor was needed to evoke chondrogenesis in the germinal cells on several avian cranial bones. The factor operated in both mobile and theoretically immobile joints (lacking articular cavities), so that gross movements appeared not to be involved. Their ideas on the factor were these: "simple pressure, the shear which accompanies either pressure or movement of any amplitude, however small, or an indirect consequence of the mechanical factor, such as a chemically active substance liberated from damaged cells."
Of the "immobile" joints, where chondrogenesis failed in paralysis or after grafting, they noted that, if pressure were the missing element, some small movement must take place in the normal bird for one skeletal element to press against its opponent. There was evidence that the degree of movement needed to stimulate chondrogenesis is slight, from the observation that in chicks recovering from paralysis, fusions had limited the range of movement, but the muscles showed signs of contraction, and secondary cartilage had formed (Experiment 8 of Murray and Smiles, 1965).

Also, in an ingenious experiment where the quadratojugal-quadrate articulation was grown in vitro, Hall (1967a, 1968d) was able to elicit secondary cartilage by manipulation of the joint sixteen-times daily. These passive movements were infrequent relative to what is likely in vivo, but still were an adequate stimulus. But placing the joint under the constant pressure exerted by the intercostal muscles connecting a pair of ribs, in the configuration devised by Glucksmann (1942), did not lead to secondary chondrogenesis in vitro; rather it encouraged the germinal cells to osteogenesis.
Hall (1968d) therefore concluded that the necessary stimuli for cartilage formation were intermittent pressure and tension associated with movement, and also that the cellular threshold of sensitivity to the stimuli altered with increasing embryonic age, and that constant pressure evoked osteogenesis.
To place these experiments in the wider perspective of skeletogenesis, Murray and Smiles (1965) remarked that the mechanical factor is but one of many evoking chondrogenesis and osteogenesis, other ostensibly nonmechanical ones being neural tube for vertebral cartilages, and whatever it is about hypertrophic cartilage, alcoholic injections, and transplants of transitional epithelium that elicits ectopic bone.

ALTMANN'S ARGUMENTS AGAINST EXTRINSIC MECHANICAL STIMULATION OF CHONDROGENESIS

In a long review building on his mentor Pauwel's (1960) work, Altmann (1964) mounted the most recent significant indictment of the hypothesis that external mechanical factors are needed for secondary chondrogenesis. But, unlike Fuchs, Altmann did not deny that the secondary cartilages were developmentally distinct from the primary cartilaginous skeleton. Hall (1970b) has already made an appraisal of Altmann's conclusions, which does only partial justice to the number of examples Altmann adduced where the mechanical factor was in doubt.
Altmann's evaluation of his own experiments on fracture repair and disturbed periosteum is certainly questionable, and those studies are of interest as further examples of secondary cartilage formation and for his descriptions of chondroid bone (see Chapter 16), but Altmann made a number of interesting points, outlined below.

W. His (1865) anticipated Roux by many years in formulating the concept that the microscopical character of a connective tissue reflects the physical factors acting upon it. His made the hypothetical distinction between forces arising from the growth of cells and external ones acting upon the cells, but noted that in practice the two would interact, so that what was primary and what secondary in nature would be very difficult to separate.

Altmann's paper may be read as an attempt to redress the balance of opinion of the school of Roux, Schaffer, Krompecher, Murray and Hall on the role of external forces in certain differentiations of connective tissues by consideration of the cells themselves as generators of their mechanical environment, as advocated by His and even more by Pauwels.
Since Roux had introduced several interrelated mechanical entities - tension, extension, pressure, and shear - as determining the differentiation of connective tissues, Altmann enlisted an engineer to advise him on the theoretical mechanics of loaded bodies and of smaller units, corresponding to cells, within such bodies.
Now, Roux (1912) himself had realized that with the give inherent to natural materials, "pure" tension or pressure could not act. For example, pulling on a tendon will narrow it slightly, introducing an element of pressure on the cells and fibers within it. Although he distinguished the one agent - tension - as primary and the other as secondary, the cells have no way of telling the effects apart: the cells experience mixed mechanical stimuli.

However, Roux wrote elsewhere as if pure pressure, tension, or shear could exist in loaded deformable materials. Roux wrote on the same topics in at least four publications, in one instance considered "secondary" mechanical consequences as important but elsewhere did not, and he did not distinguish, for example, a shearing force from strain. From all this, Pauwels (1960) and Altmann did not merely catch Roux out in the occasional contradiction, but were able to demonstrate that Roux had no theoretically clean case for mechanical factors', singly or in combination, being the agents steering cells towards chondrogenesis. From what Roux himself wrote, what had to follow from it on straightforward physical grounds, and what probably occurred in development and various clinical situations, cartilage arose because of (Altman, p. 38) a) pressure and strong shearing, b) the absence of normal pressure, c) tension with strong shearing, and d) abnormally strong tension.

If one looked at what one particular stimulatory combination of agents (tension and shearing) evoked, the predicted outcome likewise was manifold:
a) cartilage,
b) a fibrous tissue with disorderly fibers, and
c) an orderly connective tissue with the fibers nearly parallel.

At this point Altmann called attention again to what he had written earlier in the article on the physical difference between a cellular blastema and the differentiated tissue, viz., cartilage, whose present mechanics circumstances are the point of departure for a physical interpretation of its histogenesis. Cartilage can withstand loadings that would crush a much more cellular blastemal tissue, although Roux had proposed that it is such a loading that selects the cells able to adapt to, and withstand, pressure and shear.
But, could not the same kind of forces on a greatly reduced scale act on the blastema? Altmann denied this, reasoning that until such materials as fibers appeared in the matrix to give the tissue some resistance to deformation, it would react differently to the loading, and could not be compared with later cartilage or connective tissue.

Clearly what is needed here is more specific knowledge of the mechanical properties of mesenchyme and precartilaginous blastematas, and especially what attributes the early non-cartilage-specific proteoglycans confer upon the cells which secrete them. Mesenchyme is not a tabula rasa awaiting the imprint of stimuli, but is rather an established tissue in its own right already subject to stimulations, restraints, and interactions, and with a mechanical status of its own.

Altmann's argument against Roux's hypothesis that external mechanical factors acted to select chondrogenic cells was further developed along the following four lines, although the sequence Altmann followed was a little different.

  1. As outlined already, he suggested that the concepts and laws of mechanics were not properly understood by Roux and his followers; and when Roux's hypotheses were taken to their mechanically correct conclusions, no one factor - tension or shear - or combination of factors was unequivocally the cause of the chondrogenesis.
  2. Roux himself had excluded cartilage developing in early embryonic life from the scope of his mechanical hypothesis. Kassowitz (1881) had earlier drawn such a demarcation between cartilage formed because of "pressure and rubbing" and others not needing mechanical stimulation for their histogenesis, e.g., in the airway and external ear. This produced a scientifically unsatisfactory situation, where what could not be accounted for by a mechanical explanation could be referred to "inborn" or self-determining factors.
    Altmann sought an escape from this either/or kind of explanation by mustering many examples of cartilages that appeared well after the embryonic period (which means that they were secondary cartilages), and apparently in circumstances free from extrinsic mechanical influence.
  3. Among the cartilages forming after birth, several were in sites of fracture or other disturbances to normal musculo-skeletal functioning. For these, Altmann tried to argue against Roux in two ways: that either in the region of chondrogenesis, stabilization had eliminated mechanical variables, or mechanical factors were operating but were not the ones required for chondrogenesis, as stated by Roux in his hypothesis.
  4. Altmann's fourth tactic was to look not at secondary cartilages but at the primary cartilaginous skeleton, which Roux had maintained does follow biomechanical laws in its later development, stating for example that pressure and shear are needed for the maintenance of cartilage, e.g., at the epiphyseal plate.
    Again, Altmann sought to show that the most probable mechanical states could not lead to the actual disposition of the tissues seen.
Altmann's examples in these various categories are worth listing, but I shall depart from his rather higgledy-piggledy order and give first the failures of primary cartilages to behave as expected according to Roux, next the secondary cartilages formed apparently in the absence of mechanical stimuli, and then those developing where the biomechanics are highly contentious.
Thus, the anomalous primary situations are the differentiation and growth of cartilaginous skeletal primordia in tissue culture (e.g., Roulet, 1935), the development and locations of the epiphyseal plate and secondary ossification centers, the mechanics of the development and later functioning of auricular, nasal and other airway cartilages; and the apophyses, where tension might be expected to predominate (although at least some apophyseal cartilages belong in the secondary category).

Unstressed Sites of Secondary Chondrogenesis

Turning to the secondary cartilages, Altmann charged Schaffer with a partisan view in favor of a mechanical origin for all secondary formations. Altmann held that Schaffer (1930) had misquoted two authors, and had ignored contradictory evidence in citing another, on the cartilage arising in fractures; and that while Schaffer (1897) was very thorough in relating mechanical stimuli to two instances of chondroid or fibrovesicular tissue, in 1930 he wrote nothing of the biomechanics involved in seven other sites of the same tissue.
These were the retina of Petromyzon, arterial valves in fishes, the chameleon's scleral valves of a lizard, the bovine penile glans, the mammalian cardiac skeleton, and the pig's tear duct.

Altmann then introduced other locations of supposedly unstressed secondary cartilage: developing antlers, the midline of the palate and on the vomer, central enchondromas, ovaries with ectopic cartilage, cartilage nodules in osteochondromas, and parosteal callus luxurians.

Altmann's argument based on the "extraskeletal" bones and cartilages was not novel. According to Gebhardt (1903), Solger (1892) had mentioned the potential difficulties posed for the biomechanical hypotheses of Wolff and Roux by penile, dural and cardiac bones. However, for many secondary cartilages the biomechanics of the mature structure are unknown, regardless of what is going on mechanically in the structure's precursory tissues. For each secondary site, it takes only a little thought to imagine possible sources of load on the germinal cells. Thus, the deer could stimulate its antler pedicles by transmitting the movements of chewing through the scalp. The genital tubercle could be agitated by the crawling of the rat pup, or by the dam when she cleans the pup.

Each and every site of secondary chondrogenesis needs the kind of experiment involving paralysis or transplantation that allowed Murray (1957) and Hall (1967a) to show that the avian cranial cartilage requires local movement for its development. On the other hand, Glasstone (1968) found that the mouse's mandibular condyle initially develops in vitro apparently without movement. The avian scleral cartilage will grow from explanted mesenchyme (Weiss and Amprino, 1949).
Other instances of chondrogenesis in vitro without any intended mechanical intervention are the induction by bone matrix acting on epimysial cells, (Terashima and Urist, 1977; Anderson and Griner, 1977; Nathanson, Hilfer, and Searls, 1978), the inductions by avian cartilage, brain, and retina on fibroblasts (Toro, 1935), and the development of cartilage by broken chick bones (Prasad and Reynolds 1968).

Other secondary cartilages proposed by Altmann as examples of "afunctional" formations formed without outside mechanical stimulation were the cartilages growing from periosteum irritated in situ by an infected thread (Kapsammer, 1898), or periosteum implanted into the comb or wattles of the hen (Morpugo, 1899) or injected into the large veins to lodge in the smaller pulmonary vessels (Cohnheim and Maas, 1877). Whereas Altmann expressed reservations on intramuscular periosteal grafts' remaining free of forces and movement, my own guess is that the pieces of periosteum in the wattle and lung were agitated just as much.

Secondary Chondrogenesis at, but not Related to, Mechanical Disturbance

If the invariance of the physical conditions is equivocal in the above instances, it is nothing compared to the disputes aroused by claims to have eliminated particular biomechanical factors from sites of fracture healing. The latter is the aspect of Altmann's argument to which Murray and Smiles (1965) and Hall (1970b) took particular exception.

Fracture callus cartilage is but one of several post-embryonic formations resulting from an undoubtedly disturbed situation, which, Altmann argued, did not behave according to Roux's formulations, either because there actually was no mechanical stimulation, or the mechanical conditions should have led to formation of another tissue or a different distribution of tissues, or were in some other way inconsistent with the outcome.

The secondary cartilages arising in situations of mechanical disturbance were as follows.

  1. Rathcke (1898) attributed the occasional cartilage in myositis ossificans to a mechanical stimulation from the contraction of muscles. Altmann wondered why, if this were so, this stimulation did not lead to a generalized chondrogenesis in the perimysial connective tissue. That cartilage is the exception not the rule in myositis ossificans (Seeliger, 1927) can be taken to indicate either that there is usually insufficient movement for chondrogenesis, or, as Altmann thought, that the cartilage is unrelated to contractile stimuli.
  2. Schaffer (1930) viewed the development of cartilage-like cells and chondroid bone (CB II) at the tendon insertion on the Tuber calcanei as a reaction to mechanical loading. Altmann thought that at this site the transitions of one tissue into another occurred within a distance too short to provide space for enough variations in the mechanical state to have brought about the development of several specific tissues. But this was surmise on Altmann's part, for it simply is not known how much the mechanical microenvironment of one cell can differ from that of neighboring cells, but it is to factors in its own environs that a cell will respond.
  3. Altmann had a hard time faulting one experiment which strongly supports the role of mechanics in chondrogenesis: Ploetz's (1937a,b) displacement of the tendon of M. flexor digitorum in the rabbit's hind-leg (see p. 51, this volume), so that after dislocation, the chondroid region became more typically tendinous, and the tendinous part more chondroid.
    The most that Altmann could find wrong with interpreting these results in favor of Roux's hypothesis was, first, that chondrification should have occurred throughout the width of the tendon; and second, that Roux had equated sideways pressure with shear, so that only one of his two prerequisites for chondrogenesis seemed to be acting, but not both pressure and shear. Altmann was justified in expecting those who invoked physical concepts of precise meanings and interrelations to follow them to conclusions consistent with mechanical laws, but his quibbling seems not to be justified by these particular experimental conditions.
    Arguments based on statics disregard the movement to which the tendons were subjected; and whether it was sideways pressure, movement, or factors related to both or either one of these, that led to chondrogenesis can be ascertained only with further and ingenious experiment.
  4. In healing fractures cartilage is a frequent occurrence, which, although often ascribed to movement and related mechanical interactions between the bone pieces, also is seen, Altmann claimed, where either any stimulation of a mechanical kind can be excluded, or the supposedly more precise mechanical circumstances specified by Roux for chondrogenesis can be ruled out.

    Concerning the last point, Altmann, in effect, placed Roux's hypotheses in double jeopardy. As noted already, he revealed that Roux's own writings contain the idea that not one but several factors - tension, pressure, pressure and shear - caused cartilage to form, but in discussing bone healing, he held Roux to his most explicit set of conditions - pressure with shear - for the purpose of showing that this combination seems to be absent from certain sites of callus chondrogenesis, or present where cartilage is absent, for example, the fibrous union of an obliquely fractured long bone (Pauwels, 1960).

Examples of callus chondrogenesis put forward by Altmann as being without mechanical loading were:
a) the guinea pig's metatarsal broken at the time of transplantation into the back of the same animal (Wehner, 1920) (The question of the degree of movement for periosteal grafts in subcutaneous and intramuscular sites has already arisen but is unresolved);
b) in the medullary cavity of broken shafts, seen only once by him, but the phenomenon is attested to by other reports, e.g., Maas (1877), Kapsammer (1898);
c) after incomplete resections of shaft bone made by Koch (1924) and Partsch (1924) (more recently Bourne (1944) and Melcher and Irving (1962) have noticed cartilage in the callus repairing holes bored in rodents' femurs); and
d) on the healing fibula of the rat, since it is naturally fixed to the tibia.

Hall (1970b) doubted that the fibrous attachment of the fibula anchors it so well that its fragments cannot move. Also, if it is rigidly fused to the tibia, another objection can be raised against regarding it as non-functional: one that holds as well for experiments (b) and (c) above.
One can reason that if the bone is in use by the animal, it must be loaded, and the forces from this loading could be transmitted to its developing callus to an extent that is currently unknown but cannot be assumed to be nil.

In another experiment Altmann tried to ensure isolation of the fibulary callus from stresses by enclosing the bone-ends in either a boiled rat femoral shaft, or a tube fashioned out of Plexiglass but blocked off in the middle. Cartilage still appeared, indeed at four places, three of which were sites where the possibility of movement, rubbing or pressure was apparently absent.
But Hall (1970b) was skeptical of this claim, since Altmann's proposal that fusion of the distant callus to the ends of the ensheathing tube provided the stability would have left an earlier period of instability, before the fusion took place.

Altmann, in turn, had expressed doubts about the experiments of Krompecher (1937b) and Hasche-Klunder and Gelbke (1952). Krompecher fastened rods, passed through holes drilled in the ulna and tibia before their fracture, in such a way that the broken bone-ends were stable but compressed together, or stable and distracted. Altmann's criticisms were as follows.
Krompecher claimed to have eliminated movement and to have placed the callus under "pure pressure": the prerequisite for hyaline cartilage formation according to his (Krompecher, 1956) modification of Roux's hypotheses. However, the long rods intended for fixation could as well have acted as levers to aggravate the effects of any motion.
Next, according to Roux, cartilage arises from pressure and shear, but Krompecher claimed to have produced only pure pressure. More critically, Krompecher gave too few procedural details for anyone to know what tissue was placed under pressure, but it appeared to Altmann that the bone ends would have been too tightly apposed to allow a cellular blastema (the tissue supposed to respond to pressure) between them.

Hasche-Klunder and Gelbke (1952) attempted to hold the broken bone's pieces tightly against one another by a medullary nail, with a nut outside the bone. Their nailing was aimed at eliminating the cumbersome apparatus of Krompecher, thereby ensuring stability of the site of fracture.
Five of their calluses had some cartilage, which the two authors attributed to shearing forces from micromovements permitted by the nailing - a conclusion castigated by Altmann as a typical example of the backward reasoning of "cartilage now, therefore movement earlier."
Altmann also noted that biomechanical schemes based on Roux to account for the usual distribution of cartilage in angulated fractures of long bones, namely, close to the gap and within the angle, could not account for the occasional reversal of the position of most cartilage. Thus, Wurmbach (1928) illustrated tibial and radial fractures with the bulk of the cartilage over the obtuse angle, on the convex and thus supposedly tension side of the angulation.

Wurmbach's work and his own experiments brought home to Altmann the great variability in the behavior of callus tissues. In particular, the cartilage is inconsistent in its presence and, when it occurs, its position does not always correspond with the likely sites of most rubbing, pressure, and shear.

Although movement has sometimes seemed to increase the likelihood of callus cartilage, and has added to its volume in many experiments, e.g., Lindholm et at. (1970), there are other sites, such as healing cranial vaults (Girgis and Pritchard, 1958; Beresford, 1969), where one is tempted to believe that there is very little movement to evoke the cartilage, and hence to conclude, as did Girgis and Pritchard (1958), that some other factor, such as ischemia, is the chondrogenic agent.
But this is supposition and what is needed are actual micromeasurements of the mechanical and chemical events around the blastemal cells. At the gross level, measurements by strain-gauge indicate that normal biting provides enough force to move the bones of the cranial vault (Behrents, Carlson and Abdenour, 1978).

When movement can be shown to be the critical factor for cartilage formation, one should hesitate before specifying what it is about movement that is stimulatory, because to invoke pressure or tension as the property is to bring down on one an edifice of mechanical implications and constraints, as Pauwels and Altmann have taught.
The biomechanics of statically loaded bones do not lend themselves to simplification; those of the moving skeleton will not be any more easily analyzed. The nature of the intermediary signal linking mechanical stimuli to cellular responses is unsolved. Krompecher (1956) linked the chondrogenic action of pressure with chemical factors in the following sequence: in the granulation tissue on a bony stump, pressure would narrow and occlude vessels, and the resulting anoxia cause the cells to specialize in chondroitin sulfate production, thus becoming chondroblastic. More recently, other specific agents, e,g., electrical potentials, calcium ions, and cyclic nucleotides (Davidovitch et al., 1978), have been put forward.

Altmann and Pauwel's Unifying Chondrogenic Hypothesis

Altmann could have stopped and concluded that, with the marked variation in tissues seen, degree of stability, and location and geometry of the bone pieces, analyses at the gross level have left unproved the necessity of a mechanical factor for callus chondrogenesis. Dismissal of the fracture studies would have left him with the internal contradictions of Roux's hypotheses and a number of non-callus secondary cartilages to constitute a suggestive but non-experimental case against mechanical stimuli as the root of all secondary cartilage.

Altmann did not halt, but rather sought to employ the findings of his fracture studies, lacking in uniformity though they were, in support of a general theory of chondrogenesis developed by Pauwels (1940, 1960) on ideas sketched out by Kassowitz (1881).
Kassowitz (1881) had suggested that in the antler, noted for its very fast growth, where rubbing and external pressures seem improbable, the rapidly multiplying blastemal cells have an excessive Wachstumsenergie (growth energy) which, coming up against the restraining perichondrium, is converted into Wachstumsdruck. This latter is thus an intrinsic pressure derived from the cells themselves.

Pauwels (1940, 1960) developed the idea by considering the details of the pressure and likely consequences for the cells. The pressure in a soft semifluid blastema was to be hydrostatic, acting in all directions but, regardless of the nature of the external force, the only effects on the cells were a change in volume, a deformation or both. Deformation along an axis brought about fibrogenesis, and pressure without a shape-distorting deformation evoked chondrogenesis.
Altmann and Pauwels believed that in this self-generated hydrostatic pressure they had a single developmental mechanism for embryonic primary cartilage formation, and later and postnatal examples of chondrogenesis such as a callus and many other secondary formations.

As Hall (1970b) pointed out, and Altmann (1964) had already conceded, one puzzle, of how the tissue's growth energy arose, was now substituted for the older unresolved question of what exactly was involved in the self-differentiation of Roux. Altmann thought that in his fracture studies, by eliminating all external mechanical influences, only an intrinsic one - hydrostatic pressure - remained, and he was apparently unwilling to grant that no mechanical force, intrinsic or extrinsic, is acting to cause chondrogenesis. The role of inductive tissue interactions in primary and secondary chondrogenesis is now much better known (Hall, 1977, 1978).

Mechanical Determinants of Osteogenesis

Bone is another component of a fracture callus which Altmann sought to accommodate in his biomechanical account of skeletal differentiation. Fick (1857) had suggested that bone can only form where the mechanical forces are absent. But how can an absence of stimulus be a stimulation? Roux (1895) was more positive and prescribed for osteogenesis, pressure (with varying tension or without tension), but without or with only minimal shear, Pressure is incompatible with an absence of mechanical influence, so that Fick's and Roux's preconditions for bone formation cannot be reconciled.

Altmann followed Fick, but seemed to go beyond him in taking an absence of forces to be the determinant of osteogenesis rather than merely permitting it. How such a determination could work Altmann did not discuss, and the topic was peripheral to the scope of his article, but one can foresee difficulties.
For example, the quite common mixture of bone and cartilage in a callus should not arise using Altmann's hypotheses. If the cartilage forms because the blastemal cells come under pressure of their own growth, the adjacent blastemal cells could not then make bone, because his osteogenesis required absolute mechanical peace, and the claimed hydrostatic nature of the blastema would have distributed pressure throughout it.

The absolute peace of which Altmann wrote is unlikely to exist anywhere in living organisms. Moreover, Pritchard (1972) referred to experiments where osteogenesis occurred in tensed fibrous tissue apparently in response to the tension.

Determinant Stimuli Arising From Differential Growth

The notion of Kassowitz, Pauwels and Altmann requires that there is a lag in the growth of perichondrium behind the enclosed tissue, thereby placing the latter under a restraint that stimulates cells to chondrogenesis. A related but non-mechanical idea is that, at rapidly growing sites, skeletal precursor cells outstrip the growth of the vasculature, become ischemic and then respond
to their ischemia by differentiating preferentially into chondroblasts. These hypotheses are a reminder that skeletogenesis proceeds in the broad and changing context of a loaded, innervated, vascularized musculoskeletal system, but the concept of the "obliging lag" has only a toe-hold on credibility, despite its long existence.

It may be remembered from Leriche and Policard (1928) that Havers, Bichat, and other early osteologists thought of the periosteum as keeping in check the growth of its bone. Some confirmation of this comes from recent variously oriented cuts in the periosteum of growing long bones by Crilly (1972) and Harkness and Trotter (1978).
The results point to a role for the periosteum in coordinating growth within and on a bone and integrating cartilaginous (Lufti, 1974; Pritchard, 1977) with bony and muscular activities, but whether tension in the periosteum influences both cellular growth and the determination of skeletal cells awaits further inquiry.


Chapter 5 METAPLASIA I

Introduction and context
Virchow's metaplasia
Explanations of metaplasia
. Direct metaplasia
. Embryonic rests
. Displacement or migration of differentiated cells
. The undifferentiated mesenchymal cells
. Indirect metaplasia
. Defining metaplasia out of existence
. Modulation as a substitute for metaplasia
Experimental narrowing of the accounts of metaplasia

INTRODUCTION AND CONTEXT

Metaplasia comes from the Greek metaplasis, meaning transformation. There are many transformations to convert the zygote into a multicellular organism. More transformations occur in later development, physiological use, regeneration, disease, and aging. Metaplasia came into use to identify a particular kind of transformation - that of a relatively mature tissue into another mature tissue.

Virchow (1853) gave the concept its modern form and emphasis, as he sought to explain pathological phenomena in terms of the tissues' cells, as newly revealed by improved microscopes. The problems that beset metaplasia have changed little since Virchow.
To what extent is metaplasia abnormal or pathological?
Since the concept is based upon tissues, what exactly is a tissue?
How well can metaplasia be accounted for by cellular behaviors?
What relation does metaplasia bear to other transformations of tissues, in particular, to their original histogenesis or differentiation?

These general questions form the background to a discussion that will follow these lines:

  1. What Virchow saw; his conclusions and his caveats will be outlined.
  2. The observed phenomenon will be separated from the explanations offered for it, using as an example the occasional formation of ectopic bone or cartilage in the soft connective tissues.
    Five mechanisms have been proposed (two metaplastic, the others seemingly non-metaplastic at the cellular level): a direct metaplasia; an indirect metaplasia of differentiated cells; the displacement of embryonic rests; the displacement or migration of differentiated cells; and a tissue metaplasia or novel stem-cell differentiation by undifferentiated mesenchymal or other stem cells.
  3. Attempts have been made to circumvent metaplasia, either by arguing that the tissue transforming is not mature, or by regarding the transformations as modulations within one tissue.
  4. Certain kinds of experiment allow one to narrow somewhat the accepted accounts for a given situation to less than the five plus two above.
  5. Several examples of metaplasia are examined for the explanation which best seems to fit their circumstances:
  6. Connective tissue metaplasias encompass a wide variety of phenomena and situations.
    a) Examples are of cartilage and chondroid bone to bone; bone towards cartilage; fibrous connective tissue to bone or cartilage; cartilage to fibrous tissue; hyaline to fibrocartilage; and periosteai chondrogenesis and perichondral osteogenesis.
    b) The above instances lend themselves to an imprecise division into: slower, later transformations with few cells involved and little or no mitosis, as distinct from more rapid metamorphoses in the highly cellular and proliferating surroundings characteristic of regeneration and some tumors.
  7. The slow transformations in connective tissues can be reasonably accounted for by the hypotheses of direct metaplasia or modulation-within-a-tissue. The difference between these two depends on what is meant by a tissue and modulation. The concept of a tissue has to accommodate biochemical knowledge as well as the morphological. Recent biochemistry gives another dimension to cellular differentiation and changes in differentiation, whether called modulations or metaplasias.
  8. Faster transformations of connective tissues taking place early during regeneration, or involving late normal developmental switches in direction, say, by periosteal cells to produce certain secondary cartilages, are open to several explanations: to some degree modulation or direct metaplasia, but to account for the bulk of new tissue, cells able to divide must be invoked by calling on stem cells, or dedifferentiated cells (indirect metaplasia).
    a) The latter two possibilities are implied to be mutually exclusive. This is unproved because the stem cells of connective tissues and the extent of other cells' dedifferentiation are unknown quantities.
    b) Tissue-true regeneration and any indirect metaplasia accompanying it have a phase of cellular events in common, followed by a phase of varied pursuits. These two phases provide a basis for comparing the nomenclature of Yamada (1977) describing comparable cellular events in lens regeneration with the classical use of such terms as dedifferentiation, indirect metaplasia, etc.
    c) Consideration is given to how well metaplastic redifferentiations fit the hypotheses of cell lineage, quantal cell cycles, etc., derived by Holtzer and his colleagues from observing the differentiations of primary histogenesis.
  9. The matrices of connective tissues complicate the latter's metaplasia. Changes in the matrix can reflect metaplastic change in the cells, but the matrix cannot so reliably indicate that the cells are untransformed. The cells of firm connective tissues in theory can be in three situations: initially free on the surface, embedded, or freed by resorption or dissolution. Whether the cells' determination and potencies differ in these three circumstances is uncertain.
  10. Modulation requires a separate treatment embracing its origins, current meanings, applications to skeletal tissues, diagrammatic representation, validity and so on.
    The undifferentiated mesenchymal cell needs a similar individual assessment of its status.
  11. A brief conclusion has as its framework the problems still facing metaplasia, listed in the second paragraph of this introduction.

VIRCHOW'S METAPLASIA

Mid-nineteenth century microscopy showed animal tissues to be cellular and to fall into visually recognizable categories and subclasses. However, where one microscopically discrete tissue adjoined another, sometimes there was a zone of gradual transition from one into the other. Also, there were circumstances in late normal development and pathology where one distinctive tissue succeeded another at the same location. In some of the samples taken at various times from the site of succession, the tissues were seen to be linked by a type of tissue intermediate between them.

From such histological pictures and their timing, Virchow (1853) proposed his direct metaplasia - Persistenz der Zellen bei Veranderung des Gewebscharakters (the tissue changes, but the cells persist). He cautioned that one makes the interpretation from dead samples, and the microscope does not allow one to watch the transformation as it happens (tissue culture was not yet practiced).
Second, he warned (1884) that what one was doing was das Nacheinander aus dem Nebeneinander zu erschliessen, meaning that a risk existed of concluding the after-one-another from the by-one-another, in other words confusing temporal and spatial transitions.

What tissues and changes had Virchow (1853) in mind when proposing metaplasia? In normal development, he believed that some hypertrophic cartilage turned into bone, but this only became obvious in cartilage afflicted with rickets. He viewed bone marrow as the best example of a Wechselgewebe or tela mutabilis, forming by the metaplasia of cartilage or bone, then itself experiencing metaplasia between red, yellow, and gelatinous varieties.
As pathological instances of metaplasia, he cited the change from columnar epithelium to squamous, and the formation of ectopic bone and cartilage in soft connective tissues.

Virchow dealt little with cellular mechanisms of the transformation, preferring to emphasize that the same cells persisted throughout rather than to detail their participation. However, in certain instances he suggested that the cells of mature tissues proliferate and regress to an indifferent (undifferentiated) state.

EXPLANATIONS OF METAPLASIA

What Virchow and countless others have seen is that where one mature tissue was before, now there is a different one, and the transition appear to be a gradual turning of the first into the second. The succession is undeniable, but how the change of tissue comes about has been hotly disputed. To five explanatory hypotheses in contention can be added two tactics to circumvent metaplasia: disqualification of the participating tissues as being only an immature turning into a mature tissue; and distinguishing a separate class of differentiations and re-differentiations as modulations.

Direct Metaplasia
Direct metaplasia has the cells turning directly into a different mature kind, without cell division, but accompanied by necessary modifications to any surrounding matrix. This was Virchow's general idea of how metaplasia went, and ectopic bone, for example, was simply transformed collagenous fibrous tissue of the site.

Embryonic Rests
Embryonic rests were invoked as an explanation of new growths of tissues, especially those tumors composed of a tissue never normally seen at the place in question, or present there only during embryonic life. For the cartilaginous tumors growing on long bones, Virchow (1853) suggested that these arose from cartilage cells of the growth plate that had survived the process of ossification and remained quiescent, until somehow stimulated to form the tumor.
Such suggestions as this, dating from Lobstein and Recamier's (1829) publications (cited by Wolff, 1907), were gathered into a general hypothesis by Virchow's student, Cohnheim (1889), who proposed that displaced embryonic rests were the source of all tumors.

Whereas Virchow accounted for ectopic firm tissues by a direct metaplasia of tissues already present, Cohnheim suggested, for example, that testicular cartilaginous growths came from cells, originally destined to form vertebrae, that were carried along with the Wolffian duct to become part of the testes. There they remained dormant until later in life, when they formed the tumor's cartilage. This sweeping hypothesis - requiring mechanisms for both arrest of development, and for displacement - was accepted only with reservations.
Busch (1878) held that most ectopic bone arose from such displaced embryonic osteoblasts, but in a few cases, e.g., within the eyeball, a metaplasia of connective tissue had to be invoked, since errant primordia there were "unthinkable," and neither could osteoblasts from adjacent organs be the source of the bone. These ideas did not change significantly for twenty years; thus Borst (1902) held views very similar to Busch's. In that year Sacerdotti and Frattin ligated the renal vessels of a rabbit and saw that bone had formed in the calcifying necrotic kidney.
This was the first reproducible, experimental production of ectopic bone. It aroused much interest, and was repeated and modified by many scientists. Poscharissky (1905) found bone in necrotic kidneys, but not in ovaries, hepatic lobes, or spleens made necrotic by vascular ligation. That the kidney was special for something - transitional epithelium - other than its calcification escaped him, because he also studied 180 calcified lesions in diverse human organs, finding bone in 82, including the liver, stomach, and mesenteric lymph nodes. He concluded that displaced cell rests might 'explain ectopic cartilage (common in the neck and bronchiogenic cysts), but could not account for the occurrence of ossification at calcified lesions, particularly in abdominal viscera far from the skeleton, and that such rests were ruled out by the experimental evocation of ectopic bone in the rabbit.
His work finished embryonic rests as a credible explanation for ectopic bone. Although Cohnheim's hypothesis did not survive in its original form, in a way it left its own conceptual rest that is still with us - the undifferentiated mesenchymal cell.

Displacement or Migration of Differentiated Cells
Displacement or migration of differentiated cells reduced the two mechanisms of the displaced-embryonic-rest hypothesis to one. Macewen (1912), for example, proposed that already differentiated osteoblasts could be freed, say, by trauma, and wander, even using the blood stream, to lodge elsewhere and make ectopic bone. A very few periskeletal ossifications and the metastases of osteogenic sarcomas owe their existence to local displacement and vascular excursion, respectively, but otherwise the hypothesis is now given little credence.

Another version of the hypothesis of bone-derived, blood-borne precursors of ectopic osteoblasts or chondroblasts is really a variant of the undifferentiated-mesenchymal-cell idea below. It suggests that some poorly specialized stem cell travels from the marrow of normal bone to enter soft connective tissues, there to be stimulated to skeletogenesis. The hypothesis does no more than take that of the undifferentiated mesenchymal cell (UMC) and seek to explain how that cell comes to be in soft tissue. The proposition fails to address the questions of the connective tissue stem cell's existence and role (see Chapter 7), but does introduce an implication which is denied by the evidence.

Connective tissues differ in the ease with which bone can be evoked in them. The suggestion of Urist et al. (1969) that visceral tissues are unable to form ectopic bone and cartilage is not substantiated by human pathology (Poscharrisky, 1905; Willis, 1962) and experimental inductions in vitro (Nathanson, Hilfer, and Searls, 1978), but somatic connective tissues are more prone to heterotopic osteo- and chondro-genesis. Why this is so is unexplained, but a uniformity of response, rather than the observed discrepancy, would be expected were the responsive cells dispersed by the vascular system. The origin of the marrow in ectopic ossicies and ectopic sites of endochondral osteogenesis is another matter; and cells carried in the blood may play a role in establishing their populations of hematopoietic cells.

The Undifferentiated Mesenchymal Cells
The undifferentiated mesenchymal cells and other stem cells introduce the crucial topic of the way normally resident cells may bring about a transformation in a tissue. When a tissue first develops, it does so by having some of its multiplying cells slow or cease their division and become specialized in form, chemistry, and intercellular relations. Those differentiated tissues that have to replenish loss and make repairs keep some of their cells less specialized and able to divide. Progeny of these cells can then differentiate to replace lost cells.
In other words, some cellular differentiation is a regular event in the adult life of the tissue. This is thought to be the role of the basal epithelial cells of stratified epithelia, the crypt cells of simple intestinal epithelium, the stem cells of lymphoid and myelopoietic tissue, perhaps the satellite cells of skeletal muscle (Schultz 1978), and the marginal cells of the vomeronasal neurosensory epithelium (Barber and Raisman, 1978).

The less differentiated stem cell, enduring in the adult tissue, provides an alternative to the metaplasia of already specialized cells into new ones. The stem cell, instead of turning into the cell type(s) typical of the tissue, takes another pathway of differentiation to become the metaplastic type, e.g., squamous keratinized instead of mucous columnar. The metaplastic process is essentially that of normal differentiation in the adult, itself recapitulating a differentiation of initial histogenesis, and is abnormal only in that the pathway chosen is not typical of that particular stem cell.
Such reasoning introduces a train of unresolved queries. How differentiated are the most specialized cells of a tissue? And how undifferentiated are the tissue's stem cells? Do all tissues that can replenish and repair themselves have stem cells? Since some tissues of mesenchymal origin have stem cells, are these present in all mesenchymal derivatives? If so, is the cell a multipotent mesenchymal cell, or are there different and more limited ones for bone, fibrous connective tissues, bone marrow, etc. The inconclusive evidence on these matters will come up again in the separate discussions of the undifferentiated mesenchymal cell and modulation.
For now, the UMC and equivalent multipotent cells in other tissues offer one way of explaining ectopic ossification, myeloid, and many other pathological metaplasias. The attraction of the stem-cell explanation of metaplasia has been that it avoids having specialized cells of the tissue participate actively in the transformation by losing their differentiation and acquiring another, which jars with the firmly rooted idea that differentiation is the attainment of a final and stable state. The stem cell undergoes what, for it, is a novel differentiation, which on the face of it is so far removed from Virchow's definition of metaplasia that the phenomenon is better named for what it is - a novel stem-cell differentiation - and not by such names as tissue metaplasia (Yamada, 1977).

Indirect Metaplasia
Indirect metaplasia is very much a cellular metaplasia, but differs from the direct sort in having already-differentiated cells of the transforming tissue lose some of their specialization, proliferate, and then differentiate into a new specialized type. The original tissue's specialized cells thus become a temporary kind of stem cell which then differentiates anew. The key step is the dedifferentiation of the specialized cells, which denies differentiation its finality, at least for some cells of the tissue.
The process of indirect metaplasia was considered by Virchow (1871) for certain skeletal cells, but not under that name. Virchow (1871) distinguished two types of new growth, the second of which involved very numerous, rapid divisions of the differentiated cells originally present, so bringing about a large population of small cells that could again build the original tissue (a process of regeneration) or form a different, i.e., heterologous, tissue.
Elements displaying this proliferative and changeable growth were, he thought, leukocytes or mobilized connective tissue cells. Before any new line of development was followed, the population of "apparently absolutely indifferent" cells was, as he put it, in the "granulation stage," (hence the term, granulation tissue). Cells in that stage behaved like the Bildungszellen des Embryo in their potential for forming different tissues.
What Virchow was ascribing to leukocytes and connective tissue cells was what later came to be called a dedifferentiation, and already Virchow's analysis linked three events: a loss of cell specialization, a proliferation of cells, and a regained embryonic-like potential for traversing new pathways of differentiation.

One of his applications of the hypothesis was mistaken. He believed carcinoma of the epidermis to be derived from proliferated dermal leukocytes or connective tissue cells. Another application still interests us. Virchow looked on the marrow of developing bones as being a proliferating granulation tissue with hypertrophic chondrocytes as the mother cells, and bone cells as the new kind of cell into which the marrow cells changed. His sequence of a dedifferentiation of chondrocytes into marrow cells that then differentiated anew into bone cells is an example of indirect metaplasia.
Virchow did not use such a name, because he saw the sequence more as one direct metaplasia (cartilage to marrow) followed by another (marrow to bone). The idea that hypertrophic chondrocytes can survive erosion of the cartilage and enter a pool of proliferating cells is still the subject of controversy and experiment (e.g., Hanaoka, 1976; Hall, 1978), and has a parallel in the belief that osteocytes may do likewise (Young, 1962).

With regard to ectopic bone, pathologists began to notice that this often formed not only close by calcified necrotic tissue, but in a very cellular "young organizing tissue" (Paul, 1886), the granulation tissue of Virchow. The hypothesis that developed from such observations and was accepted, for example by Poscharissky (1905), held that some pathological factor causes the calcification of soft tissues; the calcified tissue acts as an irritant and provokes the proliferation of fibroblasts; then the calcium salts somehow change the young fibroblasts into osteoblasts. The fibroblasts experience a dedifferentiation, proliferation, and a re-differentiation.
Since the work of Huggins (1931) on transitional epithelium, it has been known that apart from calcified lesions, several stimuli can induce ectopic bone and also ectopic cartilage. Whatever the inducer, many, up to the present (Thorogood and Gray, 1975, inter alios), have held an indirect metaplasia of fibroblasts to be the likely source of the osteoblasts or chondroblasts, although the cells that the cited authors described have enough cytoplasmic filaments to qualify perhaps as myofibroblasts.

Defining Metaplasia Out of Existence
Defining metaplasia out of existence or circumventing metaplasia by redefinition are two ways of attempting to eliminate the concept of metaplasia, when it is defined as a transformation of one mature tissue into another. The first is to argue that the earlier tissue, although the forerunner of the later one, is an immature precursor like the mesenchyme that precedes cartilage or bone, so that what is seen is an example of delayed histogenesis, but one still of an embryonic type.

Chondroid bone and cartilage have been categorized as less mature tissues that can transform into bone (e.g., Knese and Biermann (1958)). This tactic does not so much explain away metaplasia as draw attention to how little is known of the processes of differentiation, early or late, from poorly differentiated or well differentiated precursors. Just not enough is known for connective tissues to be compared for degrees of differentiation and thereupon ordered into a hierarchy.

The second line of attack takes issue with what is meant by a tissue and is not devoid of merit. A tissue is a distinctive assemblage of cells and intercellular materials serving a common purpose. However, since the major varieties of connective tissue are sometimes linked physically by intermediate forms, such as chondroid bone and fibrocartilage, one can argue that the connective tissues are not that distinctive, but are expressions of just one tissue. Whence it follows that although collagenous tissue or cartilage might turn into bone, the transformation is not strictly a metaplasia, since it is a modification within one broad tissue category, rather than from one tissue to another.

Recent biochemical and in vitro studies have indeed revealed new patterns among and across the conventional classification of tissues. Also, an element of arbitrariness entered into the original recognition and categorization of tissues (see Chapter 6, Slow Transformations of Connective Tissues). However, neither labelling one of the participating tissues as immature, nor viewing both tissues as variants of one tissue, can conceal that a significant transformation has taken place and requires explanation in cellular terms.
The preceding arguments essentially are quibbles that the process is not metaplasia sensu stricto. The argument that certain tissues are really subclasses of a major tissue and transformations between them have a different import to ones completely out of, or into, the confines of the major tissue introduces the idea of cellular modulations to account for metaplasia.

Modulation as a Substitute for Metaplasia
The starting point of the "modulation as a substitute for metaplasia" approach to the problem of metaplasia is that there are two classes of specialized or differentiated cells: those that gain and keep one distinctive character, and others that become specialized enough to receive a histological identity, e.g., as a fibroblast, but nevertheless can alter their character within strict limits to that of another "type," e.g., an osteoblast. Taking this mere restatement in cellular terms of Virchow's conclusion that some tissues experience direct metaplasia and others do not, the modulation school then adds these stipulations:

  1. The term "differentiation" conveys the attainment of a stable cellular identity, and so, if a specialized cell can express multiple characters, these transformations need a distinguishing name - modulations - and are a different order of event from differentiation. They are "functional conversions, called 'modulations,' and indicate latitude of expression within a given cell type rather than instability of the type as such" (Weiss, 1953).
  2. The modulating cells are responsive to extrinsic controls that can not only drive them to transform in one direction, but also oblige them to return to their previously held specialized state, when the controls are reversed. This apparent reversibility was the key evidence of the lability of the cells concerned, and the prime reason for choosing from physics the name modulation.
    Were the cells to transform only in one direction, this could be construed as just a delayed final differentiation, falling back on the argument that the precursor, despite its specialization, was only incompletely differentiated. But when the cell does not maintain its final state, but can reliably be made to return to its earlier condition, this is a sure sign of its instability and responsiveness to external conditions, and that its "differentiations" or specializations are temporary (Young, 1967).
Thus, the concept of modulation recognizes two states of cellular differentiation: stable and relatively unstable. What is not established is whether the cell populations thus distinguished differ intrinsically, or whether the ones appearing to be stable appear that way only because in vivo they are always observed in environments holding them in that unchanging condition, as is suggested for example, by experiments on keratinocytes (Sun and Green, 1977).
From the standpoint of modulation, the formation of ectopic bone or cartilage involves the fibroblasts of the soft tissue exercising their option to be modulated by stimuli open to any of the related and labile cells of the connective tissues, i.e., the change is from an osteo-chondro-fibroblast to an osteo-chondro-fibroblast,

EXPERIMENTAL NARROWING OF THE ACCOUNTS OF METAPLASIA

The explanations of metaplasia - a transformation of an apparently mature tissue - are several. Almost every account says something about the transformation specific enough to be tested experimentally and perhaps proved untrue for a particular metaplasia.
  1. The explanation by cellular modulations stresses the reversibility of the transformations, so that if the cells cannot be turned back to their original state, doubt is cast on the view of the alteration as a modulation. To do so could place undue weight on negative evidence. Reasons why it might not be possible to turn ectopic osteocytes back into fibroblasts are that the dense osseous matrix might restrict the access of appropriate stimuli, and could itself be a factor maintaining the osteocytes' differentiation.
  2. If the tissue, say cartilage, or cells, e.g., dedifferentiated fibroblasts, undergoing metaplasia are not fully mature, the cells involved should carry the markers characteristic of more embryonic cells, such as certain surface coatings, fibronectin (Linder et al., 1975) and other products, such as isozymes (Schapira, 1978). Their absence might be taken as evidence that the metaplasia is direct.
  3. A novel stem-cell differentiation can account for the transformation of a mature tissue, unless one can demonstrate either that the tissue has no kind of stem or reserve cell, or that such cells, even though present, are not responsible for the transformation. Neither task is easy, as is evident from the later discussion on the undifferentiated mesenchymal cell, which is supposed to reside in connective tissues; and from the controversy over the role of the interstitial cell in development and regeneration in Hydra (Hay, 1968; Marcum and Campbell, 1978; Sugiyama and Fugisawa, 1978), despite the existence of interstitial cell-free strains of Hydra and their amenability to techniques of cloning.
  4. If a tissue or a region of a tissue, say, the interior of cartilage, has no stem cells, but experiences a transformation, this is a metaplasia, but is it direct or indirect? The direct variety has a) no cell proliferation and b) no passage through a poorly differentiated state, i.e., no dedifferentiation. The absence of mitoses can be reasonably assured by a failure to take up freely available tritiated thymidine, but the requirement that the cell not return to a less differentiated state is theoretically and technically troublesome.
In a direct metaplasia there has to be less of some existing specialized activity and the gain of another, brought about by underlying changes in the cellular programming, occurring in sequential, interrupted or overlapping fashion. Regarding an overlap, it appears that a cell may commence expression of a future identity before it has completely run down activities specific to its present status; for example, von der Mark et al. (1977a) found occasional cultured chondrocytes react with fluorescent antibodies against collagens type I and II, and developing duodenal endocrine cells contain both gastrin and cholecystokinin (Larsson and Jorgensen, 1978). In such circumstances the cell seems always to be producing specialized materials, so that it could be said to be fully differentiated while changing its type. However, there may indeed be a brief pause by the cell between programs, but the lag between transcription in the nucleus and translation in the cytoplasm (Hay, 1968) could put a brief time of change-over beyond present-day measurements.

If the cell's activity is interrupted by a fairly long delay before the implementation of the new syntheses, the reduction in the first specialized activity will bring the cell into a palpably inactive state, but a loss or reduction in an activity is not evidence of anything more than the state of that activity: inactivity is not per se an indication of a gain in potential for a new differentiation. Nevertheless, any significant gap between cessation of the first specialized activity and adoption of the second, metaplastic, one encourages those who would argue for evidence of dedifferentiation, since the cell was differentiated and now has taken on a different specialization. In effect, this is to state that there can be no direct metaplasia: the specialized cell must always return to a dedifferentiated multipotent state in a metaplastic transformation, however brief the duration of that state. The concept of modulation between specialized states was introduced partly to get away from this kind of reasoning based on a rigid definition of differentiation.

In summary, the differences between direct and indirect metaplasias are that the indirect involves cell divisions, amenable to study, and the reacquisition of an undifferentiated, i.e., multipotent, state for which neither the loss of the previous activity nor the appearance of a new one is unequivocal evidence.
Thus, while experiment may reduce the reasonable explanations of a given metaplasia to less than the total of seven, it seldom can constrain the situation to provide satisfactory proof that only one holds true. A review of the different kinds and situations of metaplasia indicates that some are fitted better by certain explanations, others by different mechanisms.
Although ectopic bone has so far served as an example, metaplasias involving the connective tissues are in several ways the most complex, and so these will be treated after some slightly more simple examples.


Chapter 6 METAPLASIA II

Examples of metaplasia
Novel stem cell differentiations ("tissue" metaplasia)
The differentiated cell as stem cell
The completeness of tissue metaplasia
Lens regeneration in newts
Lens formation by transdifferentiation in vitro: Urodela, Aves, Mammalia
Transformations in chromatophores
Glandular metaplasia of renal smooth muscle
Connective tissue metaplasias
. Fibrous connective tissue to bone
. Fibrous connective tissue to cartilage
. Cartilage to fibrous connective tissue
. Hyaline cartilage to fibrocartilage
Rapid transformations in connective tissues
The role of stem cells
Regeneration as the context of indirect metaplasia
Terminology of differentiation and metaplasia
. Dedifferentiation
. Redifferentiation
. Terminal differentiation
Slow transformations of connective tissue: issues arising
Terminal differentiation
The tissue legacy
Myoid transformations
Biochemical and morphological indices of differentiation
Degrees of metaplasia
Physiological and pathological metaplasia
"Immaturity" of cartilage
The matrix in metaplasia
Transdetermination in imaginal disks
Nuclear transplantation & cell hybridization: nuclear metaplasia

EXAMPLES OF METAPLASIA

Novel Stem Cell Differentiations ("Tissue" Metaplasia)
The bulk of the metaplasias described by pathologists such as Virchow (1853; 1871), Schridde (1909), Borst (1913), Collins arld Curran (1959), and Willis (1962), involve epithelial, hematopoietic, glandular epithelial, and other tissues that fall into Hay's (1968) labile group, i.e., subject to regular and rapid physiological renewal.
Such tissues contain stem cells: cells able to divide, giving rise to more than one kind of progeny, including more stem cells to maintain their own population (Potten, Schofield and Lajtha, 1979). There are good grounds for believing that these less differentiated cells are the source of the new cells which give the tissue its transformed character, abnormal for the site.

The phenomenon is common, is generally accepted by pathologists, and appears routinely in their textbooks under the rubric metaplasia. However, as Schulze (1929) noted, histologists in their texts have long been more wary of the term, arguing that many instances of what pathologists have called a metaplasia, while a metaplasia of a tissue, are not a cellular metaplasia, but a novel stem-cell differentiation.

Such novel differentiations or tissue metaplasia are not limited to pathology, but by experiment may be elicited by various stimuli:

1) the widespread squamous metaplasia and keratinization from:
. a) greater heat or abrasion;
. b) a lack of vitamin A (Wolbach and Howe, 1928);
. c) more direct exposure to air, by a stoma for tracheal epithelium (Matsumara et al., 1977), or by opening the egg for avian chorionic epithelium (Sawyer, 1978);
. d) by giving diethylstilbestrol to males (Arai, Suzuki and Nishizuka, 1977);

2) the intestinal metaplasia in the gastric mucosa, e.g., from the giving of N-methyl-N'-nitro-N-nitrosoguanidine (Matsukura et al., 1978);

3) the conversion of embryonic murine transitional epithelium to prostatic by transplanting it along with urogenital sinus mesenchyme (Lung, Cunha and Reese, 1979);

4) the formation of endocrine islet cells from centroacinar and other ductular cells in the rat's regenerating pancreas (Mark, Schmidt and Goberna, 1970), if the ductular cells responsible are not fully differentiated;

5) the induction of ectopic taste buds on top of the rat's vallate papillae by excess testosterone (Zalewski, 1969);

6) the glandular metaplasia of the mouse's vibrissal hair follicles in skin cultures exposed to excess vitamin A (Hardy, 1968), and the same agent's causing the chick's scale-forming skin to make feathers (Dhouailly and Hardy, 1978); and

7) a mucous, sometimes ciliated, metaplasia of chick ectoderm treated with excess vitamin A, which Fell and Mellanby (1953) clearly articulated as a stem-cell response: "The mechanism whereby different concentrations of vitamin A produce metaplasia both in the animal and culture, is completely obscure, but whatever it may be it appears to act on those cells which are basic to the growth of the tissue, in this case those of the stratum germinativum."

8) The skin over the deer's antler - the velvet - attracts this name because it is special for its hair, its sebaceous glands, and lack of arrector pili muscles, and its eventually being shed (Goss, 1987). Goss transplanted the thick periosteum from the frontal bone under where the antler would normally develop to various subcutaneous sites. Ecept on the back, nose and tail, the grafted periosteum induced a bony nodule over which the skin convered into velvet - metaplasia - as a tiny ectopic antler developed.

Some epithelial tissue metaplasia evoked experimentally can be reversed, if the offending agent is removed, as in the tracheal epithelium more directly exposed to the air by a stoma (Matsumura et al., 1977), or subjected to excess vitamin A (Murray and Smiles, 1965).
The latter authors discussed the propriety of the concept of modulation to such reversals of the tissue's character. They concluded (Chapter 8, The Connective Tissues: One Tissue?): No, the stem cells that became mucoid were not then turned back into non-mucoid cells, but yes, at the level of the whole tissue, the phenomenon could be labelled a tissue modulation. As with tissue metaplasia, the term tissue modulation is misleading for understanding what is happening to the tissue's cells. The "reversal" is the adoption by stem cells of an orthodox differentiation after their predecessors' spell of novel or deviant differentiation.
Incidentally, restoring the conditions to normal does not always result in a return to the pre-metaplasia tissue, e.g., in Hardy and Bellow's (1978) observation of vibrissal follicles transformed into glands by vitamin A, where, in its stability, the acquired glandular state "resembles a new embryonic induction rather than a modulation of the epithelium."

The Differentiated Cell as Stem Cell
It should not be assumed that the novel stem-cell differentiation can effortlessly account for tissue metaplasia. In certain transformations, for instance, of the simple squamous parietal epithelium of the renal corpuscle to cuboidal (Ward, 1970), or podocytic (Marcus, 1977), only one specialized type of cell is initially present, so that a stem cell seems to be absent, and the mature cell, like the hepatocyte in regeneration (Lajtha, 1979), acts as a stem.

Next, in a stratified epithelium, the basal (stem) cells able to divide may be quite specialized themselves, with tonofilaments, hemidesmosomes, and a secreted contribution to the basal lamina (McDowell et al, 1979). These authors note, however, that in tracheal epithelial regeneration, the indifferent reparative cells appear to come not only from basal cells, but also from mucous cells. Both kinds participate in an epidermoid metaplasia by increasing their tonofilaments and desmosomes while they divide. In consequence, McDowell et al. regarded basal and mucous cells as variants of the same differentiated cell type, which in both its expressions experiences a keratinizing metaplasia, i.e., significant reprogramming, before acting as a stem cell.
The precursor cells in intestinal crypts are likewise quite specialized (Rubin et al., 1966), although the permanent stem cells may be far fewer than was commonly thought (Potten, Schofield, and Lajtha, 1979), so that many of the cells observed may have been transit forms on their way to fully differentiated states.

The Completeness of Tissue Metaplasia
Most epithelia have a heterogeneous cellular population, with two or more such types as secretory, ciliated, absorptive, receptive, neuroendocrine (APUD), and basal (precursor?) cells. When such a variegated epithelium experiences a novel stem-cell differentiation or tissue metaplasia, can the stem cells fit out the new epithelium with all the cell kinds produced by normal development? The limited evidence is controversial, as examples from alimentary mucosal metaplasias can show.

Gastric mucosa is seen ectopically in man in around half the cases of Meckel's diverticulum - an early developmental anomaly - and in the esophagus, as Barrett's esophagus, believed to be an acquired metaplastic condition (Dayal and Wolfe, 1978). They used immunohistochemistry to search for gastrin-secreting G cells in the ectopic mucosae.

The presence of G cells in the enteric ectopic gastric mucosa, in regions where this had undergone an antro-pyloric type of differentiation, was attributed by them to:
1) the coincidence of the time of migration of precursors of neuroendocrine cells with that of diverticular development, providing the opportunity for the introduction of an appropriate stem cell into the ectopic tissue; and
2) an inductive action of antro-pyloric epithelium on the G-cell precursors. By contrast, Barrett's mucosa held mucous cells, and occasional parietal and chief cells, probably "derived from the same pluripotential undifferentiated precursors as the mucin-producing surface cells," but no G cells. Dayal and Wolfe implied that an absence of neuroendocrine stem cells was the reason for the lack of G cells in the ectopic mucosa.

But their discussion did not dispose of the difficulty that, since the Barrett's mucosa showed only cardiac-fundic differentiation, a lack of their hypothetical pyloro-antral induction could explain the G-cells' absence, allowing G-cells' precursors to be present but unexpressed. Their only hint that the matter is one of a missing stem cell was by way of a reference to Lechago, Black, and Samloff (1976), who found no G cells in the metaplastic gastric mucosa of a case of Crohn's ileocolitis, although the ectopic gastric tissue was antro- pyloric.

The implications are that when a tissue has experienced metaplasia, close examination of the constituent cells may reveal that, even as a tissue kind, the metaplasia is partial. The shortfall is explicable in the cellular terms of limits to the resident stem-cell's lines of specialization. It can produce most of the epithelium's normal complement for day-to-day turnover and regeneration, it may make certain novel kinds for a tissue metaplasia, but there are some cells into which it is unable to turn.

What is difficult about Barrett's esophagus and related gastrointestinal mucosal metaplasias is, first, the presence of argyrophil and argentaffin cells in the ectopic mucosa, and second, the lack of sure knowledge of the origin of the stem cells for the ectopic epithelium. Although G cells are missing, cells reacting with silver techniques and other methods for enterochromaffin cells are present in the ectopic gastric mucosa (Hage and Pedersen, 1972; Dayal and Wolfe, 1978). These presumably neuroendocrine cells could be de novo derivatives of some non-neural stem cell, if cells such as the gastric mucous cell and intestinal crypt cell can act as totipotent stem cells for their respective epithelia, as claimed by Matsuyama and Suzuki (1970), Cheng and Leblond (1974), and Sidhu (1979) on the unitarian hypothesis.

Explaining how neuroendocrine cells come to be in ectopic epithelia is a problem for the proposition that in initial development, cells of the ectoblast, already committed to a neuroendocrine role, join the presumptive endoderm: Pearse and Takor's (1979) modification to the earlier scheme whereby all neuroendocrine cells had a common ectodermal ancestry. Since this dual mode of development would leave no neuroendocrine stem cell, differentiated enterochromaffin cells could only be in metaplastic epithelia as holdovers from the previous tissue (Dayal and Wolfe's "entrapment"?), or by a migration of their own kind from elsewhere in the gut.

In the Barrett condition, there is some question whether the stem cell for the mucous, parietal, and chief cells may itself have migrated. If this stem cell moved from the stomach, where it would normally have given rise to those three kinds of cell, its behavior in the lower esophagus is not even a novel stem-cell differentiation. All that is unusual would be its abnormal position.

Barrett's esophagus is exceptional among the gastrointestinal metaplasias, because its proximity to the stomach brings the migratory hypothesis to the fore. Goldman and Beckman (1960) listed four means for gastric epithelium to develop in the esophagus:
1) by adaptation of a congenital residue of the embryonic columnar epithelium (shades of Cohnheim!);
2) from esophageal glands, either cardiac or submucosal;
3) a "metaplasia of squamous epithelium into columnar Barrett epithelium could be another less likely possibility;" and
4) by an upward extension of gastric epithelium in response to injury of the esophageal lining.
These authors favored the last hypothesis, which has since drawn support from clinical and experimental observations involving the denuding and repair of the esophageal-gastric junction (Hamilton and Yardley 1977, inter alios).

Accordingly, the mechanism commonly postulated for the Barrett state differs from that for other metaplasias, because the esophagus may be stripped of epithelium by refluxing gastric juice. Thus, it is not a matter of a metaplasia in an intact epithelium with its own stem cells but of a reparative migration of stem cells from an adjacent segment of the tract, which, if they do not deviate from their expected lines of development, introduce neither a cellular nor a tissue metaplasia.

On the other hand, in some cases of Barrett's mucosa, the cellular characteristics are deviant regardless of whether the stem cells have a gastric or an esophageal source. For example, according to Hage and Pedersen (1972), "the occurrence of goblet cells and patches of villous columnar cells may be interpreted as intestinal metaplasia analogous to the findings in patients with gastric carcinoma, gastric ulcer and diffuse atrophic gastritis (Rubin et al., 1966)."
Part of the Barrett picture therefore may be put on the same tissue metaplasia basis as most alimentary transformations, where the ectopic tissue is far enough from its usual site to rule out a migration, in favor of an origin from local stem cells. This one mechanism - deviant differentiation of esophageal squamous stem cells - could account for the whole Barrett transformation, and, although relegated to a minor position by Goldman and Beckman (1960), deserves attention for the economy of explanation that it offers.

The alimentary metaplasias thus demonstrate that the tissue metaplasias are still haunted by Cohnheim's hypothesis of embryonic rests, but to attribute the changes to the tissue's own stem cells only reveals how little is known of their potentials, determinants, own state of differentiation, migratory abilities, and their relation to the cells populating that locale in embryogenesis (Rubin et al., 1966; Pearse and Takor, 1979).

Lens Regeneration in Newts
The regeneration of the urodele lens in vivo from cells of the iris or neural retina is probably the most closely followed and convincing example of an indirect metaplasia. Adami (1908) and Weidenreich (1923d) drew it into their discussions of metaplasia. Reyer (1954) reviewed the early work on lens regeneration, and recent surveys are his (1977) and Yamada's (1977).

The significance of the pigmented epithelium of the mature newt's iris is that its population is static (Hay, 1968), undergoing no mitosis until the lens is removed, so that it is unlikely that any reserve cells participate in the reconstruction of the lens; nor do other cells intrude as possible lens-formers. Thus, it is the pigmented cells which lose their pigment, by a process of dilution by cell divisions as well as by active lysis and shedding (Reyer, 1977; Yamada, 1977), then commence making lens-specific proteins while elongating to take on the form of lens fibers.

Yamada's careful terminology (see p. 93, this chapter) differs from what I have chosen. Yamada calls the iridial cellular transformation a "cell-type conversion" and virtually equates it with "cellular metaplasia." Since it comprises dedifferentiation, proliferation, and a new differentiation, it constitutes plain, old fashioned indirect metaplasia.
Yamada referred to direct metaplasia as a "cell-type conversion without division," of which he was sceptical; "A short cut from one adult cell type to another adult cell type does not seem to exist." In the iris, a definite loss of differentiated characteristics occurs and six cell divisions seem to be needed for the process of re-differentiation (Yamada, 1977).

He conceded that, in invertebrates, a direct metaplasia might occur, as indicated, for example, in Hydra. Burnett (1968) had observed both a direct metaplasia of digestive cells to epidermal, epithelio-muscular cells, and an indirect metaplasia of mucous glandular cells, passing through a phase of looking like interstitial cells and dividing, to become cnidoblasts able to form nematocysts.

Lens Formation by Transdifferentiation in vitro: Urodela, Aves, Mammalia
The lentoid change in the newt's iridial epithelial cells was so clear-cut an example of indirect metaplasia that many biologists have tried to detect the phenomenon in other classes (see Reyer 1977). These in vivo and in vitro studies established not only that newts, some teleost fishes, and very young chick embryos could reform a lens from iridial or retinal pigmented epithelium, but retinal pigment epithelium could become neural retina (references in Eguchi, 1976).

Yet the conclusion that the phenomena represent an indirect metaplasia of cells has been imperilled by the chance of an experimental contamination by cells already determined for the final tissue, or of the later migration of differentiated cells.
Recent experiments of Okada, Eguchi and their coworkers have eliminated these obstacles to a metaplastic interpretation by using long-term cultures, with either cloning of identified pigment cells or close watch on pure groups of such cells. The transformed cells' production of lens crystallins was checked by an immunofluorescent method. The relevant points from their results and analysis are these:

  1. Pure clusters of urodele iridial epithelial or retinal cells form lentoid bodies, thus demonstrating the metaplastic basis for the in vivo regeneration of the lens in these animals.
  2. Avian retinal pigment cells (Eguchi, 1976), human fetal neural retinal cells (Okada et al., 1977) and pigmented cells from the iris or retina of the 12-week post-conception human embryo (Yasuda et al., 1978) can all differentiate into lens fibers.
  3. All instances of a transformation follow a period of cell proliferation and a loss of pigment (not always total), thereby meeting the criteria for an indirect metaplasia, although the number of cell divisions varies, being far fewer in the quail than in the chick.
  4. The basis for calling the cellular change a transdifferentiation was explained by Eguchi (1976) in the discussion to his paper: "'Metaplasia' is, of course, a possible term. However, we preferred 'transdifferentiation' because it distinguishes more explicitly the phenomenon at the cellular level which we have demonstrated from the phenomena of transformation and transdetermination."
    Of course, the widespread use of transformation to denote a neoplastic change now makes its retention as a specific synonym for metaplasia hazardous, unless neoplasia does not enter into consideration.
    The General Discussion I in the symposium to which Eguchi (1976) contributed gives some idea of the pitfalls of using determination and transdetermination outside the contexts of early embryogenesis and experiments on Drosophila's imaginal disks (Hadorn, 1966; Gehring, 1972) (see Transdetermination in Imaginal Disks, this chapter).
  5. Eguchi (1976) drew attention to the unidirectionality of the various ocular cellular transformations - always into lens cells. In the discussion, Wolpert suggested that the concept of a "'developmental sink' derived from work on Drosophila, might have application here".
    Something similar will arise with the connective tissue metaplasias where bone is the common end-point for many apparent metaplasias of connective tissue, fibrocartilage, and hyaline cartilage. In the section to follow, melanophores occupy a comparable terminal position in the changes in two kinds of pigment cell.
Transformations in Chromatophores
The coloration of lower vertebrates depends on cells (chromatophores) having their origin in the neural crest or medullary plate (Niu, 1954). Melanophores and melanocytes make black melanosomes, xanthophores contain yellow pterinosomes and carotenoid vesicles, and iridophores have reflecting platelets holding crystals of guanine and hypoxanthine.
The identification of melanophores is aided by their physiological responses to changes in light and temperature (Niu, 1954), alkali-treated ACTH (Ide and Hama, 1976), and MSH (Ide, 1978).

Differentiated xanthophores and iridophores are reported to transform into melanophores in vivo and in vitro. Niu (1954) grafted neural crest of Amblystoma punctatum orthotopically to larval Triturus torosus. Around 60 days later, on the host's flank, melanin appeared around the nucleus of xanthophores with still recognizable yellow bodies and processes. Ultimately the cells became heavily pigmented and indistinguishable from melanophores in looks and physiological responses.
There was no "evidence that ordinary melanophores undergo demelanization and then re-differentiate into xanthophores. Thus, the process involved in the change from xanthophores to melanophores is apparently not modulation (Weiss, 1939), but true cell formation."

Another instance occurred when the crest of the trunk had been incompletely removed. Of the few flank xanthophores seen transforming, Niu wrote: "Their slow differentiation distinguishes them from the 'reserve' propigment cells or melanoblasts." He concluded that the "transformation of xanthophores does not reveal any sign of dedifferentiation, but records a direct differentiation from one cell-type to another," i.e. a direct metaplasia.

Niu also grew amphibian cranial neural crest in culture and obtained giant cells, believed to be determined procartilage cells. When Holtfreter's medium was replaced by celomic fluid, some of the giant cells turned into melanophores, larger than those derived earlier from melanoblast precursors in the explant. Although Niu could watch individual cells transforming, studies of fish xanthophores are only suggestive of a melanotic transformation, but cannot rule out stem cells as the source of the new melanophores.

Loud and Mishima (1963) brought about melanization in xanthic goldfish scales in vitro with ACTH, and believed that a "transitional melanolipophore" was present. Of this work and their own on melanocyte inductions in goldfish organ cultures, Chen et al. (1974) commented "although . . . most of the melanocytes formed after hormonal stimulation come from stem cells, the possibility that some xanthophores can undergo melanization cannot be ruled out. This is particularly due to the close embryonic origin of pigment cells and the demonstrations that some animals contain pluripotential pigment cells, capable of synthesizing more than one kind of pigment ...." (Ide and Hama (1976) and Bagnara et al. (1979) give more references to such apparently dual-roled cells.)

The problem posed by stem cells was overcome for amphibian chromatophores by cloning iridophores (Ide and Hama, 1976) and xanthophores (Ide, 1978). In culture, the iridophores lost their platelets, made melanosomes, and took on the reactions of melanophores to treated ACTH. Although TEM showed that platelets or pterinosome and melanosomes sometimes co-exist in the same cell (perhaps in the same organelle (Bagnara et al., 1979)), the above transformations seemed to involve not a shift in existing activities but a change-over in synthetic mode, with no conversion of already formed pigment granules. The melanotic transformation of the xanthophores included the acquisition of the receptor and cyto-motor mechanisms for responding to MSH.

These metaplasias of amphibian cells in culture are direct, in the sense that the cells do not lose their differentiated products before making new ones, and the supposed hallmarks of an indirect metaplasia are dedifferentiation and mitosis. Yet the changes in the cultured xanthophores do accompany a cell proliferation, whose role in the metamorphosis Ide (1978) did not discuss, except to note that in comparison with the transformations reported for cultured avian optic cells - also of crest origin - the change came sooner, although the generation time was rather longer.

Glandular Metaplasia of Renal Smooth Muscle
Partial restriction of the blood supply to the mammalian kidney causes a great increase in the organ's glandular cells, to the point where it has been called the "endocrine kidney" (Cantin et al., 1977). The possible sources of the many new juxtaglomerular cells are the original population of juxtaglomerular cells (JGC), some kind of stem cell, or a metaplasia of already differentiated cells of some other kind, such as vascular smooth muscle cells (SMC) (Goormaghtigh, 1939). Cantin et al. offer as evidence of a direct metaplasia of smooth muscle cells:
1) the development of cells intermediate between SMC and JGC with filaments with dense attachment bodies, and specific granules and a large Golgi complex; and
2) the lack of incorporation of tritiated thymidine in JGC and the "intermediate" cells of the vessels' walls.

By contrast, the existence of cells intermediate in type between smooth muscle and secretary JG cells early and late in tho rat's renal development caused Cain, Boss, and Egner (1978) to favor a modulation-within-one-tissue interpretation of both developmental and pathological changes. They suggest that there is only one population of cells, all "bivalent," with apparently pure muscle and pure granular cells lying at the ends of a continuum. They interpret the findings of Cantin et al. (1977) not as a metaplasia but a "mobilization of specialized muscle cells," with the implication that these are special in being already bivalent but with their secretary ability as yet unexpressed. The problems arising from the concept of modulation between cells with multiple identities are discussed under the connective tissues (see Myoid Transformations, this chapter).

Connective Tissue Metaplasias

Dispersed throughout the chapters on specific sites are cited reports of three metaplasias:
a direct metaplasia of cartilage and occasionally chondroid to bone;
the rarer direct metaplasia of bone to cartilage or at least to a cartilaginous kind of bone; and
the development of some secondary cartilage from periosteum and bone from perichondrium, which might be, if not cellular metaplasias, then "tissue metaplasias."
Some other reported metaplasias of connective tissues are listed here.

Fibrous Connective Tissue to Bone
There are very many reports of pathological instances of ossification in soft tissues, and an extensive literature exists on experimental bone induction (e.g., Bridges, 1959; Ostrowski and Wtodarski, 1971). In addition, soft collagenous tissue appears to be transformed into bone within polyethylene tubes enclosing a rat's fracture (Pritchard, 1964), in "Millipore"-isolated defects in canine radii (Bassett and Ruedi, 1966), and in the posterior metopic sutures of rats made hydrocephalic (Young, 1959).
In the recovery from parturition of the guinea pig's pubic symphysis, fibroblasts seem to become osteoblasts (Ruth, 1936b). Reptilian dermal ossifications are primarily by a direct ossification (sclerification) of connective tissue (Moss, 1969).

Fibrous Connective Tissue to Cartilage
The transformation of regions of tendons and ligaments to a rather fibrocartilaginous state is described in Chapter 9, but such an event also befalls the connective tissue of: pseudarthroses (Urist, Mazet, and McLean, 1954; Pritchard, 1963); the patchy repair of osteoarthritic surfaces of human femurs (Meachim and Osborne, 1970; Little, 1973), and experimental articular lesions (Tallqvist, 1962; Pinder, 1974); the aging temporomandibular disk (Schaffer, 1930); the fibrous surface of the TMJ's articular eminence (Moffett et al., 1964; Wright and Moffett, 1974), which when subjected to abnormal loading seems to become hyaline cartilage (Meikle, 1970); nearthroses arising from deformity (Scapinelli and Little, 1970) or after condylectomy (Poswillo, 1972); calcifying tendinitis (Wrede, 1912; Uhthoff, 1975); the human ligamentum flavum (Scapinelli, 1963); the annulus fibrosus? (Vernon-Roberts & Pirie, 1977); the inner periosteum of horses subject to hereditary multiple exostoses (Shupe et al. 1979); and the developing distal interphalangeal joint (Schaffer, 1902a) of Rana pipiens tadpoles (Joyce and Cohen 1970).
Synovial soft tissue forms cartilage in osteoarthritic mice (Walton, 1977); and synovial chondromatosis is one of many human ectopic chondrifications.

Cartilage to Fibrous Connective Tissue
Examples suggestive of this transformation occur in the subperichondral costal cartilage of old rats (Dawson and Spark, 1928); the formation postnatally of the fossa nudata on the sigmoid articular surface of the human ulna (Haines, 1976), and at other sites in horse and cow; in the development of the pubic symphysis in the female guinea pig (Ruth, 1936a); in the symphysis of the mouse in old age (Ruth, 1935) and pregnancy (Storey, 1972) - for more on the interpubic ligament see Hall (1978); in articular hyaline cartilage of rats treated with testosterone (Tarsoly and Mateescu, 1972); as one of the events of osteoarthritis, which may be reflected in a change in the type of collagen produced by the chondrocytes (Gay et al., 1976); and in a lethal dwarfism (Lazzaroni-Fossati et al., 1978).

Hyaline Cartilage to Fibrocartilage
This transition is a second stage in the development of the articular cartilage of the fowl (Fell, 1925); and symphysis menti of the hamster (Trevisan and Scapino, 1976a) and rat (Bernick and Patek, 1969); in the formation of part of the intervertebral annulus fibrosus (Prader, 1947) and pubic symphysis (Zulauf, 1901); and in part of the cartilaginous nucleus on the radial tuberosity (Amprino and Cattaneos, 1936).

The range of situations in which connective tissue metaplasias have been reported thus extends from normal development to aging, regeneration to tumorigenesis, reproductive cycles to experimental transplantation and inductions. Each observation of a metaplasia of cartilage to bone or vice versa is to be examined on its merits elsewhere in this book.
However, the many metaplasias of connective tissue in general call for some comment here regarding the explanations of metaplasia outlined earlier, in order to treat certain difficulties of those explanations in more detail.

One may divide the metaplasias observed in connective tissues into two categories: rapid transformations occurring early in development and regeneration and some tumorigenesis, and slower transformations taking place later in development and healing or late enough in life to be considered as an aging phenomenon.
In the second class, the transformations of, for example, tendon and articular disk to fibrocartilage, symphyseal hyaline cartilage to fibrocartilage, and sutural connective tissue to bone, occur slowly without an obvious increase in the number of cells present or the adoption of juvenile attributes by the cells.

The overall appearance is of a direct metaplasia of an established connective tissue into another, but the warnings of Virchow apply to most of the examples: the idea of a direct transformation is indirectly based on a grading of the two tissues into one another in individual specimens, and that specimens taken in temporal sequence show one tissue to have taken the place of the other. Only very rarely has a transformation been actually observed in vitro as it happened, or tritiated thymidine been used to rule out mitoses.

Nevertheless, the slow pace of change, the small number of cells, and the dense matrices holding the cells imprisoned add up to circumstances where the observed changes may reasonably be accounted a direct metaplasia. Some of these slow transformations have otherwise been regarded as modulations, or delayed differentiations of immature tissues (see "Immaturity" of Cartilage, this chapter).

RAPID TRANSFORMATIONS IN CONNECTIVE TISSUES

The Role of Stem Cells
The first class of apparent metaplasias of connective tissues involves heterotopic tissue, e.g., bone or cartilage, forming quickly amidst numerous young and dividing cells. Such tissue is seen in fracture and soft-tissue healing, other skeletal secondary chondrogeneses, and many experimental inductions of bone or cartilage in vivo and in vitro. There are typically large numbers of proliferating, undistinguished cells, whose pedigree has proved impossible to resolve.

If the site is away from the skeleton, the ectopic cells may have arisen by a direct metaplasia of fibroblasts, or be dedifferentiated fibroblasts (an indirect metaplasia), or newly differentiated mesenchymal cells (a stem cell differentiation).
On the other hand, in a chondrogenesis by erstwhile periosteum, the chondroblasts could be: transformed osteoblasts (direct metaplasia), dedifferentiated osteoblasts (indirect metaplasia); skeletal stem cells differentiating for the first time, or, supposing the skeletal stem cell to be a separate entity limited in its potential, undifferentiated mesenchymal cells migrated from nearby in the periosteum. The latter two alternatives are stem cell differentiations. (Not knowing what to expect normally from such stem cells, it is impossible to say whether or not this behavior is novel.)

It is not necessary for osteoblasts to transform or dedifferentiate, if a population of stem cells persists in a periosteum. However, in effect this persistence postulates an undifferentiated mesenchymal cell (UMC) for bone that is open to the objections raised elsewhere (see Chapter 7) against the undifferentiated cell supposedly in other soft tissues.
While the existence of the soft-tissue UMC is questionable, if it is accepted as a component of fibrous connective tissues, then it should be present in periosteum and perichondrium. It cannot be used to account for ectopic bone and cartilage but excluded from periosteal chondrogenesis. (Ohlsen (1978) is exceptional in attributing the cartilage formed by grafted perichondrium to mesenchymal cells.) If there is no resident stem cell, then dedifferentiation, in other words, an indirect metaplasia, is obligatory for secondary chondrogenesis.

Looking at not just secondary chondrogenesis but at connective tissue transformations overall, one sees that the reason it has proved impossible to reduce the explanations for them to a manageable number rests in large part with the unknown status of the connective tissue stem cell(s). The simplest line to take is that mature connective tissues have none, so that, if a new tissue arises, it is by a direct or indirect metaplasia of specialized cells such as fibroblasts and osteoblasts. Guarded acceptance of this standpoint will allow the discussion to move on to a known dedifferentiation - that of urodele iridial cells. The discussion also will furnish a basis for reviewing the many terms now employed in the metaplastic context.

Regeneration as the Context of Indirect Metaplasia
Although the histogenesis of certain tumors may involve an indirect metaplasia, the circumstances of indirect metaplasia that are accessible to controlled studies are regenerative. In generalizing primarily from the newt's iris, we see the cellular events as occurring in two phases.

In phase I a process of transformation to seemingly less specialized cells involves two kinds of event: losses and gains. These may be partial or total, and sometimes a loss is a gain. Thus, if a cell loses its polygonal shape, it gains another, e.g., stellate. The urodele, iridial, epithelial cell loses a modest level of tyrosinase, and all its melanin pigment, but retains a modest level of tyrosinase, and a muscle cell may relinquish its arrays of myofilaments, but keep levels of actin and myosin adequate for alterations in shape, intracellular transport, and perhaps locomotion.

In general, what is lost or depleted are the materials and bodies directly related to the cell's special tasks. Their destruction may require a stepping-up of the production of lytic enzymes. Also, on the side of the gains, changes in the nucleus make it larger, with looser chromatin, and larger and more numerous nucleoli. More free ribosomes appear in the cytoplasm, so that the cell abandons the sedate nucleus and ribosomal configuration of maturity and acquires those typical of more juvenile and synthetically active cells.

Sometime after the start of these shifts in the cells' shape and constituents, the cells divide. The loss of the larger inclusions and organelles may facilitate mitosis, but proliferation need cause no major interruption in a cell's activities, as has been shown in several tissues (Hay, 1968). Division may also add to the synthesis, for the cell must lay in enough microtubules and other materials for mitosis.

Phase II follows the above changes and the mitotic increase in the number of cells involved. The cells alter their change away from breakdown, basic maintenance, and the replication of DNA for division, towards producing more specialized products and structures, acquiring a new shape and relations with other cells and losing their immature look of a large pale nucleus and more free than membrane-bound ribosomes. This second phase can run two courses:

Phases I and II (A) and (B) have been set apart to provide a clearer background for examining the choice of names for the cellular processes, particularly Yamada's (1977) (see Table III). Over the years the various names and their implications and contradictions have become embroiled in a "terminological mayhem" (Hay, 1968), which can bring any discussion of metaplasia to a standstill.
Table 111. Terminology of Metaplasia
Differentiated- - - 2 - - -> Differentiated- - - -8- - - ->Differentiated / Cell A \ Cell A (inactive) / Cell A 1/ \ \ / / \ \ /4 Stem cell \ 3 \ / \ 6\ \ Stem-like cell/ 7\ \ \ \ \ 5 \ Differentiated \ \ Cell B Differentiated \ Differentiated Cell B Cell B ----------------------------------------------------------------------------------- Steps YAMADA 1 Differentiation 2 Active-to-inactive modulation (Ml)* Transient dedifferentiation 3 Dedifferentiation, Rejuvenescence, Definitive dedifferentiation Retrodifferentiation, Entdifferent- zierung, Despecialization 3 + 4 Tissue-true regeneration, if no stem cells survive initial development 3 + 5 Indirect metaplasia Cellular netaplasia or cell-type conversion** 6 Direct metaplasia Direct cell-type converson 7 Novel stem-cell differentiation Tissue metaplasia 4 Redifferentiation Pathway of retrieval 8 Inactive-to-active M-1 modulation ------------------------------------------------------------------------------- All steps except 2 and 8 are modulations (M2). * Weiss's (1953) "operative" to "inoperative" change ** Eguchi' (1976) "transdifferentiation

Terminology of Differentiation and Metaplasia

Dedifferentiation
Dedifferentiation, retrodifferentiation, despecialization, and Entdifferenzierung (withdrawal from differentiation) have all been applied to phase I above. In Table III, depicting the relations of stem and dedifferentiated cells to two different specialized cells, A and B, step 3 corresponds to phase 1, step 4 to IIA, and step 5 to phase IIB.

What the term dedifferentiation fails to express openly is:

  1. Acquisition of the differentiated state proceeds step-by-step through many generations from the zygote, but the un-differentiation is only a partial undoing of the cell.
  2. While losing some specialization, e.g., for melanin synthesis, the cell is gaining another in the sense that it is differentiating into a blastic cell. Thus a dedifferentiating fibroblast is coincidentally a differentiating mesenchymal-like cell.
  3. Dedifferentiation implies a less restricted determination, with a matching gain in potential. To use dedifferentiation for a state of diminished activity with no loss of determination detracts from dedifferentiation as a term and is unnecessary, since an active-to-inactive shift or M I modulation properly characterizes such a change.
When phases II A and B (above) are compared as sequels to phase I:
cells taking pathway I and II B must have gained the ability to change determination somewhere in phase 1;
but if cells proceed by way of I and IIA, they come back to the original cell type.
Although this second outcome clearly does not demand that the cells ever become multipotent while en route, there is no way at present of showing that they do not. It is as probable as not that they lost their determination, but factors then operated to direct them to form cells typical of the original tissue. The newt's iridial cells are a case in point, because not all of those that lose their pigment go to form a new lens; some build new iridial epithelium. That some of the participating depigmented iridial cells can turn into lens-formers implies that all the cells lost their determination. Some then were caused to regain their earlier determination, others got a new specialization.

What Yamada (1977) defined from these events conflicts with what he implied - "data on the present system suggest that the cellular processes underlying both types of dedifferentiation (with and without reduced determination) are identical or closely related. . . ." The two types of dedifferentiation were defined elsewhere as a diminished manifestation of specialization without a change in determination (transient dedifferentiation) and with such a change (definitive dedifferentiation). As Yamada defines it, transient dedifferentiation is not a dedifferentiation, but an active-to-inactive shift (less of the specialized activity).
However, if, as he justifiably suggests, the cellular processes early in the regeneration are identical, then all the participating iridial cells dedifferentiate both definitively (because their determination is lost) and transiently (because the loss is not permanent). The difference is that cells engaged in the tissue-true regeneration give no sign that their determination was temporarily in abeyance. Only with change in cell type is there some evidence of a loss of determination, and so the only cells offering an indication that a dedifferentiation occurred (a transient one, incidentally) are the ones that take the definitive, i.e., definitely metaplastic, route.
In Table 111, A cells taking the route 2 + 8 cannot be distinguished from ones going by 3 + 4, since neither inactive A cells nor stem cells express themselves.

If the detection of dedifferentiation poses difficulties in cells as distinctive as iridial pigmented cells, the obstacles are greater for the fibroblast and other connective tissue cells. When the fibroblast is quiescent in the dermis it is "singularly featureless" (Breathnach, 1978). Its major products are not chemically unique (although components of its surface coating may prove to be). It is not unlike the mesenchymal cells that originally gave rise JUMP if the site is away from the skeleton, the ectopic cells may have arisen by a direct metaplasia of fibroblasts, or be dedifferentiated fibroblasts (an indirect metaplasia), or newly differentiated mesenchymal cells (a stem cell differentiation).

On the other hand, in a chondrogenesis by erstwhile perioseum, the chondroblasts could be: transformed osteoblasts (direct metaplasia), dedifferentiated osteoblasts (indirect metaplasia); skeletal stem cells differentiating for the first time, or, supposing the skeletal stem cell to be a separate entity limited in its potential, undifferentiated mesenchymal cells migrated from nearby in the periosteum. The latter two alternatives are stem cell differentiations. (Not knowing what to expect normally from such stem cells, it is impossible to say whether or not this behavior is novel.) It is not necessary for osteoblasts to transform or dedifferentiate, if a population of stem cells persists in a periosteum. However, in effect this persistence postulates an undifferentiated mesenchymal cell (UMC) for bone that is open to the objections raised elsewhere (Chapter 7, this volume) against the undifferentiated cell supposedly in other soft tissues. While the existence of the soft-tissue UMC is'questionable, if it is accepted as a component of fibrous connective tissues, then it should be present in periosteum and perichondrium. It cannot be used to acqount for ectopic bone and cartilage but excluded from periosteal chondrogenesis. (Ohlsen (1978) is exceptional in attributing the cartilage formed by grafted perichondrium to mesenchymal cells.) If there is no resident stem cell, then dedifferentiation, in other words, an indirect metaplasia, is obligatory for secondary chondrogenesis. Looking at not just secondary chondrogenesis but at connective tissue transformations over-all, one sees that the reason it has proved impossible to reduce the explanations for them to a manageable number rests in large part with the unknown status of the connective tissue stem cell(s). The simplest line to take is that mature connective tissues have none, so that, if a new tissue arises, it is by a direct or indirect metaplasia of specialized cells such as fibroblasts and osteoblasts. Guarded acceptance of this standpoint will allow the discussion to move on to a known dedifferentiation-that of uro'dele iridial cells. The discussion also will furnish a basis for reviewing the many terms now employed in the metaplastic context.

JUMP to it and other skeletal cells. Its nondescript form is one into which other cells may lapse, if their needs in vitro or in vivo are not attended to. For instance, Irving and Durkin (1965) described the inactive osteoblasts under the scorbutic epiphyseal plate as "fibroblast-like;" Tonna (1975) made the same comparison for the periosteal osteogenic cells of mice.

The difficulty for the tissue culturist of the fibroblastic form assumed by such cells as macrophages was acknowledged, but not fully heeded, by the early workers, Carrel, Maximow, and Bloom (see Chapter 7, Maximow and the Fibroblast). After a similar warning by Levitt and Dorfman (1974), Pastan and Willingham (1978) still find it appropriate to write, "The terms 'fibroblast' or 'fibroblastic cell' are used rather indiscriminately to describe a flattened cell that looks something like a cultured mature skin fibroblast. However, flattened cells propagated in tissue culture are often derived from nonfibroblastic cells."

Thus, one is in a poor position to assign a state of determination when the cells' "fibroblastic" morphology cannot give assurance that the cells are fibroblasts.

Redifferentiation
Redifferentiation is another term applied in the setting of indirect metaplasia. Most commonly, it signifies the traversing of pathway II B. However, it could equally describe passage via II A back to the original cell type. Taking the whole metaplastic sequence, redifferentiation has also been made to encompass phase I (dedifferentiation) combined with II B. Transdifferentiation (e.g., Eguchi, 1976) conveys the same sense.
Lastly, redifferentiation has another meaning, if used for a direct metaplasia with no dedifferentiation. Therefore, the circumstances of its use always need to be carefully defined.

Terminal Differentiation
Differentiation - the process - leads to a relatively stable or differentiated state. An older view held that immature phases of limited duration preceded the ultimate attainment of differentiation. Regression one step back from this last step of differentiation was dedifferentiation. Opinion has shifted, regarding all or most of the steps towards the final state as differentiations, which necessitates a specific identification of the last step as a terminal or final step differentiation. This term makes the meaning of dedifferentiation even vaguer than it was, and highlights the contradiction between a terminally differentiated cell and its ability to take on new differentiations by direct or indirect metaplasia.

The experiments comprising the context for the concept of terminal differentiation were on initial histogenesis. One now has to ask how well the terms and "rules" of embryonic differentiations fit metaplastic transformations involving older tissues, especially the connective tissues?

Terminal differentiation conveys a fact, but is also associated with a hypothesis. Production of certain specialized cellular molecules starts at a definite time during differentiation. This onset is an all-or-none or quantal event. Henceforth, the cell is said to be terminally differentiated. (The synthesis of the products may be very slow through a "proto-differentiated" phase, before full-blown production is established (Rutter, Pictet and Morris, 1973).)

The hypothesis is that the nuclear rearrangements of a particular cell division - that preceding the expression of the special molecules - are mandatory for initiating the manufacture of the mRNAs specific to the luxury molecules, the "quantal cell cycle" needed to bring in "terminal differentiation" (Holtzer and Abbott, 1968).

From work on myogenesis, chondrogenesis, and erythrogenesis, Holtzer and his colleagues set out certain rules that differentiations in these systems seem to follow, including ideas expressed tentatively in Holtzer and Abbott (1968), e.g., "the succession of transitory phenotypes in a cell lineage ... probably is dependent upon mitotic activity," but more categorically in Dienstman and Holtzer (1975). Their predictions for differentiation in general are that:
1) all embryonic cells are in lineages;
2) cellular synthesis in each "compartment" of a lineage differs from that in other compartments, i.e., the cell always has a measure of differentiation;
3) the sequence of compartments is obligatory; and
4) no single cell type can give rise to more than two new types as immediate progeny.
In this scheme, there is not one but a series of quantal cell cycles, each ushering in an altered program for a different, i.e., differentiated, state, only the last of which is the ultimate or terminal one.

Do mature cells experiencing metaplasia, i.e., a new differentiation, abide by the above scheme for differentiation? One can start by asking if such an expectation is even justified, since Holtzer and his co-workers derived the rules from the behavior of embryonic tissue. One would have to answer yes, if the metaplasia is indirect. The process of dedifferentiation appears not only to take the cells back to an embryonic morphology, but in certain instances their "rejuvenescence" is evidenced by antigenic products typical of embryonic cells (Ibsen and Fishman, 1979).
If the cells have returned to a degree of embryonic condition, they may be expected to conform to any "laws" of embryonic differentiation. However, the principles of lineage, obligatory lineal sequence, and only binary options are hard to square with the reported metaplastic behavior of skeletal and other cells.

Any metaplasia contradicts the idea that the ordering of events in one fixed lineage of cell types is needed to arrive at a specific mature cell. Thus, lens can be derived not only via ectoderm from lens vesicle in the normal manner, but also through cells of the iris or neural retina (Reyer, 1977; Yamada, 1977); osteoblasts develop diversely from somitic and neural crest mesenchyme directly, probably via fibroblasts and muscle cells in ectopic osteogenesis, and perhaps through chondrocytes as a direct metaplasia.

The quantal-cell-cycle hypothesis also proposes that the progeny of a particular cell division can be one of only two cell types: "the single cell that serves as a progenitor for myogenic and fibrogenic cells will not yield chondrogenic cells" (Dienstman and Holtzer, 1975). The cells of the connective tissues, thought to be derived from a common stem cell and evincing an ability to transform into one another, are many more than two: Pritchard's comment to Young's paper (1967) introduced the osteochondro-fibro- hemocyto-osteoclasto-genic cell, which gives an idea of the potentials, but the ability to form elastin (and synovium?) should be added, and the osteoclast deleted.

Even when held to only two options per division, one cell can arrive at six destinations after only three mitotic cycles, few among the many observed in initial skeletal histogenesis and regeneration. However, does the bipotential rule even hold within the connective tissues? The question can hardly be framed, given the difficulty of establishing what a cell type is for the skeleton. How dissimilar are the synovial lining cell (B) and the fibroblast? Are a uninuclear osteoclast and a monocyte different cells? Are chondroid cells, elastic, hyaline and fibrocartilage cells all separate entities? Should osteoblasts of woven bone be considered separately from those of lamellar bone? Are cartilage, bone and chondroid bone three distinct tissues from the standpoint of the cells programming to form them? If the compartments are not yet demarcated, the question of how many divisions are needed to enter them, or move from one to another, is premature.

The delineation of compartments revolves around the queries. What is a tissue and what is a cell type? These questions apply even more to the slower transformations of connective and other tissues, because such direct metaplasias involve tissues, e.g., pigmented, already related to one another in some ways (Bagnara et al. 1979), and they may be incomplete, leading to the formation of ambiguous intermediate forms. But before attempting the tissue problem, one must consider the application of the concept of terminal differentiation to the slow transformations.

SLOW TRANSFORMATIONS OF CONNECTIVE TISSUES: ISSUES ARISING

Terminal Differentiation
If the metaplasia is direct, as it appears to be in certain connective tissues (see Connective Tissue Metaplasias, p. 87 ), by definition there should be no sign of any recovery of embryonic attributes, when these are sought by the appropriate means. Another difference between direct and indirect metaplasias is the length of time over which the transformations occur. The indirect metaplasias of regeneration happen in periods comparable to those of fetal histogenesis. However, in the connective tissues some of the transformations of late development or healing, e.g., hyaline to fibrocartilage, take much longer. Nevertheless, if the rules emerging from observations on embryonic differentiation truly reflect the ways cells institute new programs, then the later, slower transformations should be likewise constrained.

For established mature tissues, the concept of terminal or ultimate differentiation, based on qualitative chemistry, becomes unsatisfactory in various ways. First, it fails to consider
a) the later supramolecular ordering, e.g., fibrillar architecture, imposed by the cell on its products (Knese, 1963; Pritchard, 1977), as the names woven and lamellar bone so well express, and other changes as the matrix matures; and
b) the slow drift in cellular chemistry that occurs with aging, for example, the increasing production of keratan sulfate by chondrocytes (Bayliss and Ali, 1978). In these ways the terminal embryonic state is not terminal.

Second, in some sites hyaline cartilage may become fibrous, or tendon cells chondrocytic. When the tendon cells initially become fibroblasts they are terminally differentiated, but the ultimate nature of this differentiation is annulled by their going on to become another recognizable cell type, the chondrocyte.

Sometimes one can identify a terminal state from which no further differentiation is possible. Epithelial and blood cells are shed or destroyed. The newt's iridial epithelial cells can become anucleate lens fibers, and in the connective tissues, if a cell becomes an osteocyte, it is possible but not certain that it will die in situ (Enlow, 1966; Tonna, 1977), or when the area of bone is resorbed. Thus, terminal differentiation needs further qualification: doomed for the lens fiber and keratinized cell; and interim for the iridial cell, fibroblast, etc. (with the intervening period before any major instability of the cell type manifests itself usually extending a lifetime, but allowing minor slow changes in the morphological and biochemical phenotype).

The direct metaplasia of tendon fibroblasts, for example, described as occurring without mitosis, contravenes the principle that reprogramming of a cell requires a cell division. Of course, as has been pointed out, a mitosis must have preceded any change in a cell's phenotypic expression, so it is possible that two determinations were effected at the time of mitosis, one determination expressed soon thereafter, the other remaining dormant until awakened by some stimulus. This resembles the picture presented by Tsanev (1975) for the developing hepatocyte, where the cell type is acquired in an early burst of proliferation by foregut endoderm, and the later developmental changes are "cell-cycle-independent but depend on the inductive presence of different hormonal, nutritional and other environmental factors."

The Tissue Legacy
What did a vertebrate tissue have in order to be identified as a discrete entity by the early histologists? In general, a tissue had some bulk and other eye-catching features; it endured in a stable state; if not widespread, at least it occurred consistently at particular sites in various species; and forms transitional between it and other tissues were uncommon.

At the time, a direct metaplasia was thought to be commonplace for some tissues, so that transitional forms such as chondroid bone raised no obstacle to drawing a distinction between cartilage and bone: each was a separate tissue, and chondroid bone was inevitable as one turned into the other. (For the subsequent interpretation of chondroid bone's significance in the context of the connective tissues, see Chapter 8).

Chondroid bone I was not included as a type of tissue, probably because it was minute in amount, itself graded into bone and/or cartilage, was short-lived and appeared to be a temporary modification of another tissue. Of other fleeting tissues of development, the mesenchyme was extensive and could not be denied the status of a tissue, whereas the sparse dental stellate reticulum was and is omitted from schemes of tissues.

Although their bulk already had made them known to the gross anatomists, elastic and fibrocartilage initially had similar difficulty in receiving recognition. For example, Meyer (1849a) distinguished the hyaline variety as "true cartilage" as opposed to the other two kinds, which were a gemischte Gebilde of fibrous tissue and cartilage. However, their extent and persistence won them recognition, which in the case of fibrocartilage remained grudging until Schaffer (1930) and later.

For similar reasons, enduring calcified cartilage (CB II) failed to attain the status of a separate tissue. Its persistence was not widely acknowledged (perhaps because, to oust the metaplastic theory of osteogenesis, stress was laid on the destruction of calcified cartilage); it was limited in extent; it was a modification of an existing tissue that left the tissue with its major morphological hallmarks intact; and lastly, the cartilage's role in calcification was regarded as passive and non-cellular.

The idea of tissues derived from gross dissection and observation by hand-lenses was absorbed by the microscopists able to see the cellular (and extracellular) nature of the tissues. But, instead of providing a basis for a radical re-classification of the tissues, the concepts of the cell and cellular identities have had to conform to the senior entity - the tissue.
Early on, one problem lay with the matrices of connective tissues: were these some huge, intracellular inclusion (an idea persisting until Haggqvist, 1929) or were they extracellular? If the latter, did the cell processes become the fibers? More recent examples of the failure of the cell theory totally to supplant the tissue theory are:
1) the competing, and in a way, conflicting concepts of tissue and cellular metaplasia;
2) the emphasis on a change of tissue, with rare exceptions (Busuttil, 1976), has denied the oncocytic transformation of epithelial cells recognition as a kind of metaplasia, because only some of the gland's cells are involved and no new tissue is formed;
3) along the same lines, it is rare that major modifications to singl-celled organisms are thought of in metaplastic terms (Pearson and Willmer, 1963); and
4) the insufficient notice given Willmer's (1960) approach to analyzing tissues by their content of representatives of three classes of cell: epitheliocytes, mechanocytes and amebocytes.
For example, the belief that the cells in a tissue were of a tissue delayed until very recently the appreciation that bone and cartilage, as mechanical tissues, may have as an integral element ameboid cells, osteoclasts, from another tissue, namely marrow. Langerhans cells (Silberberg-Sinakin et al., 1977 inter alios) and the stratified epithelia in which they lie may prove to be another example of cells of very different origins combining to form one tissue.

Although the concept of metaplasia stems from the time when the idea of the cell was in its infancy, and the emphasis still lay with the tissue, the cell and its behavior have to be kept in the forefront of any assessment of the phenomenon aspiring to a connection with today's research. The myoid metaplasias bring out the point, although some of them involve an indirect rather than a direct metaplasia.

Myoid Transformations
For many years muscle seemed to be a tissue distinct from connective tissue and epithelium. The muscular epithelial cell (Kolossow, 1898; Zimmermann, 1898) or myoepithelial cell constituted one of the rare instances of apparent overlap, but it remained controversial for many years.

Now it is common knowledge that cells of all tissues have filaments of actin and myosin chemically similar to those forming the basis for muscular cells' powerful contractions (Clarke and Spudich, 1977; Korn, 1978). Together with auxiliary organelles the filaments serve intra- and extra-cellular transport, contraction and locomotion. By contrast, during normal development, arterial smooth muscle has a secretory role, making elastin and collagen, and behaves like a connective tissue cell. Some uterine smooth muscle cells also can make collagen (Wu et al. 1978).

The myoid transformations involve metaplasias towards muscle cells by non-muscular cells, which appreciably increase their filamentous contractile apparatus. Conversely, certain smooth muscle cells reduce their filaments and take on another or an additional specialization. An example of the latter change is the juxta-glomerular conversion of renal arteriolar cells (see Glandular Metaplasia of Renal Smooth Muscle, p. 87, this chapter).

Other instances exist of a partial metaplasia away from smooth muscle cells. Hansen's (1899) brief reference to the muscle of diseased arteries becoming connective tissue has had subsequent intermittent support from pathology (inter alios, Hata and Hosada, 1979). The condition of experimentally provoked canine arterial dysplasia offers another example (Sottiurai, Fry and Stanley, 1978), where smooth muscle cells transform into myofibroblasts.
Next, as the chick forms its smooth feather muscles and their tendons, myofibroblasts occur at the myotendinous junction, leading Drenckhahn and Jeikowski (1978) to suggest that smooth muscle cells might be turning into fibroblasts to construct the elastic tendon.

Transformations from other tissues' cells towards muscle occur in regeneration. The fibroblasts appearing in a repairing wound have secretory and contractile characteristics, hence gaining the name myofibroblast (Gabbiani, Ryan, and Majno, 1971; Guber and Rudolph, 1978). A myoid transformation also affects the epithelial cells as they are covering the wound (Gabbiani, Chaponnier, and Huttner, 1978). Not only do epidermal cells experience an accentuation of the filaments typical of another tissue - muscle - they also make more macromolecules of a class - collagen - characteristic in general of yet a third tissue (Stenn, Madri, and Roll, 1979).

The above observations, the last in particular, further undermine the dominant belief that the customary classification of the tissues into four major, mutually exclusive categories reflects the natural order. Despite isolated examples of cells that seem to lie astride two of the principal tissue categories, experimenters seeking to establish that cells sometimes do not respect the major lines drawn between the tissues have faced a very sceptical audience.

By bio- and histochemistry, it has proved not too difficult to show that materials such as actin and collagen, thought to be characteristic of a single tissue class, are widespread (see Hall, 1978). But to demonstrate that cells of one vertebrate tissue can transform into those of another principal class rarely has been possible.
One example is amphibian limb regeneration, and by no means everyone is convinced, as Hall's (1978) detailed analysis indicates. Following amputation of the urodele limb, the tissues of the stump form a cellular blastema. Not only do cartilage cells dedifferentiate and then differentiate anew to make a new skeleton for the limb, but some chondrocytes may contribute to the new skeletal muscle, an idea for which Desselle and Gontcharoff (1978) have fresh support. Other evidence from the injured amphibian limb indicates that certain dedifferentiated muscle cells, or satellite cells, can turn into chondroblasts.

These various myoid transformations of fibroblasts and chondroblasts can be viewed in the context of the affinity known to exist between muscle and connective tissue cells, reflected in Willmer's (1960) grouping them as expressions of a common mechanocyte. Based primarily on the cells' behavior in tissue culture, Willmer (1960) and Hall (1978) propose that the embryonic parting of the ways yields stem cells for three, not four, classes:
mechanocytes as the formative cells of muscle and the connective tissues;
amebocytes for blood cells and osteoclasts; and
epitheliocytes for epithelia and glands, with neural elements as a special subdivision.

How such a cellular classification conflicts with the classical separation of tissues was taken up earlier. For now, the concept of the mechanocyte might seem to diminish the significance of conversions between muscle and connective tissue cells to the order of intra-tissue-class changes, such as from a columnar to a stratified squamous epithelium. Taking such a line is, however, to yield to the temptation of dismissing a metaplasia as "only" some event mistakenly believed to be more readily explicable, e.g., by a histogenetically based affinity; and it overlooks the breaches of even a threefold division of the body's tissues evidenced by the secretory epithelioid transformation of renal smooth muscle, the possible production of cartilaginous matrices by tumorous myoepithelial cells (Doyle et al., 1968; Takeuchi et al., 1978), the synthesis of collagen by epithelia (e.g., Sakakibara et al., 1977), and the transformation of glial cells into skeletal muscle (Lennon, Peterson, and Schubert, 1979). Furthermore, one would miss the biochemical pointers to an opportunity to place the many metaplasias on a common cellular footing.

Biochemical and Morphological Indices of Differentiation
Biochemical attention to a cell's products provides another, and in some ways less equivocal, index of its type and state of differentiation, in addition to the microscopic morphology of the cell and its environs. To show this, one may contrast the various ways in which the red blood cell (RBC), neuron, skeletal muscle, and connective tissue cells express their differentiation.

First, certain cells make cell-specific products, such as hemoglobin (RBC) or acetylcholine (neuron). These have their own chemical stability and do not experience any direct supra-molecular ordering by the cells that make them.

However, the cells develop other structures to hold accumulations of their specific products in particular places. The developing RBC makes actin for participation in filaments (Lux, 1979) to help constrain the mass of hemoglobin into the typical biconcave disk of the mature RBC. The neuron develops vesicles and other devices for transporting and releasing its neurotransmitter, and has the further functions of migrating, growing processes, and communicating with other cells for correctly placing its attachments. The elaboration of filaments, microtubules, vesicles, membranes, etc. constitutes another aspect of the cells' differentiation; one evident in morphology, but underpinned by syntheses, both general and specific, e.g., for components of the cell's surface needed for cell-to-cell interactions. Although the programs for the production of many macromolecules and organelles are common to all kinds of cell, deployment of the organelles and the proportions of the macromolecules require special instructions for each cell type.

By contrast with the RBC, the most plentiful macromolecules of muscle - actin and myosin - are not unique to the tissue. Lacking the chance to show its differentiation by making large amounts of a qualitatively distinctive chemical, muscle's character is more confined to the second aspect of differentiation. (Muscle's actin does differ by at least 25 amino acids from other cytoplasmic actins (Vandekerckhove and Weber, 1978).) What matters is the amount of the non-unique macromolecules made, and their ordering into imposing intracellular filamentous arrays, accompanied by other precisely stationed organelles.

In a similar vein, the connective tissues are distinctive not so much for the uniqueness of their principal macromolecules, but for the sheer quantity of them, and the precision with which collagen and proteoglycan are combined into oriented extracellular matrices. It is true that collagens made by epithelia and connective tissues differ somewhat; and cartilage cells make a unique proteoglycan (von der Mark and Conrad, 1979), and a collagen that in the chick (Toole and Linsenmayer, 1977) may be shared with the vitreous body, but maybe not in the cow (Schmut, 1978). But most connective tissues share their predominant macromolecules with each other and, to a more restricted extent, with smooth muscle.
For these tissues the quantitative differences provide the index for distinguishing the tissues at the biochemical level. Biochemists hold that it is the relative proportions of the macromolecules made or aggregated that constitute the significant differences between fibroblasts and smooth muscle cells (Mayne, Vail and Miller, 1978), mesoblastic and chondroblastic matrices (Toole and Linsenmayer, 1977), and tendon fibroblasts and other collagen-makers (Schwarz and Bissell, 1977). As the last authors put it, "We define a differentiated cell in culture not only in terms of its ability to express tissue-specific function but also in terms of its potential to express function at a level and percentage comparable to those in vivo. Quantity of synthesis is not just an arbitrary definition of differentiation. This definition reflects the fact that, for a cell to function normally in vivo, it must regulate precisely both the type and quantity of its products."

Schwarz and Bissell's specification of "cells in culture" reminds one that it is often not possible for cells in vitro to follow through with another phase of differentiation; the ordering of their products into the larger patterns detectable with microscopy. Indeed, in culture work, even cellular form is quite often divorced from the synthesizing aspect of differentiation, for example, in chondrocytes (von der Mark et al., 1977a), and transformed fibroblasts (Pastan and Willingham, 1978).

In summary, a cell's differentiation is many-sided:

This simplistic analysis (the typical mammalian cell makes around 5000 proteins) demonstrates that there are at least six aspects to differentiation (Weiss, 1953), and serves to reinforce two points. It almost goes without saying that because cells are different from one another, in detailed terms their differentiations cannot be equated: one cell is multinucleated, another matrix-rich, etc.
Although one may be inclined to make an across-the-board hierarchy of tissue subdivisions and then equate, say, the types of skeletal muscle with the types of keratinizing epithelia, the controls for the two tissues' differentiations may be on very different levels.

Second, the analysis is to be kept in mind when considering how far a mature cell has gone with a change of differentiation, i.e., a metaplasia, and indeed what to construe as a metaplastic conversion, since cells differ in the options open to them to express and alter their differentiations.

Degrees of Metaplasia
Three aspects of a metaplasia need to be kept apart: How far has the cell changed its differentiation? What lines are open to it to express its differentiation before and after the change? How is detection of the change in differentiation limited by the technical means available? The evidence for a metaplasia so far has relied heavily on demonstrating the production of unique products and the classical morphological means of distinguishing tissues, aspects 1 (b), 2(b), and 3(b) of cellular differentiation (p. 102).

Cells that cease production of a unique material, e.g., a pigment, and start forming another, a lens crystallin or another pigment, are readily recognized by histological and biochemical measures. Another cell, such as renal smooth muscle, making no special materials, can start synthesizing renin to show that it has taken up a new activity. In addition, the cell probably retains a low level of actomyosin to provide service organelles for secretion rather than as the major working organelle. Although relocated and reattached within the cell, the major change in the actomyosin is a quantitative chemical one, and proceeds not to the elimination of actomyosin from the cell, but to the attainment of an amount appropriate to an epithelioid cell. Thus, the smooth muscle cell reveals its metaplasia in two ways: one clear-cut, the other more of degree.

The slower metaplasias of the connective tissues introduce difficulties in that the changes may involve no new macromolecules, only altered ratios, harder to detect by immunohistochemical means; and the existing matrix may hinder or prevent the cells from imposing new patterns on the matrix. Thus the cells are denied two ways of revealing modifications to their differentiation. Although the metaplasia may seem to be partial, when compared with that of other tissues, the question to be asked is: Is the new state a properly functioning response to new circumstances, adequate and complete in the context of the tissue? The metaplasia may be complete when judged by its success in arriving at a cell functioning in a new and stable role.

In assessing a metaplasia many aspects of the cells' structure and behavior go unnoted. The striking and the measurable naturally dominate. In the connective tissues these have been fiber size, calcification, canaliculi, metachromasia, etc. Now added to the measurables are many biochemical indications of cells' activities, revealing in particular the proportional rates of synthesis which partly define cellular differentiation and changes in it.

Although it might seem easier for a cell to alter the balance of current activities than to add a new product to its repertoire or to delete one, this remains a subjective evaluation until the coordination of the controls on gene expression is better understood.

Attempts to estimate the ease with which a mature cell changes its character have been made in order to help distinguish metaplasia from modulation, modulation conveying the sense of easy, reversible, normal transformations, and metaplasia referring to the infrequent, difficult, bizarre, and pathological, without reference to any reversibility. Thus, Weiss (1939), in developing the concept of modulation, wrote of the formation of ectopic bone by mesenchymal cells, "a transformation of this order (my emphasis) is known in pathology as metaplasia."

More recently, Hall's (1970b) diagram of modulations identifies in the key that between the chondrocyte and osteocyte as a metaplasia; and Johnson (1964) also categorized certain of his modulatory transformations as metaplastic. The point is that, not knowing what the stimulus to change is, and how the genome reacts to it, we cannot yet gauge the order of the event.

It seems safest to cast the metaplastic net wide and then throw back some of the catch. At the same time, this approach holds some answer to the "tissue metaplasias" based upon novel-stem-cell responses, and to the question of the immaturity of such tissues as cartilage that often seem to experience metaplasia. Accordingly, a metaplasia will be regarded as any enduring change by a cell of a mature tissue, involving: 1) an altered ratio of the major materials produced, with or without microscopically demonstrable further assembly; or 2) the manufacture of a new material, whether or not it receives any supramolecular ordering, and regardless of whether the production of other cell-specific materials ceases.

By this definition, I include among the metaplasias many normal transformations called by others modulations, and follow Willis (1962) in designating these as physiological metaplasias.
The definition's inclusion of endurance is to omit from consideration the briefer physiological fluctuations in cells, to which modulation is now often and aptly applied. Physiological metaplasia then denotes the more permanent normal alterations in differentiation, be they perceived as great or small, from tissue to tissue, e.g., muscle to epithelium, or between tissue subtypes, e.g., type I skeletal muscle to type II.

PHYSIOLOGICAL AND PATHOLOGICAL METAPLASIA

Since metaplasia for many has suggested only a pathological process, it now becomes necessary to illustrate what is meant by a pathological metaplasia. The examples also serve to illustrate the broad scope of the above definition of metaplasia, and to introduce cellular changes that meet it, but for which a more traditional term exists and is to be preferred.

The transformation of cartilage towards bone in fracture repair and the growing antler, if it occurs, is physiological, but four instances can illustrate the restricted meaning given here to pathological metaplasia.

Many kinds of glandular epithelial cell can turn into the striking oncocytes (Tremblay, 1969), packed with mitochondria but almost no other organelles, and equipped with a small heterochromatic nucleus. In becoming oncocytic, the epithelial cell develops to excess a service organelle, which because of the nuclear shut-down has no major cell-directed function to subserve. As a metaplasia, the change is unusual in being to become an ostensibly purposeless cell (although Raikhlin and Smirnova (1976) suggest one), and one perhaps more under the control of the mitochondria than the nucleus.

In another pathological metaplasia - the formation of a myxoma - connective tissue cells synthesize proteoglycans in amounts typical of chondrocytes (Bashey and Nochumson, 1979), but the cells keep their fibroblastic form. The metaplasia expresses itself only by a quantitative change, if one accepts that the myxoma cell is an altered fibroblast (Enzinger, 1965; Feldman, 1979). (Others see it as a derivative of undifferentiated mesenchymal cells (Lagace, Delage, and Seemayer 1979, inter alios)).

The conversion of a fibroblast to a neoplastic bone-forming cell in the formation of an extraskeletal osteosarcoma is a pathological metaplasia, but one introducing a general point. The difficulty arises with the first step, the premalignant dedifferentiation of the fibroblast, and holds for any dedifferentation of the fibroblast, and applies whether the subsequent behavior of the cell is pathological or physiological. Dedifferentiation involves an altered profile of activities, and at least sometimes the expression of juvenile enzymes and surface carbohydrates: it too is a metaplasia, as the term has been broadly defined above, if one overlooks the promptness with which many cells leave the dedifferentiated state.
And by so regarding it, one may have the means to bring the "tissue metaplasias" or novel-stem-cell differentiations (see Novel Stem Cell Differentiations ("Tissue" Metaplasia) p. 79, this chapter) into harmony with a definition of metaplasia based upon the cell rather than the tissue.

It was earlier noted that the stem cells in tissues have strictly limited potentials and in considerable measure are already differentiated. Their differentiation is expressed by unique molecules for communication, 1 (a), and specific patterns of service materials and organelles, 3 (a) (b). These change and new elements come into play as the cells undergo final differentiation, so that the changes constitute a metaplasia, if this is broadened to include any significant long-lasting change of products-ratio or product-type by cells in a mature tissue. In this way, the common tissue metaplasias of pathology can be regarded as a special instance of cellular metaplasia, the deviant differentiation of a mature tissue's stem cells in response to stimuli, abnormal in kind or degree.

On the other hand, for the normal course of stem-cell differentiation for tissue renewal and repair, the concept of metaplasia may be kept in mind, but the term withheld in favor of differentiation. Likewise, dedifferentiation is best retained for the first events of an indirect metaplasia or a neoplasia, keeping in mind the direction and brevity of the change. Otherwise, one ends up in the situation of modulation (Table III) where each and every change in a cell becomes a modulation, and the term becomes imprecise to the point of uselessness.

To disquiet and challenge the histologist, the above definition of metaplasia encompasses changes, pathological and physiological, that may have little matching morphological counterpart. Examples are the production of ectopic hormones by endocrine and tumor cells (Shields, 1977, 1978), e.g., human chorionic gonadotrophin by chondrocytes (Mack, Robey, and Kurman, 1977) - a pathological metaplasia; alterations in the enzymatic profiles of skeletal muscle with exercise (e.g., Guth and Yellin, 1971; Jannson, Sjodin, and Tesch, 1978) and aging; and changes in the proteoglycans made by aging chondrocytes - physiological metaplasias.
Skeletal muscle fiber types do differ morphologically (Gauthier, 1969, 1979), but many of the differences are subtle, e.g., in fibril packing density (Howells, Jordan, and Howells, 1978). And, like chondroid bone, certain muscle fibers lie astride the typical classes defined by morphology and histochemistry, e.g., the rabbit's semitendinosus fibers (Appell and Hammersen, 1979); and some type II A fibers in the adult rabbit's anterior tibial muscle possess both fast and slow myosin (Lutz et al., 1979).

"IMMATURITY" OF CARTILAGE

The slow transformations of hyaline to fibrocartilage or cartilage to something very like bone have attracted the argument that these are not metaplasias, but rather delayed differentiations of a tissue with a "lower order of differentiation" (Meyer, 1849a; Moss, 1958). Knese and Biermann (1958) also viewed neither chondroid nor cartilage as completely differentiated. They put forward their own findings at tendinous insertions, together with several classic instances of a claimed osseous metaplasia, e.g., in antlers, as evidence for a transformation of cartilage into bone. They quibbled with calling the change metaplasia, because "the concept postulates individual determined tissues with no potential for further histogenetic differentiation." Their contrary view was that "Cartilage tissue does possess a specific mechanical ability, but is histogenetically not fully differentiated, i.e., unipotent, and has the possibility of further tissue development ... Perhaps it is the fate of cartilage finally and eventually to 'umdifferenzieren' to bone."

Although they did allow that a matrix-rich tissue is doing something useful at the time of observation, aside from any prospective and "provisional" purpose, by being there in order to turn into bone, there are other measures of the fullness of the differentiation of cartilage. Some fetal cartilaginous structures regress in large part, e.g., Meckel's cartilage, and clearly are not present to become bone.

Next, their contention that bone represents the final stage in the differentiation of cartilage is contradicted by the occasional instances of an apparent metamorphosis of bone towards cartilage (Chapter 21), and the persistence in the body of much cartilage for as long as any other mature tissue.

Another difficulty is that this treatment of the metaplasia of cartilage is selective. The labelling of immaturity is not applied over the gamut of connective tissue transformations. For example, although fibrous connective tissue also appears to become a kind of bone, the former has not been viewed as therefore a poorly differentiated tissue.

The functional state of cartilage and fibrous connective tissue does not conflict with their ability to transform, i.e., to develop further, as is also true for, say, epithelia undergoing metaplastic change. When metaplasia is broadly conceived as any significant durable cellular change in an established tissue, the late transformations of chondroid, cartilage, and fibrous connective tissue fall within its scope, along with the novel differentiations of stem cells. The issue of immaturity can be laid to rest, while attention is given to the questions of how and to what the transforming cells respond.

THE MATRIX IN METAPLASIA

The extensive extracellular materials introduce considerations to the metaplasia of connective tissues that apply hardly or not at all to the other tissues. Changes in the matrix lag behind those in the cell. It may not be in the power of the cell to transform its enclosing matrix. The cell can have various relations with its matrix: building it, maintaining and at the same time being confined and stimulated by it, and escaping from it.
The matrix may alter, not from the actions of its own cells, but because of extraneous agents and conditions, e.g., lytic enzymes from phagocytic cells (Horton, Tarpley, and Davies, 1978), mechanical disruption, hypercalcemia, etc.

The matrix serves as an index of changes in the activities of its cells, but how reliable a measure is it? If the matrix alters, and factors extrinsic to the tissue can be eliminated, the cells may be held responsible. One difficulty is that with a low cell-to-matrix ratio, the cells may have to persist for a while in their new role to achieve detectable alterations in the matrix. Thus, there will be a lag.
Another complication is that a cell may differentiate in other ways before making its principal products that go to form the matrix. The matrix itself cannot reveal these early differentiations or similar changes in cells involved in a metaplasia.

If the matrix remains unchanged or alters only a little, is this worthwhile evidence that the cells have not transformed? No; the stability of the matrix may balance or offset the lability of the cells, for matrix materials have some stability of their own, as is seen in the persistence of bone and dentin after the cells have died.

If a smooth muscle cell becomes glandular, what needs to be diminished is its contractile apparatus, within itself and readily under its control. If an osteocyte should become a cartilage cell, how reasonable is it to expect it to transform its surrounding matrix to that typical of cartilage? Concerning the connective tissues, two questions need to be kept separate: Has the cell changed its programs of synthesis and destruction? How much of the pre-existing matrix is affected by these changes?
In considering the second question one keeps in mind:
the ability of the untransformed cell to rework its matrix - considerable for cartilage, e.g., Lohmander (1977), but far less, if any, for bone (see Chapter 21 on osteocytic osteolysis);
changes brought about by extraneous agents;
the period that has elapsed;
the cell-to-matrix ratio; and
what is happening immediately around the cells.

Taking these factors into account, one may reason that if osteocytes appear in form and staining reactions to have become like chondrocytes, and the nearby pericellular matrix is more cartilaginous (Chapter 21), these changes are evidence for a cartilaginous metaplasia. Although the tissue taken over-all is still partly bony by virtue of unmodified matrix and is a kind of chondroid bone, the cells themselves appear to be cartilaginously transformed (they should react with antibody against collagen type II), and to have taken the dead weight of the matrix as far in the cartilaginous direction as can be expected.

Another aspect of the reworking of a connective tissue's matrix to fulfill its metaplasia concerns any distinctive orientation of its fibrils and fibers before the metaplasia. Jones and Boyde (1977a) propose, from studying osteoblasts in vitro, that their movement in only one direction is a factor necessary to the alignment of the collagen fibrils in bone (Boyde, 1972). If an established fibrous connective tissue, say, fibrocartilage or tendon, attempts a metaplasia into bone, it would be unable to bring the collagen into a pattern characteristic for bone if its cells remain immotile and unable to spin out new collagen. The apparent direct metaplasias of connective tissues all may therefore be limited to this extent, that a bone-like ratio of collagen to mineral, for example, may be achieved, but the collagen lacks the order and alignments typical of bone formed de novo.

Last, circumstances have been conjectured in which chondrocytes or osteocytes are no longer imprisoned and burdened by their matrix. If live cells were to be liberated by erosion, they could traverse new pathways of specialization free from the task of modifying their old matrix, and free from any stabilizing influence the matrix might have on their differentiation.

This notion of escape serves as a reminder that differentiation and metaplasia cannot be completely reduced to the activities of single cells. For example, since osteocytes have little ability to destroy bone matrix, the liberation of osteocytes to a new life as surface osteocytes (Jones and Boyde, 1977b), or perhaps stem cells for other lines, requires the cooperation of resorptive osteoclastic cells.
Aspects of bone's differentiation such as the pattern of trabeculae and the weave and thickness of its fibrils require that the osteoblasts coordinate their activities. (Other tissues may lack matrices, but have comparable supracellular expressions of their coordinated differentiation, e.g., the dendritic branching and location of synapses of neurons.)
The induction of some cartilage - somitic (Hall, 1977) and extraskeletal - is one of many examples of tissue-to-tissue interactions needed for differentiation. In such inductions many cells achieve effects by pooling their product to reach a threshold level.

In conclusion, a study of metaplasia in connective tissues has to give separate attention to the cells, the matrix, the matrix with the cells in situ, and the cells freed from the matrix. (The matrix with the cells dead or emigrated is no longer vital and is therefore ineligible for metaplasia; but it might act as chemical stimulant and oriented substrate for other cells.)

Turning to the other extreme, there are enduring changes in gene repression and derepression meriting consideration as metaplasias, where the non-mammalian cells involved have no major products to encumber their change of state, or the nucleus is not even hindered by its cytoplasm.

TRANSDETERMINATION IN IMAGINAL DISKS

The development of the fly occurs in two principal steps: the prompt differentiation and growth of organs such as the gut and nervous system needed immediately by the larva, and a later construction, at metamorphosis, of those adult parts such as the eyes, antennae, and wings. The development of these special organs of the adult form starts in larval life with a few cells which multiply to form distinctive superficial clusters or imaginal disks within the epidermis. Although the late larval disks are unstructured collections of indifferent-looking cells, the cells are by no means totipotent, but rather they are already tightly restricted as to their as-yet-unexpressed roles.
Hadorn (1965) and his co-workers demonstrated this state of cellular determination by culturing various imaginal disks, or regions thereof, inside the abdomens of adult flies, where the hemolymph provided nourishment. Such cultured disks, or reaggregated mixtures of their cells, could be implanted into larvae, whereupon they underwent metamorphosis into the adult structure appropriate to the disk, or disks, of origin.

Thus, the construction of the eye or leg, for instance, involves a period of multiplication of cells determined for the structure early in larval life, followed by a phase of differentiation, triggered by changes in larval hormones, whereby the determination expresses itself as the synthesis and ordering of chitin, visual pigments, etc. Hadorn (1965) concluded that "in the imaginal discs determination occurs long before differentiation sets in," but he noted that in vertebrates the stem cells of marrow and the spermatogonia exhibit two similar phases of development.

A surprising observation after serial cultivation in abdomens, followed by larval reimplantation and metamorphosis, was that in addition to the structures expected of the genital or optic disks (autotypic), "sooner or later also antennae, legs, palpus, wings and thorax arise as allotypic differentiations." Following from this Hadorn (1965) stated: "Since the allotypic organs appear in the offspring of cells which have been first autotypically determined, a change in determination must be postulated. We call this event transdetermination."

Although it involves an alteration of determined rather than differentiated cells, transdetermination appears to have the character of a metaplasia, by being a change from one stable, restricted genetic state to another. In consequence, the event raises the same questions as does metaplasia regarding the possible participation of stem cells, the stability of the unorthodoxly acquired state, and also the role, if any, of mutations.
Gehring (1966, 1972) explained why transdetermination must involve fully or partially determined cells rather than undetermined reserve ones. Hadorn (1965, 1966) presented the evidence against transdetermination's being a somatic mutation, but his suggestion that transdetermination may share a common mode of action with the homeotic mutations is considered doubtful by Karlsson (1979), principally because she found transdetermination to lack position-specificity. Transdetermination is stable enough to result in the production of differentiated structures upon metamorphosis; however, blastemas of allotypic cells may undergo further transdeterminations, and reversals can occur with varying frequencies. Nevertheless, occasional non-reversible stable blastemas can arise by transdetermination (Gehring, 1972).
In conclusion, since both phenomena involve a more or less durable alteration in the activity permitted genes, transdetermination can be regarded as a special case of cellular metaplasia.

NUCLEAR TRANSPLANTATION AND CELL HYBRIDIZATION: NUCLEAR METAPLASIA

A cell's identity involves the nucleus as the principal keeper of its genetic programs, and membranes, filaments, microtubules, and the cytoplasm as the agents to carry out selected programs. The agents also participate in the nuclear-cytoplasmic-extracellular interplay that decides the choice of programs, i.e., what character the cell expresses. The nucleus can be exposed to decidedly new cytoplasmic surroundings by being transplanted by itself, or by cell fusion to make a hybrid cell. That the nuclear genetic activity changes is evident in the emergence of cells differing in type from that contributing the nucleus. What occurs is a kind of metaplasia - a nuclear, rather than a cellular metaplasia, if you will.

The most thorough experiment is Gurdon, Laskey, and Reeves's (1975) transplantation to enucleated frog eggs of nuclei from cells active in, or determined for, keratin synthesis, obtained from adult frog skin. Serial transplantation of nuclei from the resulting blastulae to other enucleated eggs resulted in some tadpoles with functioning, well differentiated muscles, brain, eyes, etc.
Each tissue's cells had nuclei genetically marked by their one nucleolus as originating from those nuclei transplanted out of skin cells. Gurdon et al. discounted the possibility that stored mRNAs in the enucleated egg were responsible for the later embryonic development and cellular differentiations seen. Briggs (1979) offered other considerations favoring the conclusion that the tadpole's differentiations express an altered genetic activity in the transplanted nucleus.

He went on to consider the related circumstances of hybrid cells, where the performance by a differentiated cell is switched in another direction as the genes of two nuclei are placed in competition for control of the cell, or a nucleus comes under the influence of a nucleus-free cytoplast.

These experiments on nuclear transplantation and cell hybridization clearly differ from the metaplasias of tissues in situ, with their cells intact. Yet, they provide a means to alter the gene expression of differentiated cells and hence to investigate metaplasia.


Chapter 7 UNDIFFERENTIATED MESENCHYMAL CELL

Introduction
Maximow's concept of UMCs
Maximow and the fibroblast
Maximow and Bloom's textbook
Doubts regarding UMC and its role in ectopic ossification

INTRODUCTION

If specialized cells, such as fibroblasts, may be unable to lose their specialization and do something different, one way around the difficulty is for unspecialized cells that can become the osteoblasts for the ectopic bone to already be in the connective tissues. The undifferentiated mesenchymal cell is introduced with this ad hoc phrasing, because it bears the stamp of a cell put forward to overcome a theoretical impasse.

Although one often sees Maximow's name associated with the proposition that perivascular undifferentiated mesenchymal cells are the precursors of ectopic osteoblasts (Ham, 1977, inter alios), this is a fusion, and a confusion, of two ideas that Maximow kept apart.

One is the issue of whether cells continue to exist in an undifferentiated multipotent state in mature connective tissues. Since these tissues came from mesenchyme, the cells would be undifferentiated mesenchymal cells (UMC) (Maximow, 1905). The second matter deals with the origin of ectopic cells, such as osteoblasts in soft tissues, which could arise either by a metaplasia of differentiated cells, for instance, fibroblasts, as Maximow himself (1906) thought, or from undifferentiated cells, if these exist.

Since Maximow's time, this matter of the origin of ectopic cells has been turned around to become, for some, a source of confirmation that UMCs are present. Thus, heterotopic osteogenesis happens, so that its osteoblasts are evidence for either fibroblasts' metaplasia or the presence of UMCS. Those denying metaplasia are left with the UMC. This apparent support for the UMC has become more significant, as an increasing knowledge of lymphoid cytology has diminished Maximow's own arguments in support of a UMC.

To be treated first are Maximow's experiments and ideas, which after his death were expounded for the English-speaking world by Bloom. Then, doubts concerning the UMC's current standing will be listed, especially treating the cell's contribution to explaining ectopic osteo- and chondrogenesis, and noting that Maximow and Bloom actually proposed two kinds of UMC.

MAXIMOW'S CONCEPT OF UMC

He (1906) and a colleague repeated Sacerdotti and Frattin's experimental induction of bone in the rabbit's kidney and concluded that the ectopic osteoblasts came from renal fibroblasts, but his chief interest then and thereafter lay in the origin of the ossicle's ectopic marrow and the general relations between the cells of blood, marrow, lymphoid, and soft connective tissues.

His undifferentiated mesenchymal cell (UMC) initially (1905, 1910) was the lymphocyte in all its various forms. It was the indifferent stem cell for all hemocytopoiesis and, in the connective tissues, such cells, in the form of lymphocytes, wandered without any special relation with the walls of vessels. Three of his later reviews (Maximow, 1924, 1926, 1927a) throw more light on his conception of the UMC, why it has endured so long, but also why it is an insecurely based relic.

Except for what would now be regarded as eccentric behavior on the part of the lymphocytes, Maximow's (1926, 1927a) accounts of what the cells of connective tissues do in inflammation are not markedly out of step with modern interpretations. But, if the ubiquitous lymphocyte was able to act as a UMC, what was the evidence for another kind of UMC? And, if Maximow did not have ectopic osteogenesis in mind for the undifferentiated mesenchymal cell, what phenomena did he seek to explain?

Maximow's (1926) German paper most clearly reveals his thinking, wherein he:

  1. equated the lymphocyte with both the hemocytoblast and the motile or ameboid undifferentiated mesenchymal cell (UMCa);
  2. listed several situations where, according to him, their lymphocyte-hemocytoblast-UMCa alone cannot account for all the new cell kinds appearing;
  3. proposed an additional fixed, undifferentiated mesenchymal cell (UMC);
  4. adduced evidence eliminating the fibroblast, endothelial cell, and histiocyte from contention for the role of UMC;
  5. concluded that in hemopoietic tissue the fixed UMC is represented by a non-phagocytic syncytium with pale oval nuclei;
  6. suggested that the pericytes seen most readily around omental vessels are UMCS; and
  7. proposed that a similar UMC, but one not to be distinguished from fibroblasts, is present in all loose connective tissues, including adipose tissue.
Since, in effect, he proposed a need and then searched for a cell to meet it, one can start by questioning the requirement for a fixed UMC. He saw such cells as forming:
1) histiocytes and lymphocytes in germinal centers;
2) new myeloid cells in some pathological situations;
3) histiocytes in the omentum and arachnoid;
4) perhaps lymphocytes in the same two places;
5) lymphosarcomas;
6) fat cells in adults;
7) myelocytes in extramedullary myelopoiesis;
8) the reticulum of ectopic marrow;
9) some of the histiocytes in the reaction to injected foreign proteins; and
10) free mesenchymal cells.
The sizeable number of instances does not contribute to the argument for the existence of a fixed UMC, for, with the controversial exception of fat-cell histogenesis, none involves a situation where a motile stem or progenitor cell could not have arrived via the blood or lymph.

While the need for stem cells in myelo- and lymphopoiesis is obvious, that such stem cells are mesenchymal to the extent of also being able to form fibroblasts (and skeletal cells) and fat cells was, in so far as he considered the matter at all, only a conjecture by Maximow and was based primarily on the culture of leukocytes, not of connective tissues (Maximow 1927b, 1928). (The only instances above bearing even a potential connection with fibroblasts and fibrogenesis are the lymphosarcoma and marrow reticulum cell: both still are very obscure entities (Leder, 1978).)
With regard to item 6, that fat cells are formed from fat-free precursors in adults, this is unsettled; and if they are, tissue culture work suggests that fibroblasts could be their source. A line of cultured murine fibroblasts (3T3-L 1) can differentiate into adipocytes (Green and Kehinde, 1975; Chang and Polakis, 1978).

If it is accepted that some kind of stem cell (which for lymphocytes, histiocytes, and myelocytes need not be as undifferentiated as Maximow's) is circulating and migrating, then the circumstances listed by Maximow, 1-9 above, do not constitute a need for a separate, fixed variety of undifferentiated cell (UMC).

Despite his conviction that such fixed cells existed, Maximow had difficulty finding them. In lymphoid and myeloid tissues, he believed they were represented by the non-phagocytic syncytium, now known to be discretely cellular but still troublesome for the discernment of the different cells responsible for phagocytosis, fiber-formation, lymphopoiesis, etc.
Other tissues presented more of a problem but one Maximow thought solveable by the comments of Herzog (1916) on the behavior of perivascular cells in the omentum. Zimmermann (1923) gave these cells, adherent to the walls of minute vessels, the name pericyte. Maximow held, "It may surely be that a large proportion of the pericytes are UMCS. This makes itself evident in an especially convincing way in the omentum." The pericytes, thus, were derivatives of neither histiocytes nor endothelial cells, but were the indifferente perivasculare Keimlager of the title of his review.

However, Maximow went on to write that, in the usual diffuse, loose connective tissue, the presence of the UMC is harder to demonstrate, principally because the cell's number is smaller and there is no clear difference between its structure and that of fibroblasts. Nevertheless, "they may well be found dispersed everywhere in connective tissue, preferentially along small vessels." This conviction on the part of Maximow, in essence, was an extrapolation from the circumstances of the vessels in the irritated omentum, already questionable in itself as evidence for pericytes as perivascular progenitor cells; the passage of leukocytes through the vascular wall could have been readily mistaken for a mobilization of pericytes.

MAXIMOW AND THE FIBROBLAST

The fibroblast was peripheral to Maxiniow's main concern with hematopoiesis, inflammation, and infection, but he saw it as related to the cells of blood and soft connective tissues, in particular with the UMC, in two ways. One, we have seen, was that his fixed U MC may take the form of a fibroblast. The second way concerns what cells can form bona fide fiber-forming fibroblasts.

Since fibroblasts seem only to participate late in defense and then only to form a capsule or fill defects, Maximow relied mostly on tissue culture for information on the fibroblasts' relations with other cells. The experiments involved the culture of white blood cells in plasma, where these eventually took on a spindly, fibroblastic form. The evidence that the cells had anything to do with fibroblasts lay solely in their morphology, and Maximow (1924) acknowledged the absence of a critical item of evidence thus: "Of course, production of collagen by these spindle-shaped cells, originating from white blood corpuscles, has not been shown. The cells, however, were exactly similar to the common fibroblasts, which can be cultivated from connective tissue and which also, during many years of in-vitro existence in Carrel's laboratory, have not been seen to produce collagen. We have to conclude, therefore, that a transformation of fixed and free histiocytes and of certain types of white blood corpuscles - the lymphocytes and monocytes, which cannot be sharply separated from each other - into common connective tissue cells, fibroblasts, is possible. At any rate, there are no facts directly contradicting this hypothesis." His last sentence betrayed his hesitation in accepting his own conclusion, at that time.

The subsequent three years allowed several more investigators to culture blood cells, but it was his (1927b) and Bloom's experiment on the buffy coat from guinea pigs that finally convinced Maximow (1927a) that small lymphocytes could turn "into a polyblast (Macrophage) and finally into a fibroblast."
His conclusion is still open to the objection that, whatever the number of the experiments evincing it, the transformation seen was solely morphological.

An indication of the slight importance he attached to this supposed mode of origin of fibroblasts is that although he accepted the evidence of appearances in tissue culture, he did not list white blood cells as the source of any of the fibroblasts growing in a site of inflammation: "they are the progeny of the preexisting local elements of the same kind; only a small part of them may arise from the endothelium of the small vessels. . . ."

Regarding the fibroblast's own potential, Maximow (1926, 1927 a,b) was very frugal: it could proliferate, but was "highly differentiated" and had none of the multipotentiality attributed to it by Mo1lendorff and his school.
In reaching this verdict, Maximow forgot his much earlier proposition that the fibroblast is the source of the osteoblasts in experimental ectopic ossification (Maximow, 1906); but that hypothesis was not germane to the kinds of cell which Mollendorff would argue arose from the fibroblast and it only saw the light again in Maximow and Bloom's (1930) textbook of histology.

MAXIMOW AND BLOOM'S TEXTBOOK

The undifferentiated mesenchymal cell of the connective tissues presented in almost every English-language textbook of histology since the nineteen-thirties was launched by Maximow and Bloom's (1930) book. Bloom wrote the textbook on the basis of their joint research, a partial manuscript left when Maximow died, and from Maximow's earlier book in Russian.

In the section on connective tissue, the book proposed two kinds of undifferentiated mesenchymal cell: one called that (UMC) and discussed in a paragraph under the matching subheading; the other kind appeared in the paragraph on "ameboid active wandering cells," of which he wrote, "many of them have originated in the embryonic mesenchyme and stayed there ... There is no reason or basis for distinguishing hematogenous and histogenous cells among them; they all have the same prospective potencies."

Thus the second (lymphocyte-like) kind of cell was held to be derived from mesenchyme, but still, in mature tissues, to be multipotent - in other words, it was an ameboid undifferentiated mesenchymal cell (UMCa). The book's section on fibroblasts held that fibroblasts are highly differentiated and their potencies of development are restricted; how far restricted is indicated elsewhere in the book, where fibroblasts give rise to cartilage by metaplasia (Maximow and Bloom, 1930, p. 156) and to osteoblasts in the course of normal ossification (p. 175).

One has to conclude that to the end Maximow had two kinds of undifferentiated cells in mind, saw neither as the source of ectopic osteoblasts, and believed that fibroblasts could experience a metaplasia.

It is ironic that Maximow introduced his article (1928) by deploring the way "the usual erroneous statements make their way from one text book to another. In special papers these misstatements are accepted uncritically as basic self-evident facts."
Despite his wary phrasing - "However I pointed out long ago that the fibroblasts are not equally differentiated in all species and in all places of the body. Some of them may perhaps remain in an undifferentiated mesenchymal condition, without necessarily showing structural differences, as compared with true fibroblasts" - the existence of the fixed UMC has become bald assertion, which is incautious, if not erroneous.
Also out of keeping with Maximow, ectopic osteogenesis has somehow become evidence for the UMC's presence. The UMC of current histology texts still is used as an explanation in research papers, e.g., Owen (1970), who wrote "this concept has never been seriously disputed." Serious or not, there are grounds for not accepting the premise that mature connective tissues contain undifferentiated mesenchymal cells.

DOUBTS REGARDING UMC AND ITS ROLE IN ECTOPIC OSSIFICATION

  1. Maximow (1927a) considered diffuse, loose, irregularly arranged connective tissue to be a residue of the original mesenchyme, "which remains more or less unchanged after the various other connective tissues have been formed." Although he went on, "of course, the name 'mesenchyme' in this case is not quite suitable, as this term was originally proposed as the designation of the embryonic connective tissue only," the damage to clear thinking was already done. If adult connective tissue is "mesenchyme" and experiences mesenchymal reactions, a mere renaming of the tissue has prepared the mind for the cells of connective tissues to be mesenchymal in form and potency.
  2. The term UMC itself holds an inherent contradiction. If cells have progressed in embryonic growth to the point where they can be said to be mesenchymal, they are already partly differentiated (Knese, 1963) and some are producing collagen and proteoglycans (Toole and Linsenmayer, 1977).
    This point is perhaps made clear by comparing the mesenchyme of the jaw, thought to give rise to odontoblasts and osteoblasts and to be derived from migrated neural crest cells (de Beer, 1937; Le Lievre, 1978), with the primordia for the cranial ganglia also derived from migrated cells. The neural crest cells that have become mesenchymal (von Kupffer's mesectodermal) already are significantly different, i.e., differentiated, from other crest cells that have become neuroblasts.
  3. The arguments brought earlier against Cohnheim's displaced embryonic rests, summed up in the 1920's by Nicholson (1950), also hold true to counter undifferentiated mesenchymal cells:
    . a) in normal connective tissues, they cannot be identified;
    . b) rests of less differentiated cells survive development for a while, but their usual fate according to embryologists and pathologists is autolysis or resorption. Therefore, since mechanisms exist to do away with superfluous tissue in some way (Saunders, 1966), how do UMCs escape the autolytic or scavenging systems;
    . c) an adequate cause must be found for the initial "error of development": why did the UMC fail to differentiate further while its surrounding fellows turned into specialized cells;
    . d) is there an adequate reason for its suddenly embarking on further differentiation?
  4. Why was the UMC proposed? The first cell so designated was the lymphocyte. By crediting it with a wide potential for differentiation Maximow had a stem cell for the forming blood cells and, he believed, for the production of new macrophages and perhaps fibroblasts.
    Lymphocytes are now known to lead complicated lives, to divide and to transform, into plasma cells, but they are differentiated to a degree that excludes broad mesenchyme-like potencies. The life of the lymphocyte was not understood when Maximow and Bloom's (1930) textbook appeared, in which lymphocytes of the connective tissues were called ameboid active wandering cells and were still accorded a multiple developmental potential to account, for example, for transformations then believed to occur in vitro and in inflammation. This lymphocytic cell was the UMCa.
    Now that macrophages are known to be migrated monocytes, and the potentialities and limitations of the lymphocyte are better known, there is no good reason for thinking of lymphocytes as undifferentiated mesenchymal cells.
  5. Maximow and Bloom (1930) muddled the notion of a UMC greatly by suggesting that another, fixed, cell of connective tissues, specifically called the undifferentiated mesenchymal cell (UMC), had the same ability to transform as they thought the lymphocyte had. The story of the UMC has been interpreted above as a superfluous hypothesis in search of a cell. But accepting for argument's sake the premise that in the connective tissues there is an undifferentiated kind of cell, what cells are present that could so act?
    The lymphocyte (UMCa) was the first proposed, and to offer a second seems to be hedging one's bets. Maximow and Bloom (1930) suggested that some of the small fibroblasts are UMCS, as are some pericytes.

    For the fibroblasts, to take only some of the members of a population is a facile device for generating a class of cells for another role, but one with snags. Fibroblasts produce fibers and ground substances. The claim that an adventitial fibroblast is a UMC leaves unexplained how the matrix, sparse but present in the vicinity of that particular cell, was formed, since, as an undifferentiated cell, it would not be making much in the way of fibers.

    The pericyte still has no certain task (Weibel, 1974), so that it can have roles foisted upon it. The notion, however, that the pericyte is the cell that is induced by, say, bone matrix or transitional epithelium, to become an ectopic osteoblast can be questioned for two reasons. First, why does its enclosing capillary basal lamina not insulate the pericyte from the inductive chemicals or cell contacts? Second, the hemangiopericytoma (McMaster, Soule, and Ivins, 1975; Enzinger and Smith, 1976), of which the pericyte is the chief constituent, throws some light on the cell's potentials, for example, to divide, form reticular fibers and basal lamina, but only occasionally bone and cartilage. Such behavior is not an adequate basis for attributing broad mesenchymal potencies to the normal pericyte, which remains a puzzling cell, apparently sui generis.

  6. To credit one class of tissues arising in mesenchyme with retaining broadly potent cells must create anomalies between the connective tissue classes. Even within one class, the hypothesis confers a dual origin on at least some cell types. For example, Breathnach's (1978) ultrastructural study of dermal development included the thought that a relatively undifferentiated cell endured, which could be a potential macrophage or perhaps a precursor of Langerhans cells. The second role suggested reflects the general tendency to seize on the UMC for the presently inexplicable; the first echos Maximow's thinking. However, since macrophages come from monocytes, to suggest as a second source an unidentifiable cell is to introduce a distracting and untestable speculation.

    The UMC hypothesis is applied inconsistently to the various connective tissues. The hypothesis does not make it plain whether and how the cell participates as a resident stem cell in the later development, maintenance and repair of soft connective tissues. On the other hand, some cells covering bone and cartilage are allowed to function as stem cells, but are left outside the UMC category.

    Whether any connective tissue - soft, firm, medullary, or lymphoid - has multipotent mesenchymal-like stem cells is uncertain, and has to be set against the possibility that the untoward transformations seen could occur by dedifferentiation.

  7. Since the UMC cannot be distinguished from fibroblasts and pericytes, it holds a position singular among the cells of the body. The UMC is the 'wallflower' cell that may wait a lifetime and never be asked. Not only may it not be asked to differentiate by a physiological stimulus; it cannot be asked by the investigator - he is unable to identify the cell in order to experiment with it.
  8. There is a gap in the experimentation concerning the UMC and ectopic osteogenesis. Those authors, e.g., Anderson (1976) and Buring (1975), who account for experimentally provoked and pathological ectopic bone in terms of UMCs, credit cells of mature connective tissues with a mesenchymal response, but, to my recollection, have not shown mesenchyme itself to behave in the osteogenic manner attributed to the UMCs.
    In other words, they have not implanted bone matrix (Urist, Hay, Dubuc and Buring, 1969) or urinary epithelium into mesenchyme, or grafted or cultured the two tissues together, and shown that definite mesenchymal cells can be induced to become osteoblasts by materials osteogenically effective in adults.

    One such exercise has now been done (Kahn and Simmons 1977 a,b), but as a control for a different experiment. They grafted demineralized rat bone matrix obtained from Urist to the chorioallantoic membrane of chick embryos. No bone formed and this "suggests that the mesenchymal cells of the CAM lack the capacity to differentiate into osteoblasts. . . ."
    Of course, this is only one negative finding, and mesenchyme can be induced to abnormal chondrogenesis by other, embryonic, tissues, for example, the otocyst (Benoit, 1960) and avian epithelium (Silbermann et al., 1977).

    Urist's own thinking on the UMC appears to have changed, for, when Nogami and Urist (1974) induced bone and cartilage in cultures of minced muscle from full-term rat fetuses, they described the responding cells as "amoeboid mesenchymal," but recently Urist et al. (1978) have written, "the fibroblasts morphologically dedifferentiate into amoeboid mesenchyal", but recently Urust et al. (1978) have written, " the fibroblasts morphologically dedifferentiate into amoeboid mesenchymal cell".
    Although the tissue explanted was fetal, how mesenchymal was it is unclear; and there is also the chance raised by Wlodarski (1978) that satellite cells of muscle might be another source of osteogenic cell. Nathanson, Hilfer, and Searls (1978) were not able to demonstrate the latter event in the rat, but chick skeletal muscle cells were induced to chondrogenesis by bone matrix.

  9. On a superficial level, as man and the long-lived animals age, the name mesenchymal cell sounds less and less fitting for an enduring element of their tissues. Accompanying the formation of bone in the recent abdominal scar of a man of seventy (Classen, Wiederanders, and Herrington, 1960), fibroblasts proliferate and form the collagen of the soft region of the scar. Are the nearby osteoblasts derived from proliferating fibroblasts or from UMCs? The UMCs would be undifferentiated, seventy-year-old mesenchymal cells.
    But, before the discord in the name prejudices one against the UMC, one wonders how old are fibroblasts that yesterday formed by the division of seventy-year-old fibroblasts? A riddle addressed tangentially by Hayflick (1976) but beyond the scope of this review.
In conclusion, as a cell to explain ectopic ossification, the UMC is surrounded by more evasions than is the fibroblast. The unanswered questions are:
Can we see UMCs in normal tissues? No, they are too few; they are unevenly distributed, and, anyway, they are not to be distinguished from fibroblasts. These widely enunciated characteristics are illogical
If the cells cannot be identified, how can one know their number and distribution?
Why did the UMC not differentiate?
Why is the UMC not destroyed along with other rests?
Why does the UMC remain inert for many years?
Why is it that the cell that has long been refractory to any stimulus is the one to respond to the osteogenic stimulus?
Does the same factor trigger proliferation and differentiation? (Since UMCs are supposed to be sparse, they would have to multiply to give enough osteoblasts to form an ossicle.)
Why is the UMC's differentiation to a tissue novel for the site?

Maximow was concerned with hemopoiesis and inflammatory reactions, in which he conceived of free and fixed undifferentiated cells as participants. The postulated existence of a fixed UMC throughout loose connective tissues was a generalization from a particular pathological situation, to meet a need developed more from theory than experiment.
Even in hypothesis, the fixed UMC had less to do with the formation of fibroblasts than with generating histiocytes, and to claim that the UMC gives rise to ectopic osteoblasts is to stretch to the implausible what was already one of Maximow's more tentative notions.


Chapter 8 MODULATION

Introduction and summary
Modulation: Weiss's concepts and modern usage
Schemes of modulation for bone
. McLean and Bloom
. Bloom, Domm, Nalbandov, and Bloom
. Willis
. Young
. Hall
Schemes of modulation for connective tissues
. Schaffer
. Pritchard
. Johnson
. Hall
. Willmer
. Willis, Moss, Klaatsch, etc
Variations between schemes in the cells participating
Formative-destructive schism
Osteoblast-to-osteocyte modulation
Reticular cells in bone modulations
Osteogenic cells of marrow: experiments
Consequences of omitting a cell type
Average cellular condition
Stability of the modulated state
Diagrams of modulation: implicit simplifications
Modulation 2 by chondrocytes and skeletal muscle
The connective tissues: one tissue?
Modulation and metaplasia

INTRODUCTION AND SUMMARY

Modulation, as used biologically, is a confused concept, and in its application to skeletal tissues numerous difficulties have arisen. The chief question is, since modulation concerns the transformation of specialized cells (and this is one way secondary cartilage and chondroid bone may arise and one change to which they may be subject), do the term and the reasoning behind it embody truths not considered by the older metaplastic ideas of the metamorphosis of tissues and cells?
No is the conclusion reached by an argument whose principal elements are set out briefly in advance.
  1. The modern usage of modulation has its basis in Weiss's (1939) concept, which embraced two kinds: active state to passive state (M1), and specialized to specialized (M2), reversible cellular transformations.
  2. There are a multiplicity of schemes of modulation for bone alone, and bone along with other connective tissues.
  3. Agreement is lacking on which cells participate and the kinds of transformation executed. For example:
    a) There is a potential schism in many schemes between formative and destructive kinds of cell.
    b) The meaningfulness of an osteoblast -> <- osteocyte modulation is doubtful, when the theoretical and observable points of transition and the likelihood of reversal are heeded.
    c) The inclusion of the reticular cell as a participant in skeletal events is questionable. Reasons for omitting the cell include:
    . i. the ambiguous nature of the cell;
    . ii. the lack of a good reason ever to have included it;
    . iii. its restriction to endosteally exposed bone; and
    . iv. transplantation and other experiments suggesting that marrow may have both inhibited osteoblastic cells and inducible osteogenic cells, but not proving either kind normally to have intercourse with the bone
  4. If a participating cell is inadvertently omitted, or distinct cell types lumped together, the ability to interpret kinetic data - a supposed strength of modulation - is destroyed.
  5. The original concept's postulated "mean state" cannot be ascertained. It is not chondroid bone or the undifferentiated mesenchymal cell.
  6. Questions posed for metaplasia have not been rephrased in modulatory terms, e.g., the role of mitosis in effecting a change in synthetic program.
  7. Modulation places the onus of maintaining the cell's determination on extrinsic factors, but some skeletal cells appear to outlast the inducers or determinants of their differentiation.
  8. Where the change in specialization is drastic, upholders of modulation from Weiss onwards excuse the cells involved and take refuge in metaplasia.
  9. The simplifications inherent in diagrams of cellular modulations conceal underlying weaknesses in the concept.
    . a) Cells' identities are not easily defined or, in practice, recognized.
    . b) The convention of arrows implies a tram-line rigidity that does not square with an arrival at intermediate states such as chondroid bone or myofibroblasts; does not distinguish between M1 and M2 modulations; gives a misleading impression of symmetry among the transformations; and suggests that time goes backwards.
    . c) It is not made plain that modulation requires that individual cells, rather than populations, experience reversibility.
    . d) Transformations between uninuclear and multinuclear cells require but do not receive special accommodation.
    The diagrams lend the concept of modulation a strength which is undeserved, since the above points involve matters more basic than mere problems of diagrammatic representation.
  10. In other tissues, such as skeletal muscle, the M2 concept of modulation, temporary reversible modifications of specialization, may be valid and useful. However, modulation has mostly brought to skeletal biology a confusing amalgam of two ideas and an imposition - reversibility of any changes - for which there is little evidence.
It is suggested that modulation be confined to its M1 sense involving short-term changes in activity, and that M2 modulations be called reversible metaplasias, when verified experimentally.

MODULATION: WEISS'S CONCEPTS AND MODERN USAGE

Weiss's (1939) adoption of modulation for biological use appeared first as a personal communication to Bloom (1937). It was introduced to distinguish between the believedly irreversible process of differentiation, whereby cells become specialized, and the process of modulation for those reversible transformations in the appearance and behavior of cells not involving a loss of specialization.

Modulation thus was a technical noun denoting transformations in already specialized cells. Nowadays the word is in vogue in anatomical, physiological, immunological and other fields, applied variously as:
a noun in its original two senses;
a transitive verb - modulate- roughly synonymous with control or mediate; and
as an intransitive verb, as when osteoprogenitor cells modulate to osteoblasts (Hall, 1970b).

What did Weiss (1939) mean by modulation? To quote: "It is by no means easy to arrive at an agreement as to what to consider as the end point of differentiation. Change is inherent to life, and so long as the living activity lasts, the cell is in a state of flux ... The physiologically active cell at the end of development fluctuates about an average condition without deviating permanently from this state ... The differentiation reached by a given cell is understood to include all the specific reactions of which the cell is capable."
An example of a reacting cell of changeable appearance was the pigment cell of lower vertebrates in its contracted and relaxed states, giving respectively less or more dark color to the skin in response to a light or dark background. (HI Nielsen's (1978) paper illustrates the variety of cells and reactions.)

On the basis of Bloom's (1937) review, Weiss suggested that some mesodermal cells could experience a modulation to a very different physiological state, from a fiber-forming, sessile reticular cell to an ameboid phagocyte, and back again. His next illustration of modulation was between the inactive and active states of the glandular cells of the seminal vesicle in castrated rats, intact rats, and castrated rats given hormone. His last example, also derived from Bloom's work, concerned bone cells and, like the other mesodermal example, involved to-and-fro changes from formative to destructive states.

From the outset, therefore, modulation had two shades of meaning. One (M1) for the physiological and matching morphological cycling between inactive and active, or contracted and relaxed, phases of glandular, pigmented, and muscular cells; hence the current widespread use of "to modulate" meaning to control.
The other shade of meaning (M2) is more complex: a transformation of a cell from one form already active, fiber-forming, to another cell active in a very different way, phagocytic, as in Weiss's examples of the change from reticular cell to macrophage, and of a bone cell to a macrophage.

This second kind of modulation overlaps the first in that the activity first pursued ceases, i.e., an active-to-inactive change occurs, but the first activity also appears to be lost while a second and new specialization is gained. Not so, said Weiss; the first specialization, or the potential to indulge in it, is not lost, because the transformation to the second activity can be reversed; and, since the irreversible acquisition of a specialization is differentiation, modulation serves to mark off such a reversible change in specific activity.
Thus, reversibility of the changes in the cell's activity and morphology is the key criterion of modulation in both its meanings.

How has the concept of modulation, in particular modulation 2, been applied to bone and other skeletal tissues?

SCHEMES OF MODULATION FOR BONE

McLean and Bloom
McLean and Bloom (1937) briefly reported the histological effects on bone cells of giving excess parathyroid hormone to growing rats. The experiment gave the basis for one example of modulation (Weiss, 1939), and was discussed in a later paper on changes in pigeon's medullary bone during the egg-laying cycle (Bloom, Bloom, and McLean, 1941).
To describe the cell transformations apparently taking place in the parathyroid hormone-treated rats, Weiss proposed that modulations (of the second meaning) occurred between macrophages, spindle-shaped connective tissue cells of the fibroblast type, and osteogenic bone-producing cells.
Later, the simplifications inherent in diagrams of cellular transformations will be presented, but to save space and facilitate comparisons the modulatory schemes limited to bone for the present will be represented by just such diagrams. Weiss (1939):
reticular cell -- macrophage -- spindle cell -- bone-producing cell

Bloom, Bloom and McLean (1941) from female pigeon's bone:

osteoclasts -- reticular cells -- osteoblasts -- osteocytes; and osteoclast - osteoblast
The principal evidence for these transformations was the observation of cells described from light microscopy as transitional, for example, between active osteoblasts and what were called "resting" or "modulated" osteoblasts on the surface of the bone.
These resting osteoblasts "cannot be distinguished from reticular cells of the adjacent marrow." Although the experimenters were distinguishing them by position, they pooled resting osteoblasts with reticular cells in a single "reticular cell" category. They believed, again from "transitional forms," that osteoblasts fuse to become osteoclasts, but had no evidence for a reciprocal transformation. They incidentally noted numerous mitoses in reticular cells and osteoblasts.

Bloom, Domm, Nalbandov, and Bloom
Bloom, Domm, Nalbandov, and Bloom (1958). examined the medullary bone of laying hens to find that "cells intermediate between osteoclasts and osteoblasts may occur at every stage of the laying cycle." They were confirmed in their belief that "reticular cells, osteoblasts, osteocytes and osteoclasts are temporary functional states of the same cell type, with no change in developmental potency." The authors then credited osteoclasts with an ability to become osteoblasts, and proposed that some osteoblasts are derived from osteocytes liberated by the osteoclastic resorption of bone.

Their scheme can be depicted thus:

                       reticular cell

                     / /          \\

                osteoclast -- osteoblast -- osteocyte
The evidence, influential in their belief in the reversible transformations, was the numerous "transition" forms, the rarity of degenerating cells, and the general lack of mitotic figures. In presenting this last observation in confirmation that new cell kinds arise by modulation rather than division, they neglected to comment on the many mitoses reported in the (1941) paper on the pigeon.

Willis
Willis (1962) noted that human bone destroyed pathologically in hyperparathyroidism, fibrous dysplasia, Paget's disease, and at the margins of many primary and secondary tumors in bone, is replaced by fibrous tissue. He maintained that "normal and pathological histology show that the osteoblasts, osteoclasts and fibroblasts are not immutable self-reproducing species of cells, but only functional variants of cells of the same kind."

Willis called the transformation to osteoclasia or to osteogenesis a "physiological metaplasia," and did not write of modulations. His cells' potentials can be depicted in this way:

                        fibroblast
                        //       \\
                  osteoblast --  osteoclast
Young
Young (1962) envisioned, for endochondral ossification in young rats, modulations between four types of cell which he defined as:
an osteoprogenitor cell "which includes the spindle and reticulum cell of other authors, and the mesenchyme cell of Kember, and Young;"
osteoblasts, resting on bone or cartilage and with abundant basophilic cytoplasm;
osteocytes, within lacunae; and
osteoclasts, as multinucleated cells.
From the numbers of labeled cells seen at different positions at various times after injection of the mitotic label, Young concluded that osteoprogenitors could form osteoclasts and osteoblasts directly, and that there might be a turnover of nuclei within osteoclasts with some loss of nuclei: an hypothesis of the hedding of individual nuclei.

By substituting the macrophages, spindle-shaped cells, and osteogenic bone-producing cells of Bloom, Bloom and McLean (1941), one has the modulating entities put forward by Young (1962) to account for the changes in appearances, populations, and positions of tritiated thymidine-labeled cells by, on, and in the bone of young rats.

Young's radioautographic work gave fresh impetus to a concept of modulation that has influenced the present-day thinking of many concerning how bone cells behave.

Hall
Hall (1975b) offered a critical evaluation of Young's work and related experiments, considering, in particular, evidence on the formation and fate of osteoclasts. Hall's modulating entities in his Figure 2 are actually nuclei, but the cells in which they may reside are osteoclasts, osteoblasts, macrophages, and osteoprogenitor cells.

SCHEMES OF MODULATION FOR CONNECTIVE TISSUES

The schemes of modulation below are the principal ones involving other tissues in addition to bone.

Schaffer
Schaffer (1888) wrote, in endorsement of Wolffs earlier idea, that osteoblasts, odontoblasts, and chondroblasts are essentially similar: "Bildungszelien, whose physiological distinction appears to lie in still unknown differences in their chemical-formative processes. Holding fast to this view we can also cope quite well with sudden changes in the type of tissue, e.g., where the formative tissue for a long while produces bone then suddenly cartilage so that both stand in the most intimate spatial relation. But here no-one is required to think of a transformation of one tissue into the other, although the reason is today still unknown why a seemingly equal, germinal tissue forms now bone, now cartilage."
Schaffer's hypothesis of a modulation of the germinal tissue was echoed by Murray and Smiles (1965).

Pritchard
Pritchard (1956) believed that the following cells are intertransformable: osteoblast, chondroblast, osteocyte, osteoclast, marrow reticulum cell, resting osteoblast, and spindle cell.

Johnson
Johnson (1964) analyzed the relations of skeletal, medullary, and vascular cells from the standpoint of the pathologist and morphologist. From the behavior of the cells in various diseases and their normal content of some fat, Johnson proposed a lipo-skeletal series of cells with the fat cell as a "reserve cell" of adult bone from which osteoblasts and osteoclasts are recruited.
Other cells participating in this particular mesenchymal modulation (he postulated two other modulations for cells of mesenchymal origin (his Fig. 33)) were chondroclasts, fibroclasts, fibroblasts, chondroblasts, and myxoblasts that specialized in making polysaccharides: "The change of cell activity state - modulation - is imposed by the field in which the cells are active. Modulation involves a time-specific and place-specific accentuation of one of several capacities."

Hall
Hall's (1970b) scheme had transformations between fibroblast, chondroblast, osteoblast, chondrocyte, osteocyte, chondroclast, osteoclast, and also allowed mononuclear leukocytes to contribute to osteoclasts, but without osteoclasts' giving rise to macrophages: a possibility that he raised later (1975b).

Willmer
Willmer (1960) wrote of osteoblasts and chondroblasts that "it is even possible that they are interchangeable, i.e., one may be a modulation of the other; both are modulations of the mechanocyte."
He subdivided his mechanocytes, based primarily on the behavior of cells in tissue culture, into myoblasts to become muscle, and "myxoblasts" (not the myxoblast of the pathologist, Johnson (1964)), which shared the property of secreting significant amounts of mucoprotein.
Willmer's myxoblasts as a class encompass fibroblasts, chondroblasts, osteoblasts, synovial cells, and odontoblasts, of which he regarded the last two types as "more aberrant," while the others are "rather closely related genera."
Willmer placed osteoclasts in a completely distinct class - the amebocytes - a class complementary to mechanocytes and having representatives in each organ to balance the activities of the mechanocytes present.

Willis, Moss, Klaatsch, etc.
Other writers sharing some of Willmer's broad view of cell classes and relationships are Willis (1962), whose concept of physiological metaplasia, discussed earlier for osteoclasia, also allowed cells of callus to form bone, fibrous tissue, cartilage, and synovium; and Moss (1964a), whose "scleroblasts" (Klaatsch, 1894) are cells of many sites, in various classes, able to form bone, cartilage, dentin, enamel, and intermediate tissues, as the particular circumstances of development dictate.

The point of each of the above schemes of modulating cells is that, for the author, it accounts for the development, turnover, normal alterations, and sometimes for pathological changes, of skeletal tissues, in terms of populations of several kinds of cell (which are, at the same time, expressions of one cell) that can transform reversibly into one another, under the influence of various factors.
As one type of cell turns into another, the greater numbers of the second kind result in more of a particular activity, and less of another, and the tissue changes. However, the application of Weiss's concepts of modulation to skeletal tissues has stumbled in various ways.

VARIATIONS BETWEEN SCHEMES IN THE CELLS PARTICIPATING

Since the schemes deal with a limited set of tissues, why do the plans not agree on the kinds of cell that can transform? It is not just a matter of the first group of authors looking only at bone, the second's including cartilage, synovium, and other soft connective tissues.
There are fundamental differences.
One, Willmer's (1960) plan has a barrier, in other words, no modulation, between osteoblasts and osteoclasts.
Two, Hall (1970b) and others have osteoblast-to-osteocyte and osteocyte-to-osteoblast modulations, whereas Willis (1962) excludes osteocytes.
Three, Hall (1970b, 1975b) omits the reticular cell of marrow as a reversibly transforming entity, several others include it.

These three discordancies will be taken in turn.

Formative-Destructive Schism
Several recent authors, cited by Hall (1975b, 1978), suggest that osteoclasts come from uninuclear precursors distinct from those giving rise to osteoblasts, and that osteoblasts do not transform into osteoclasts. Were osteoblasts not to form osteociasts and vice versa, most of the schemes listed would be split asunder.
However, modulatory transformations might occur between the cells in each of the two resulting sections of the original scheme, for example, between the formative cells, as suggested by Willmer.

This issue is further complicated by the possibility that some formative cells have a destructive ability and degrade the materials that they synthesize. For example, some fibroblasts of the periodontal ligament appear to be so active in degradation (Ten Cate, 1972; Frank et al., 1977) that they have attracted the name fibroclast (Ten Cate, Freeman, and Dickinson, 1977); and the osteocyte has a weak synthetic potential (Baylink and Wergedal, 1971; Melcher and Accursi, 1972) but has also been cast in the role of bone-resorber.
For the fibroblasts of the periodontium, it seems that the same cell is performing both synthesis and destruction concurrently, thereby placing the cells astride any formative-destructive barrier and avoiding a switch in specialization. But, in other fibroblasts and perhaps the osteocyte, if the processes were to run successively, this would entail the now unlikely crossing of the barrier.

Osteoblast-to-Osteocyte Modulation
The osteoblast-to-osteocyte modulation illustrates a fundamental problem concerning the transformation of tissues and cells, whether called a differentiation, modulation or metaplasia. The same intermediate tissues or states of a cell that are evidence for the transformation introduce a continuity between the starting tissue or condition and the final one, which has the effect of obliterating their existence as separate entities.
Thus, the chondroid bone which suggests a metaplasia of cartilage to bone also supports the argument that cartilage and bone are essentially one. The difficulties are psychological, philosophical, and practical. The visual system's ability to detect contrasts and contours rather than subtle gradations has profound effects on our thinking.

When one thing turns into another in a smoothly flowing manner, one tries to identify highlights or stages in the smooth progression. But a "smooth progression" contradicts itself. One can try to arrest and specify some "frames," and from these imagine how they would blend, if the frames were run together in correct sequence. But in a truly fluid progression, the idea of a frame or stage is human fancy. Our terms - progression, procession, sequence - falsely imply that we discern one thing coming after another. Although we can see the participant in the process, if the process is smoothly continuous, we do not see discontinuities in it; or if we insist on the discontinuities, we arrest the motion - the paradox of the successive positions of the arrow in flight recognized by Zeno of Elea (490-430 B.C.).

What this philosophical musing has to do with Modulation is to cast doubt on our ability to distinguish an osteoblast from an osteocyte and hence to discern whether an osteocyte is able to revert to an osteoblast. The extreme states are recognizable - the small, mature, buried osteocyte, and the mesenchymal cell at the site of future ossification - but to separate "stages" in between may in some respects be impossible.

The key criterion for differentiating osteocytes from osteoblasts has been that the latter lie on the bone, the former are enclosed. The compartments thus demarcated have no significance for cellular differentiation. If the osteocyte is young, both it and osteoblasts are active in forming bone, and the difference in locations is of no consequence to the cells' differentiation: an active osteoblast is a very young osteocyte.
An early detectable event in osteoblastic specialization is the deposition of osteoid, which is by an active osteoblast no different in its determination from an osteocyte, which the osteoblast gradually becomes. It therefore makes no sense to talk of osteoblast-to-osteocyte modulations since they amount to the same cell.

The one significant discontinuity occurs when the precursor or progenitor cell acquires the ability to form bone, but until it expresses this potential, its new state cannot be detected. Thus, one modulation (inactive to active (M1) or Weiss's (1973) "covert-to-overt" transaction cannot be separated from the earlier M2 acquisition of a specialization for making bone; which in turn means that reversibility in each of the two transactions is undetectable.

The inability to assess the state of determination of the small, apparently inactive cells on bony surfaces is reflected in the variety of names for them: resting osteoblast, surface osteocyte, osteoprogenitor cell, osteochondroprogenitor cell, reticular cell, etc.

Reticular Cells in Bone Modulations
There are four reasons for doubting the eligibility of reticular cells for the schemes of skeletal modulations:

  1. The nature of the reticular cell is itself a puzzle. The light microscopy of marrow revealed fat cells, hemopoietic cells, endothelial cells, and "reticular cells," together with supporting reticular fibers manufactured presumably by some cells present. Phagocytosis took place, but the cell responsible could not be identified with certainty.
    Bloom and Fawcett (1975) listed fat cells, endothelial cells, "primitive reticular cells which retain some of the pluripotentiality of embryonic mesenchyme. These cells are not actively phagocytic but can give rise to fixed and free macrophages." Ham (1977) described a richer marrow with fat cells, endothelial cells, macrophages, reticular cells, fibroblasts, and osteogenic cells (these last based on marrow's behavior rather than the cells' morphology). Ham accordingly has the marrow equivalent of his "relatively uncommitted mesenchymal cells" in other connective tissues already turned into osteogenic cells and fibroblasts, while Blood and Fawcett (1975) have multipotent reticular cells not yet differentiated into other kinds.
    When TEM is applied to the non-myelopoietic elements, Weiss and Greep (1977) have identified only endothelial cells and adventitial fibroblastic cells, but the adventitial cells can become fat cells. Westen and Bainton (1979) histochemically distinguish fibroblastic from phagocytic reticular cells. Therefore at present the reticular cell means too many things to histologists to help explain skeletal behavior.
  2. There never was a good reason for including marrow reticular cells as participants in osseous events. The inclusion of the reticular cell of marrow in skeletal modulations can be traced to Bloom, Bloom, and McLean (1941), who equated them with the "resting" osteoblasts actually on bony surfaces, because the two looked alike and had processes in contact.
    The authors went on to write that, from the existence of "transitional forms," reticular cells became active osteoblasts, although it could as well be argued that what they saw were "resting" osteoblasts on the bone becoming active (merely an M1 modulation), and the similarity between the osteoblasts and reticular cells was the fortuitous result of both kinds' being small and relatively inactive.
  3. Hall (1975b) drew attention to a limitation of several of the schemes of modulating cells, namely, that they deal only with endosteal events. The range of a scheme incorporating marrow reticular cells is to begin with confined to the interior of bones; but reshaping may eventually bring some endosteal bone to lie at the periosteal surface.
    Moreover, as bone trabeculae are reshaped, bone and its cells can come to occupy positions earlier held by marrow; and the extent of the migrations of cells between the marrow and vessels, and to and along the surfaces of bone, is unknown. For example, Gong's (1978) proposal, that the hemocytopoietic stem cell proliferates at the endosteal surfaces of bone in a form resembling a small lymphocyte, would require some migration.
  4. The idea that marrow participates on the formative side of schemes of skeletal modulation draws support primarily from experimental transplantations of marrow, reviewed by Burwell (1964) and Owen, (1970). The topic has a long and controversial history (see below), but it can be argued that the experiments demonstrate only that marrow cells, like those of other connective tissues, can become osteogenic, not that they join in the normal commerce of the bone, aside from the monocytic contribution to osteoclasia.
Osteogenic Cells of Marrow: Experiments
For the student of bone, marrow is a nest of hornets and no gain. No agreement can be reached on the nature of its stromal cells; its cells wander; it introduces blood-borne cells into the interior of bones; and bone and it have an inductive relationship. In one regard it is obliging - approximately half of the transplants of marrow to such extramedullary places as under the skin or in the eye, reviewed and tabulated by Burwell (1964), have formed bone. While this bone might be induced by marrow from local cells, experiments using heavy irradiation of the site for transplantation or radioactive labeling of grafted or host cells lead one to see the marrow as containing cells already primed for osteogenesis (and occasionally chondrogenesis, e.g., Bruns, 1881).

Bruns grafted marrow and noted that the place of the fat cells and hemopoietic elements in the graft was taken by proliferating spindle-shaped cells. Later, bone developed, which he attributed mostly to osteoblasts that were present, he claimed, in normal marrow, had survived transplantation and begun osteogenesis. He specifically denied a connection between the osteoblasts and the spindle-cells.

Could the bone arising in his grafted marrow have come from endosteal osteoblasts? Bruns took special note of this possibility and believed that he had excluded it on two counts. First, he met bone trabeculae in some of his transplants, but there was no new bone in their vicinity. Second, when he deliberately included more trabeculae by taking marrow and spongiosa from nearer the epiphyses, he found no osteogenesis, but rather a massive resorption of the trabeculae by giant cells (presumably osteoclasts).
Pfeiffer (1948) and others also took particular pains to transplant only marrow, but nevertheless bone appeared, leading to the conclusion that marrow itself contains osteogenic cells.

Bruns (1881) and several successors (see Burwell's table, 1964) called the osteogenic cells osteoblasts, from their ability to make bone - which also means after the fact of their having done so. Can these cells be identified in normal marrow? And if not, should they be named osteoblasts before they have demonstrated an osteogenic ability?
The answer to the first question is no; and, following from this, the answer to the second question is a matter of personal choice.
For what one could call a covert marrow osteoblast, Friedenstein (1973) preferred "determined osteogenic precursor cell (DOPC)" of marrow, and Urist, in his comment on Friedenstein's article, viewed as functionally an "osteoprogenitor cell .

If the medullary cell is determined, i.e., is an osteoblast, why does it not form bone? This question must also apply to many resting cells on the periosteal and endosteal surfaces of bone. I am not setting out to trace the history of the idea of inhibitory controls on osteogenesis, but note that Pfeiffer (1948) suggested that it was not estrogen, as he first suspected, but other factors that limited the in situ production of endosteal bone, and the transformation of reticulum cells to osteoblasts (he accepted Bloom's and others' (1941) hypothesis).

Burwell (1964) was more explicit on the possible role of inhibition in marrow cells' behavior: "Though it may subsequently be shown that primitive marrow cells have an inherent tendency to become osteoblasts, which is normally kept in check by local inhibitory factors. . ." Pfeiffer and Burwell thus moved the postulated inhibitory block on the osteogenesis by normal marrow back from being a restraint on the activity of a differentiated osteoblast to being an obstacle to the differentiation of a precursor into an osteoblast.

If the osteoblasts or their precursors are present in marrow, and bone is to keep its lightness and space to house marrow, one can see a need to restrain their activity, but is there substance to the notion of an inhibition?
Boyne, in remarks to Friedenstein's (1973) contribution, noted "in some autogenic bone-marrow grafts that, after a short spurt of osteogenic regeneration, there is a reversion of the graft material to a hemopoietic function." Spurts are generally typical of the period immediately after a release from inhibitory control, but his comment hardly establishes the existence of an inhibitory breakdown.

Another difficulty intrudes in the form of extramedullary hematopoiesis. If the enclosing bone is the source of a factor preventing marrow cells from depositing bone, when marrow is without bone (as in fetal life, certain animals' lymphoid organs, and some human diseases (Ward and Block, 1971)), why is this extramedullary marrow not osteogenic in situ? A way to keep the notion of an inhibitory action of bone on medullary osteogenesis would be to postulate an interlocked dual mechanism, whereby bone inhibits osteogenesis by certain nearby soft tissue cells, because it has induced those cells to be osteoblastic.
By this reasoning, naturally occurring ectopic marrow is not subject to the inductive action of nearby bone and therefore has no "osteoblasts" needing an inhibitory control. Following from this, one would predict that transplanted extramedullary marrow should not form bone.

In addition, such an experiment might provide additional evidence of the contribution of endosteal cells. These could not be the source, should grafted extramedullary marrow make bone. A cursory survey of the literature has not disclosed such a transplantation. Chalmers, Gray, and Rush (1975) were troubled by the absence of osteogenesis in extramedullary marrow, but they proposed that its cells are already osteogenic, though restrained by some inhibitory control.

Another kind of experiment which at first seems to rule out an inhibitory action by bone on its marrow osteoblasts is Friedenstein's (1976) culturing of stromal fibroblast-like cells from marrow but failing to obtain bone. However, since these cells form bone when transplanted back into a living animal, their failure to do so in culture may be attributed to an inadequacy of the medium or some other condition of the culture.

Bone matrix has an inductive action on certain connective tissues (Rohlich, 1941a,b; Urist, 1965) so that if marrow holds the same kind of cell, this cell may well be induced to be an osteoblast and then require inhibition. In Rohlich's (1941a) experiments, where he removed the marrow from the humerus of rabbits, the granulation tissue growing into the empty cavity formed trabecular bone.
From the association between an increased erosion of the Haversian spaces and medullary osteogenesis, Rohlich suggested that the erosion liberated an osteogenic agent to act upon the fibroblast-like cells of the granulation tissue. Later, the new bone was resorbed and the cavity taken over by hemopoietic marrow.

The experiments considered so far intimate that although medullary marrow lacks morphologically identifiable osteoblasts, there are osteogenic cells present of which these questions can be posed:
Does marrow hold cells that, while phenotypically fibroblastic, already by their determination are osteoblasts?
Does it contain cells not yet so determined, but able to differentiate into osteoblasts?
Or does it have both differentiated osteoblasts and some other kind subject to conversion into osteoblasts?

There is some foundation for this last alternative in the work of Burwell (1964, 1966), although this was not his personal interpretation. He noted that only about half of autogeneic grafts of marrow produced bone. When he placed the autogeneic marrow for grafting within heterogeneic bone, the incidence of osteogenesis greatly improved.
He advanced several arguments for his conclusion that the organic component of bone induces some cells of the marrow to become osteoblasts. However, he did not concede that cells of the marrow might have already come under this inductive influence from their own enclosing bone, causing their differentiation into osteoblasts, whose activities remain suppressed while they stay inside the bone.

That autogeneic marrow transplanted with heterogeneic bone forms bone more consistently than marrow grafted alone could be ascribed to the marrow's new relation with bone. The bone is under immunological and other attack, perhaps resulting in an inductive recruitment of osteoblasts from marrow cells that somehow had not earlier been transformed to osteoblasts by the enclosing autogeneic bone. On this hypothesis the stroma of marrow includes osteoblasts (Ham's (1977) osteogenic cells) together with some other kind susceptible to osteogenic induction.

The line actually taken by Burwell (1964) to explain the formation of bone by grafts of marrow was more roundabout than the sequence outlined above, although he did consider that some marrow cells might be inhibited osteoblasts.

His postulated steps were the following:

  1. Normal cortical bone releases catabolic products.
  2. These pass into the marrow's vessels and are picked up by phagocytic littoral cells lining the sinusoids.
  3. When marrow is transplanted, some of it experiences a necrosis resulting in the liberation of the bony breakdown materials from the littoral cells.
  4. This material has an inductive action.
  5. Other littoral cells have freed themselves from the sinusoidal wall and become primitive wandering cells.
  6. The materials of breakdown induce these cells to become osteoblasts.
This sequence is unappealingly complicated, and runs counter to some of the evidence. Pfeiffer (1948) maintained that it was the healthy grafts that produced bone, suggesting that necrosis of the marrow is not helpful to ossification.

In conclusion, the results of transplanting medullary marrow can be explained, using the known inductive properties of bone, by the presence in bone marrow of inactive, hence covert, osteoblasts, and some other kind of cell amenable to osseous induction.
The identity of the second cell raises exactly those difficulties associated with osteogenic (and chondrogenic) induction in extramedullary connective tissues.
Investigators favoring frugality would identify it in marrow as the cell that has already shown an aptitude for making the stromal collagen, i.e., a marrow fibroblast;
some scientists would stay with the primitive reticular cell as the equivalent in marrow of the undifferentiated mesenchymal cell;
yet others, such as Burwell (1964), follow Trueta (1963, 1968), Keith (1927) and some of the early histologists in the belief that endothelial cells of sinusoids and capillaries can give rise to osteoblasts;
and finally, Friedenstein (1973) has modified the old idea of Macewen (1912) and others of blood-borne osteoblasts to an hypothesis that his inducible osteogenic precursor cells (IOPC) can migrate via the vasculature.
Friedenstein's foundation for this concept is based on the maintained osteogenic response of connective tissue to implanted urinary mucosa after strong X-irradiation of the host site. However, Maniatis, Tavassoli, and Crosby (1971) and Friedenstein himself (1976) noted the ability of stromal cells to withstand high doses of X-rays; their equivalent in other connective tissues could have the same fortitude.
Also, from the well-known resistance to therapeutic irradiation of the cells in osteogenic sarcomas, it cannot be assumed that irradiation eliminates the local precursors of induced osteoblasts.

Returning to the cells to be included in schemes of modulation, neither of the cells of marrow postulated above can be identified without histochemistry or TEM in normal marrow. Thus, including a "reticular cell" in a scheme might introduce a covert osteoblast, a multipotent cell, a fibroblast, or a phagocyte; or, as Westen and Bainton's (1979) work shows, more probably some combination of these, viz. a "fibroblast-type" and a "macrophage-type."
Lastly, the possibility of an inhibition of marrow's osteogenic potency suggests that the precursor and "osteoblastic" cells of marrow may, in normal circumstances, be prevented anyway from participating in appositional events in the enclosing bone, thereby withholding them from schemes of currently active skeletal modulation.

Consequences of Omitting a Cell Type
If a particular cell type, say, the reticular cell, is participating in the skeletal modulations, but one neglects to consider it, or, in practical terms, to count the number of its cells that are labeled, what are the consequences?

Where the plan focuses on the events of a particular skeletal site and claims rigor in accounting for the numbers of cells involved in the transactions, a failure to count cells that are participating would invalidate any kinetic interpretation of the data.
This would also be the result of pooling into one figure the numbers of populations that are actually distinct, as the precursors of osteoblasts and osteoclasts may be (Wong and Cohn, 1975; Owen, 1978). A hypothetical, but not altogether ludicrous, extreme would be for all the cells under consideration to turn or "modulate" into the omitted entity and disappear beyond the predictive power of the scheme.

AVERAGE CELLULAR CONDITION

The original definition of modulation had activity fluctuating about an "average condition." However, Weiss (1939) claimed neither that there was a mean state for the M2 modulation, where the cell goes from one specialized activity to another, nor that such a state was represented by the undifferentiated mesenchymal cell under that or another name. Very little has been made of this point since then, although when the determining stimuli balance or are absent, a mean intermediate or unexpressed state is to be anticipated. In several schemes of skeletal modulation no cell occupies anything like a pivotal position. In some bone-only schemes, the osteoprogenitor seems cast in that role, for example, where Young (1964) had an osteoblast undergoing "despecialization" before transforming into an osteoclast.

STABILITY OF THE MODULATED STATE

When cells are stimulated to experience a metaplasia to another kind, e.g., osteoblasts, does the stimulus have to continue, if the osteoblasts are to be held in their specialized state? In modulatory terms there is no query, only a prediction. By definition, a given state of modulation is the response to particular stimuli, and the cell's state should change when the stimuli change. For an M2 modulation from a connective tissue cell to an osteoblast, the change expected would be back to a fibroblast or whatever gave rise to the osteoblast. If the cells do not transform back again when the stimulus is withdrawn or reversed, this would mean that modulation is not involved.

Owen's (1970) contention that ectopic osteoblasts cannot outlast the inducing stimuli can be challenged. First, in human and animal pathology, ectopic ossicles and pieces of cartilage can be longstanding, composed of mature tissues, e.g., with osteons, but unrelated to any other clearly abnormal tissue. No sign of an original osteogenic stimulus remains. Second, some experiments bear directly on the question. Zalewski (1961) induced bone by transitional epithelium that itself succumbed to the immune response, but the bone continued to live. The cartilage and ensuing endochondral bone provoked by amniotic WISH cells could outlive the xenogeneic cells by several weeks (Wlodarski, Hinek, and Ostrowski, 1970).
Their results do not support the statement in the summary concerning "bone tissue, which was gradually resorbed within two months." Although osteoclasts were active in the induced bone, their Table I has two examples each of bone and cartilage present 60-72 days after grafting.
In a different situation - the clitoris of rats - the ectopic bone brought about by the neonatal injection of testosterone lasts for many months (Glucksmann and Cherry, 1972; Beresford, 1973).

However, there is an explanation for the induced bone's persistence that does not diminish the modulation hypothesis. Modulation was an idea founded on the cell, unlike metaplasia, which originated with the tissue and was then adapted, not altogether successfully, to cellular terms. Hence, modulation said nothing about the tissues and their non-cellular components.

For bone induction, the osteoblasts originally induced soon become osteocytes, around which the matrix may insulate the cells from the stimuli and itself have a bone-inducing property, so that the matrix and its cells in effect become self-perpetuating. Hall (1970a, 1978) discusses experiments that show the matrix to be a stimulant for the synthesis of more matrix materials by skeletal cells.

DIAGRAMS OF MODULATION: IMPLICIT SIMPLIFICATIONS

There is a tradition of schematizing cytodifferentiation by diagrams resembling those of an evolutionary tree, having a few less specialized cell types to which the names of a greater number of differentiated cells are joined by arrows representing the process of specialization and its direction. Such diagrams have been modified by the device of double-headed or paired arrows to include modulations or reversible transformations of cells. An example is von Mollendorff's (1927) Figure 31. It has 11 types of cells, 20 arrows (9 double-headed), and 5 question marks.

The diagrams can lead one's thinking into treacherous paths.

  1. As the osteoblast-to-osteocyte transition exemplified, one can give separate names to apparent stages, when there is not always a theoretical justification for the existence of distinct entities. When there is (for example, between a dormant osteoblast and a uninuclear osteoclast), they may not be distinguished easily by the means now practicable (Vilniann, 1979);
    or, one name, e.g. reticular cell, may encompass separate kinds of cell (Westen and Bainton, 1979).
  2. The diagrams have an overall symmetry which tempts one to think of the cells as having a uniformity of response, and each and every transformation as occurring with equal facility. As just mentioned, each cell type may be more heterogeneous than the one name conveys (particularly when biochemical variability in the phenotype is brought in, as in fibroblasts (Narayanan, Page, and Kuzan, 1978)). For example, there is an implication that any chondroblast, if stimulated appropriately, would modulate into an osteoblast, but in practice chondroblasts of elastic cartilage very rarely display any inclination to form bone, and endosteal osteoblasts seldom produce cartilage. Of course, there may well be no proper stimulus, but these particular cells may in themselves be reluctant to change.
  3. The lines of a diagram imply very restricted pathways that differentiating cells follow like a train on a railroad. A similar metaphor which has been applied to the differentiation of skeletal tissues is of travel along a valley which forks into two, one arm representing cartilage, the other bone (Murray and Smiles, 1965).
    Both the valley and the railroad images of cellular differentiation fail to accommodate those cells which appear to be on two tracks at the same time, for example, chondroid bone cells, myofibroblasts, the mixtures of cartilage and muscle found in invertebrates (Person and Philpott, 1967), and the intermediate glandular and contractile juxta-glomerular cells.
    A better metaphor for cellular behavior may be a sail-boat with some steering on board, but sometimes steered by the wind and tide to various in-between destinations.
  4. Where double arrows are used to indicate a reversible transformation there is nothing to indicate whether the modulation is of the active-to-inactive variety (M1), e.g. of an osteoblast, or one involving a new specialization (M2), say, to chondrogenesis.
    If destructive processes are, for the moment, excluded and only formative ones considered, are the modulations of skeletal and connective tissues in practice only of one kind, viz. M1? Following others such as Bassett (1964), Hall (1970b; 1975b) suggested that bone and cartilage are chemically very similar, so that osteoblasts, chondroblasts, and fibroblasts are the same kind of cell - a scleroblast (after Moss), which produces a range of products including collagens and proteoglycans. Thus, if a newly active osteoblast becomes a chondroblast, its spectrum of chemical activities undergoes only quantitative changes - more proteoglycans formed, less collagen - so that the levels of activity alter, but there is no change of specialization, and an M2 modulation does not occur.
    This attractively simple hypothesis has sustained setbacks. For example, Okayama, Pacifici, and Holtzer (1976) reported that chick chondroblasts synthesize unique sulfated proteoglycans, not formed by fibroblasts or other skeletal cells, or presumptive chondroblasts.
  5. The cells participating in resorption give additional significance for the arrows. If a macrophage or uninuclear osteoclast (Boyde, 1972) is already destroying or able to destroy bone, its fusion with others to form a multinucleated osteoclast may be a tactical change, minor when compared with the earlier gaining of a lytic potential.
    Such a fusion of cells could be viewed as yet another kind of modulation, except that no one knows (Hall, 1975b) that the process can run exactly in reverse to free whole uninuclear cells rather than just nuclei. How reversible does a transformation have to be for it to be a modulation? Since, by definition, it must be exact, another difficulty arises.
  6. Weiss specified that the individual cell experienced the modulation, not merely that there could be reciprocal transformations between two classes of cell. This requirement for the individual cell does not emerge from diagrams of modulation. It applies to all skeletal cells between which a modulation is claimed and its demonstration would be especially difficult for the multinucleated osteoclast, should it have a dual origin (Hall, 1975b).
    Showing on separate occasions that a) osteoclasts become monocytes, and b) monocytes turn into osteoclasts, does not establish that it is those osteoclasts derived from monocytes that undergo the reverse transformation. Other factors making it hard to trace the fate and reversibility of individual cells are the propensities of various skeletal cells to divide, migrate, fuse, and take on nondescript forms.
    If neither mitosis nor migration occurs, then the new cells must be the existing cells transformed. One example studied does not favor the hypothesis of M2 skeletal modulations. In the Japanese quail, S. Miller's (1977) recent counts reveal that, during the egg-laying cycle, endosteal osteoclasts do not vary in number nor do the nuclei per osteoclast. Instead of depending on an M2 modulation of cells to and from osteoclasts, a variation in bone resorption appears to be achieved by the osteoclasts' altering the activity of their bone-facing membranes, i.e, expressing only an M1 modulation.
    While his findings were limited to quail, Miller in his introduction seems also to cast doubt on the story of osteoclast-to-other-cell transformations hypothesized by Bloom et al. (1941, 1958) for the hen.

    Little mention so far has been made of evidence favoring the reversibility of cellular specialization: the M2 modulation. Chondrocytes in vitro and skeletal muscle in vivo provide legitimate examples of the phenomenon, which serve also to illustrate some of the experimental difficulty in demonstrating the cells' initial state, the new one, and the critical return to the first.

MODULATION 2 BY CHONDROCYTES AND SKELETAL MUSCLE

Toole and Linsenmayer (1977) describe the chick's limb precartilaginous mesoblastic cells as forming type I collagen. As the matrix around them becomes metachromatic, they shift to synthesizing type II, and after a while make exclusively this variety of collagen. However, when the cartilage cells become hypertrophic at the ossification front, they seem to revert to the production of type I collagen (von der Mark and von der Mark, 1977b).

There are other situations where chondrocytes, previously producing their typical type II collagen, start making I, as, for example, in many cultures of chondrocytes, in osteoarthrotic cartilage (Gay et al. 1976), and maybe in salamanders' regenerating limbs (Toole and Linsenmayer, 1977). If the assumption is made that the chondrocytes arose from cells making first type I collagen, as in the chick's skeletal primordia and the mouse's (Elmer and Selleck, 1975), then each of the above instances is a completed modulation 2 or a reversible metaplasia. (Most cellular transformations called modulations are at best only half-modulations, because the cells do not subsequently traverse the route in reverse.)

Reversible shifts in chondrocytic expression emerge from Pacifici et al.'s (1977) experiments on the production of type IV proteoglycan. They transformed chick chondroblasts with Roux sarcoma virus. At 36 C the cells changed to synthesizing type I collagen and stopped production of type IV proteoglycan, but when warmed to 41 C they reverted to making type IV proteoglycan. A lowering of the temperature brought a reversion to the metabolic behaviors of transformed chondroblasts.
A comparable kind of switching of the collagen phenotype has been obtained by introducing or withholding calcium from the medium in which rabbit's articular chondrocytes are cultured (Deshmukh, Kline, and Sawyer, 1977).

Skeletal muscle is divisible into subclasses according to its physiological properties - fast- and slow-twitch - which correspond quite well with typings based upon fibrillar architecture and histochemistry (Ranvier, 1874; Gauthier 1969, 1979; Needham, 1971). The types represent specializations which are not immutable. After a skeletal muscle fiber has differentiated, it responds to changes in its innervation. Denervation promptly makes it inactive. But later events, after denervation or the experimental transposition of nerves, constitute a modulation 2 - a reversible change of specialization.

Buller, Eccles, and Eccles (1960) contrived to innervate fast muscles by nerves originally serving slow muscles and vice versa in kittens of various ages and in adult cats. The muscle then changed its physiological characteristics in the direction of those of the muscle normally served by that nerve, for example, slow or tonic nerve fibers converted the fast Flexor digitorum longus (FDL) to slow reponses.
They commented, "Since muscles of fullgrown cats were transformed just as effectively as those of kittens, the neural influence must be continuously exerted by nerve cells throughout life. Thus we are introduced to a new concept, that of a continuously operating differentiating principle."

The evidence for reversibility in such transformations or "redifferentiations" is more recent. Schmidt and Stefani (1976, 1977) denervated the frog's pyriformis muscles by crushing the sciatic nerve. The all-or-none responses of which the amphibian slow pyriformis muscles are normally incapable began to appear during the early period of natural regenerative re-innervation by (foreign) fast-conducting motor axons. Beyond 60 days after crushing, the slow muscles gradually recovered their inability to generate action potentials: in the same period, the slowly conducting axons were re-establishing themselves in the muscle, although not to every now-slow muscle fiber. This last finding suggested to the authors that the slow nerves' influence on the muscle was independent of their main synaptic function.

In sum, the pyriformis muscles went from slow type to fast and back to slow, meeting the criteria for a modulation 2. What is missing is histochemical or morphological evidence of the reversibility to corroborate the neurophysiological, but findings in the nature of those of Ellisman et al. (1978) may be forthcoming.

Ellisman et al. switched the nerve of the FDL to the rat's soleus. Nine to 12 months later the soleus muscle fibers had not only acquired histochemically discernible ATPase reactions and a fiber-type grouping typical of fast muscle, but the end-plate sarcolemma now had the "square array" of particles characteristic of fast-twitch muscle. It will be interesting to learn whether a restitution of slow innervation can reverse these morphological characters, and what these techniques of examination will reveal in frogs recovering from denervation.

THE CONNECTIVE TISSUES: ONE TISSUE?

As belief waned in the metaplastic hypothesis in general, and of metaplastic osteogenesis in particular, intermediate tissues such as chondroid bone remained to confound the separate identities of the connective tissues. However, at the same time the cellular basis for histogenesis was becoming better known, and it held an answer to the several intermediates, such as chondroid bone, osteodentine and fibrocartilage. These were to be the products of the same population of germinal cells that make bone, cartilage, dentine, etc. Hence, the connective tissues, including the intermediate expressions, were linked by a common cell of origin (Schaffer, 1888; Muller, 1863; Klaatsch, 1894).

The idea of a unity among the connective or supporting tissues already had a foundation in the supposed identity of fibroblasts and osteocytes (Virchow, 1851), and the ability of morphological components to combine to form mixed tissues, e.g., chondrocytes and collagen fibers to make fibrocartilage (Meyer, 1849a).
The common nature of the connective tissues subsequently acquired a chemical dimension. Schaffer (1888) and Hansen (1899) suggested that the tissues represented the same chemicals in different morphological expressions, and that a basic physicochemical process underlay the various modes of formation of the matrices. But it was not until after Weiss's (1939) positing cellular redifferentiations as temporary modulations, more evidence from various secondary cartilages of the lability of the precursor of chondroblasts and osteoblasts (Murray, 1957), and the following by radioautography of connective tissue cells from one state to another (Young, 1962), that the biochemical evidence for unity among the connective tissues fell into place.

For a while, the weight of the findings obtained in vivo and in vitro favored considering the connective tissues as really only one tissue, whose cells do more or less the same activities, resulting in various manifestations of the tissue. Thus hyaline cartilage and bone, for example, are recognizable and common expressions of the tissue, but ones whose important differences are only quantitative.

From such a standpoint, the expressions seen and given recognition by the classical tissue names are highpoints - solely from the aspect of abundance - on a continuum, for which intermediate forms such as chondroid bone and fibrocartitage are evidence.

Even at the time, the idea of the connective tissues comprising one collective entity was not without its difficulties. There was no name for the collective, and any attempt to identify it by all the cell types believed to be interconvertible would have been very cumbersome, and ran head-on into another obstacle. Weiss's (1939) formulation of modulation allowed cells greater freedom to express themselves than a differentiation commonly conceded. But how the cell's abilities related to the various connective tissues was never satisfactorily worked out. The lack of agreement on which cells participate was dealt with in this chapter, p.

Another aspect of the confusion between tissue and cellular events (see Chapter 6, The Tissue Legacy) centered on those tissue transformations effected by germinal cells. Murray and Smiles (1965) identified the problem. These authors noted the behavior of certain germinal cells on the cranial bones of chicks paralysed for a while in vivo. From the germinal population came first cartilage, then bone when paralysis supervened, and again cartilage upon recovery of mobility.
They wrote: "When the germinal cells at the sites here studied formed bone instead of cartilage or cartilage instead of bone the effect was not reversible, the newly formed cartilage or bone persisted. But this was not true of the germinal cells which formed the bone or cartilage; the kind of tissue which was produced changed in either direction, when the stimulus changed, and so the change in the germinal cells was a 'modulation' and not a true 'differentiation'."
After discussing the similar influence of vitamin A on the germinal cells of epithelia, Murray and Smiles assessed the two situations thus: "At first sight it does not seem that changes of this sort fit very comfortably into the category of 'modulations,' but the difficulty depends on whether one's attention is fixed on the germinal cells or on the cells which have completed their differentiation."

They went on to view the germinal cells as standing near the fork of the valley. Once they have set off along a fork, they are differentiated.
To change, for example, from chondrogenesis to osteogenesis then would involve climbing over the ridge between the valleys, i.e., a metaplasia, in whose existence for skeletal tissues Murray and Smiles hesitated to believe, notwithstanding the chondroid bone seen earlier by Murray (1957, 1963).

There is no question that Murray and Smiles were sensitive to the difficulties of matching the theoretical constructs, modulation, differentiation, and metaplasia, with the behavior of the cells in their experiments, but is there really evidence for any modulation? Can the tissue composed of germinal cells have a property that is not exhibited unequivocally by any of its component cells?
The germinal cells like other populations of stem cells do two things: some differentiate, in this case into chondroblasts or osteoblasts, and as Murray and Smiles noted, these cells give no sign of reversibility or changing their course; the others stay undifferentiated and later able to be steered towards either bone- or cartilage-formation. But, is the bipotentiality of these latter undifferentiated cells evidence of modulation?
The presumption seems to be that in their labile state they can be forced by a stimulus into one direction, but until they reach a "point of no return," their commitment can be turned around. This phase, of being committed but unexpressed and reversible, interpolated between the undifferentiated and differentiated states, is hypothetical. Because neither the differentiating nor the undifferentiated progeny of the germinal cells can be proved to experience modulation, i.e. reversal, the germinal tissue composed of those cells cannot usefully be regarded as exhibiting modulation.

Last, the lumping together of all connective tissues on the basis of their sharing common macromolecules was a reductionism unjustified even on biochemical grounds; for instance, only some connective tissues mineralize; and not all connective tissue cells make elastin. Beyond that, the chemistry left unheeded the patterning subsequently imposed on the macromolecules; Knese (1963), for example, identified six orders of architectural complexity in bone.

The era of the connective tissue collective turned out to be short-lived, as several kinds of finding came to show 1) a heterogeneity among the connective tissue macromolecules; 2) the production of these macromolecules by other tissues; 3) quantitative chemical differences as distinguishing tissues rather than providing a basis for amalgamating them as one; and 4) the possession by connective tissue cells, e.g., fibroblasts, of materials typical of muscle, which would require the chemically based connective tissue collective, already unwieldly, to enlarge to encompass muscle.

When the various expressions of the connective tissue change chemically, say, when hyaline cartilage calcifies or becomes fibrous, or fibroblasts become myoid, the transformation - quantitative or qualitative - has to be accounted for, whether or not one names each expression as a separate tissue. There is a difference between before and after, and such differences are what demarcate tissues and comprise the stuff of differentiation. Viewing all the connective tissues as one and calling the transformations "modulations" in a way deny the tissues and events their significance, and overemphasize what is true for any tissue, that it has much in common with other tissues and its stability is not absolute.

The positive aspects of the notion of the connective tissue collective were two. First, it offered recognition and places in the one spectrum to neglected entities such as the kinds of chondroid bone, which physically join and merge with the commonly acknowledged tissues.
Second, by stressing that the various expressions of the tissue form a continuum, a question was brought into focus for those molecules that are common to part or all of the continuum: do the cells making those expressions release their genes from repression in a continuously variable way? Or do the cells have several separate programs, but the continuity with which one tissue expression merges with another mirrors a regular gradient of post-translational and extra-cellular influences on the products of the programs?

MODULATION AND METAPLASIA

The concept of modulation has invigorated study of the transformations of mature cells, in particular by its emphasis on cellular behavior as a response to extrinsic stimuli, which reflects how limited is the stability of the differentiated state in normal cells. Virchow (1863) had recognized these properties, for example, in normal marrow, where his metaplasias, e.g., from cartilage cells to marrow, and fatty marrow to red, were in reaction to changed physiological circumstances. But metaplasia fell into disfavor, and the idea of the immutability of cellular differentiation took hold. Modulation's contribution was to facilitate a second recognition of how immaturely labile many mature cells are, in situations not strictly pathological. But, as metaplasia was burdened by the failure to reconcile cellular with tissue events, modulation has had its own handicaps.

On the basis of now unconvincing reports of histiocyte-to-fibroblast transformations in tissue culture (see Chapter 7, Maximow and the Fibroblast), Weiss extended modulation from denoting short-term physiological responses of glandular and pigment cells to include far-reaching changes of specialization. He contended that bringing in the claimed formative-to-destructive transformation (and vice versa) did not involve events of a substantially different nature, in the belief that reversibility of the transformation was evidence that the cell kept its determination throughout.
It is equally tenable to hold that after any one of the M2 transformations the acquisition of the new identity is evidence that the old one is lost, i.e., as thoroughly suppressed as all the other specializations that a differentiated cell does not express.

By its emphasis on reversibility - its one innovation - modulation places a theoretical limit on its appropriateness for particular circumstances, without there always being the practical means to test for reversibility. Calling the metamorphoses of connective tissue cells, e.g., of fibroblast to osteoblast or chondroblast, modulations clarifies no more than metaplasia, but automatically attributes reversibility to the event.
Even for tissues without a matrix, such as skeletal muscle, proving a reversal is not easy (see Modulation 2 by chondrocytes and skeletal muscle, this chapter). For the connective tissues, the matrix may make it impossible to get the cells to turn back, although the behavior of chondrocytes in culture (see Modulation 2 by Chondrocytes and Skeletal Muscle, this chapter) may give some kind of answer. Metaplasia is not restricted in this way, and was retained by Weiss himself and others to describe certain transformations to bone.

Questions that were posed for the older notion of a one-way change of specialization, metaplasia, although pertinent and unanswered, have not been rephrased for modulations. Thus, if a fibroblast can modulate to an osteoprogenitor cell, does the latter have to divide before it can give rise to an osteoblast? It will be remembered that Bloom et al. (1958) regarded a lack of mitoses to indicate the occurrence of a modulation.

Even though the dividing line is not hard and fast, two classes of event are recognizable: brief ones in the day-to-day physiological cycle of a cell (M1 modulations); and longer-lasting ones involving a change in the nature of the basic activity in which the briefer fluctuations take place (a metaplasia). Removing cells from their usual context, e.g., manipulating chondrocytes in culture, may change the time scale but the distinction remains discernible.

Because modulation from the start had two meanings, M1 and M2, and has since spawned transitive and intransitive verbs, the term now brings more muddle than enlightenment, Loutit's (1976) criticism. Following current physiological practice, modulation can be restricted to the short-term changes in activity, leaving metaplasia for longer-lasting changes in a cell's character, recognizing that these are also under extracellular control by factors both normal and pathological.


Chapter 9 LONG-BONE PERIOSTEAL AND INSERTION-STRUCTURE CHONDRIFICATIONS

Introduction
Endochondral bone's subperiosteal chondrogenesis: Zawisch-Ossenitz
Other instances of subperiosteal chondroid bone
Reports with long-bone chondroid bone absent
Circumscript periosteal attachments
Tibio-fibular fusion in rodents
Schaffer's chondroid tissues
Fibrocartilage-vesicular tissue distinction
Cartilage in tendons and ligaments
Cartilaginous metaplasia in tendons
Other peri-skeletal metaplasias

INTRODUCTION

The best known secondary cartilages are on membrane bones. Why then take long bones as the first site to examine in detail? Three factors determined the choice. The first secondary cartilages to attract notice were on long bones. Next, one of the later writers on the more peculiar aspects of long-bone subperiosteal osteogenesis was Zawisch-Ossenitz (1927), whose descriptions of chondroid bone have had considerable influence. Third, I wanted to tackle chondroid bone II early, and that required prior discussion of the tissues at the skeleton's attachments.

Every so often anatomists remind their colleagues that in addition to areas clad in articular cartilage and periosteum, a significant part of a bone's surface takes the attachment of tendons and ligaments. According to Schneider (1956), Laurentius Heister (1771) regarded tendon attachments as being free of periosteum. Following Ko1liker (1852) and Matschinsky (1889), more recent writers, including Weidenreich (1923b), Biermann (1957), Haines and Mohuiddin (1968), and Knese (1978b) emphasize that periosteum is lacking at the insertion structures.
Some of these authors make the qualifying point that, while the insertion is to a degree specialized and different from periosteum, on cellular and functional levels they have much in common. Thus, the tissue formed by both periosteum and insertion can sometimes have a cartilaginous nature; both can add mineralized tissue to the surface of the bone, thus subserving growth of the skeleton; and of course, periosteum is also a means of attaching soft structures to bones.

The tasks are to review reports of chondrogenesis or chondroid bone formation by both periosteum and tendinous and ligamentous insertions, and then to add these obscure secondary cartilages and the extraskeletal cartilages to the better known secondary cartilages, the primary cartilaginous skeleton and the permanent cartilages, to arrive at a knowledge of all the cartilages in which longstanding mineralization, i.e., CB II, (see Chapter 10) might be found.

ENDOCHONDRAL BONE'S SUBPERIOSTEAL CHONDROGENESIS: ZAWISCH-OSSENITZ

Apart from periosteum-free attachment regions towards the epiphyses of long bones, can the periosteum proper make anything like cartilage during normal growth? In several circumstances, the answer is yes. As Muller (1858) noted, the first bone laid down on the cartilage of an endochondral precursor is partly deposited on living cartilage. Along this interface of initial periosteal bone and living cartilage is a narrow strip of tissue where the matrices of bone and cartilage blend together, providing an atypical environment for the outermost chondrocytes and the innermost osteocytes.
This anomalous layer is very limited in its thickness. The periosteal apposition continues and, in certain regions in particular species, what is deposited may be a quite extensive layer (up to several cells wide) of bone with a cartilaginous cast.

Virchow (1871) described the cells at the surface of growing bones as being cartilage-like, which was the idea expressed by Schaffer (1888) in naming early mandibular periosteal bone chondroid bone. Virchow also mentioned that in very actively growing regions on long bones cartilage could develop, but, as a rule, the matrix turned into osteoid and then calcified before the tissue was truly cartilaginous. In describing some bone as more cartilage-like than other new bone, Virchow alluded to human subperiosteal chondroid bone. For no clear reason, this observation apparently did not receive systematic attention until Zawisch-Ossenitz's three papers (1927, 1929a, 1929b).

Although sometimes used as a source of information on chondroid bone, her papers are not easy to follow, because:
1) she tried to give full justice to the variety of cells and other basophil elements that she saw in developing femurs;
2) as a colleague of Schaffer she chose terms that would not imply that cartilage could experience metaplasia; and
3) her interpretation used certain ideas of his, eccentric for the time, such as a destructive role for osteocytes, and that if cells were not retractile, they were not chondrocytes.

Zawisch-Ossenitz (1927) found the developing femora of small domestic animals, bats, and rats have cells, areas of matrix, and lines, staining basophilically with hematoxylin, thionin and safranin. The various kinds of line, e.g., cement lines, are discussed in her 1929b paper. She used such lines, and the differences in physical dimensions between embryonic and older bones, to establish that some basophil cells and islands of basophil matrix in the diaphyseal bone were not cartilaginous remnants of the epiphyseal growth zones, but were periosteal formations.
The periosteal site most responsible was that around the shaft just short of the epiphysis - a region she termed hypepiphysar, and later renamed telodiaphysar (1929a).

In the first animals she studied, cat and rat, the cortical islands of basophilic matrix were absent in embryos and postnatally varied so much in size, shape, and the adjacent cells that she was at a loss to attribute the basophilia to a particular kind of cell. She noted in an islet one cell that was very large and cartilage-like. When she looked at newborn and two-month-old guinea pigs, she saw in a nodule on the middle of the shaft and more regularly at the ends by the epiphyses the periosteum was making cells showing all variations between cartilage cells and osteocytes. The basophilia of the tissue came from some of the cells, parts of the matrix and from capsules around many of the cells.

She called this tissue chondroid bone, but was she applying the term as Schaffer (1888) had to young large-celled bone, or is the tissue truly cartilaginous in some way? Two features of certain cells were suggestive of chondrocytes: a rounded or ovoid shape and a basophil capsule. (She made a partial distinction between the basophil cells of capsule-builders and ground substance-builders.) But the lacunar wall around osteocytes stains distinctively and contains proteoglycans (Heller-Steinberg, 1951; Matesz, Foldes, and Modis, 1975).
What Zawisch-Ossenitz seems to have seen and perhaps paid over-much attention to is the considerable variety in form and chemistry of osteoblasts (Knese, 1964) and osteocytes. Nevertheless some of the cells were very like cartilage cells; she herself pointed out their marked resemblance to chondrocytes in the epiphysis nearby. Her stated reason for calling the chondrocytic cells Pseudoknorpelzellen was that, unlike true cartilage cells, these filled their lacunae completely, i.e., they had not retracted.

Another reason, probable but not expressed, is related to the fate of the tissue in which they lie. The bone with the basophil elements becomes overlaid by more bone and comes to lie deeper in the shaft and further from the epiphysis. This bone with basophil islands is her Mischknochen. It is resorbed, and replaced first with woven bone, and eventually with lamellar bone. The cells of the Mischknochen are more like osteocytes, and basophil cells are few, but there are basophil islands present.
Zawisch-Ossentiz believed that before its resorption, the chondroid bone laid down initially altered in two ways. Some cells underwent dissolution and contributed degenerate material to the basophil matrix. Other cells, derived as osteoblasts from the periosteum, were incorporated from the start in the tissue, depositing new more bone-like matrix. Thus, she had two grounds on which to avoid the conclusion that a tissue with plentiful cartilage-like cells - chondroid bone - had turned into a more bony one (Mischknochen), i.e., a metaplasia in Virchow's sense had taken place.

First, the more bony nature was to be attributed to the reworking of the matrix by those cells that were osteocytic from the time of their inclusion. Second, the cartilaginous cells that might become some of the osteocytes were false rather than true cartilage cells. She viewed the periosteum as vacillating between forming osteoblasts and chondroblasts, but while allowing the periosteum to form osteoblasts, she termed those of the other tendency only pseudochondroblasts, and wrote of the chondroid bone that it must be viewed on a lower plane of differentiation, since its conversion to bone indicated a persisting embryonic capacity.

Why should the periosteum show this lability? In animals and man, the chondroid bone was seen in only particular places and at restricted times. Her ideas as to the reasons are conjecture, but of some interest. In the guinea pig, needing mobility from birth on, the tissue is more prominent at term and thereafter than in other small animals, because prenatal growth has to be more rapid, requiring chondroid bone.
From the greater amount on the anterior face of the human femur than on the posterior, she postulated that movements in utero were significant in the later months of gestation, and the anterior side was more heavily loaded. She linked the lesser amount of chondroid bone on the humerus than the femur to a difference in stress between the two. Against her speculation, however, on a mechanical cause, three months after birth, when skeletal stresses would be expected to be mounting, the formation of chondroid bone ceased.

Her two papers (1929a, b) on chondroid bone in the human femur added more classifications to the already complicated ones she had earlier derived (1927). For example, in the chondroid bone there were these classes of cell plus transitions between them: capsule-builders, large pseudo-cartilage cells, callus cells, and true cartilage cells. The overall result was a jungle of overlapping categories.
The fibers of bone were: fine, fine-to-middling, middling, middling-to-coarse, and coarse.
Bone was: lamellar, or woven in its four forms of early embryonic diaphyseal, late embryonic diaphyseal, islet-bearing or mixed bone, and filling-in or connecting.
Chondroid bone was fiber-free to fine-fibered, or coarse-fibered.
Bony tissues served preparatory, provisional, and principal roles.
Her point that bone is subject to great variability in its cells, matrix, origin, and purpose was lost in the complexity and went unheeded by many.

For now, one need only note that for her all bone is bone, and some chondroid bone is conventional bone or cartilage. Thus, her coarse-fibered chondroid bone has cells which, though basophil, have processes, and are osteocytes. It is bone found by tendon attachments, but she stressed that it is bone and not what Weidenreich termed ossified connective tissue.
Some of the chondroid bone (beta) around cartilage canals that she mentioned in reference to Hintzsche's (1928) work is probably small-celled eosinophilic cartilage (see Chapter 10).
On the other hand, fiber-free and fine-ribered chondroid bone occurs briefly on the developing human femur at the ends of the diaphysis. Her illustration of it in her Figures 8 and 20 (1929b) shows a seam of large-celled bone looking exactly like the new bone observed under the periosteum at some distance from a newly healing fracture. This similarity is further brought to mind by Zawisch-Ossenitz's likening some of the cells of chondroid bone to Kalluszellen, although whether she meant the cartilaginous cells near a fracture gap or osteoblastic ones further away cannot be known.
In her view of human chondroid bone the cells were mostly smaller than the Pseudoknorpelzellen of animal chondroid bone, and while they had a well-stained capsule, it is likely that they were large osteocytes. Nevertheless, both in human and animal fine-fibered or alpha chondroid bone seen by her, some cells were large, vesicular and like chondrocytes. The presence of such cells barely justifies classifying the tissue as true chondroid bone type 1, while one acknowledges that the bias in the human tissue is more towards bone than cartilage.

OTHER INSTANCES OF SUBPERIOSTEAL CHONDROID BONE

The true chondroid bone seen under the periosteum of normal growth probably has not been reported more often because a) it develops only in certain species for a brief time during gestation or infancy; b) it is confined to relatively small regions of the bone; c) and it is soon resorbed, if it does not give the appearance of experiencing a metaplasia to a more bony state.

Confirmation of the presence of chondroid bone comes from several sources. Before Zawisch-Ossenitz, Bidder (1906), in describing the knee of the eight-day-old rabbit, drew attention to the surface of the Crista anterior tibiae just below the tubercle. Bidder saw a picture, which, he maintained, could easily be misinterpreted as a metaplasia. The cartilage on the surface (his Figure 13) blended gradually into the underlying bone. Bidder himself thought that both the cartilage nucleus and the CB I joining it to the bone were neoformations by the periosteal germinal cells.
He went on to compare this tibial sub-periosteal cartilage with such accessory cartilage nuclei (secondary cartilages) as the mandible's.

Weidenreich (1930) illustrated (his Figure 48) a chondrocyte-like cell in the periosteal bone of the vertebra in a guinea pig. Lipp (1954b), working with Zawisch in Graz, noted that one of the kinds of large cell between osteocyte and chondrocyte, the encapsulated ground substance-builder, seen by her (1929a) in developing bones, was sometimes present under the adult periosteum, e.g., of the rib (his Figure 18a), or within Haversian bone.

A clearly described formation of chondroid bone I is at the ventral region of the neonatal human minor trochanter adjacent to periosteal bone (Knese and Biermann, 1958). Here, "between the cartilage-forming zone and the zone of purely periosteal bone a tissue develops, that can be named chondroid bone (Schaffer, 1888) from its intermediate position between cartilage and bone." That they meant intermediate in nature, as well as in position, is indicated in the legend to their Figure 12 (my Figure 4): "a tissue in which the cells closely resemble chondrocytes, while the intercellular substance displays marked bony characteristics." In this region of the trochanter, "the pluripotency of the periosteum results not only in the reversible possibility of forming various tissues (cartilage or bone), but also in the making of tissues, that can be ranked systematically between cartilage and bone. Thus is seen the whole breadth of play of the supporting tissues continously connected and with corresponding transitional forms.

Knese and Biermann (1958), while describing the direct formation of a chondroid bone by periosteal-perichondral cells, also observed another event early in development that gave rise to a kind of chondroid bone. Examination of the minor trochanter at age seven (their Figure 13) revealed some bone trabeculae containing not the remnants of cartilage matrix typical for endochondral ossification but cells grouped, as chondrocytes are, in their nests. They took this to indicate that in the lateral growth of the trochanter, bone was formed indirectly by a direct metaplasia to bone of a primitive cell-rich cartilage that they called a chondroid tissue.
They described the latter as having large cells and little matrix, but their Figures 20 and 21 show a definite matrix staining darkly with azan. (Knese and Biermann maintained that in the fetal period the whole minor trochanter is composed of this chondroid tissue, and it is also present on the inferior anterior iliac spine (their Figure 21).) However it is named, the primitive fetal cartilage appeared to turn into bone, with the intermediate, chondroid bone, necessarily involved in the transformation. Fig 5

Another "endochondral" site of a possible periosteal chondrirication is in the facial skeleton. The ethmoturbinal bones develop in cartilage, but then require extensive deposition and resorption to keep up with the changing shapes, size, and relations of the other facial bones. In the kitten, certain edges contain cartilage that in places seems to be developing from the germinal layer enclosing the bone (Figure 5). Later, some projecting margins bear chondroid bone with bone matrix enclosing chondrocytes (my Figure 6).

Another explanation for the chondroid bone is Schaffer's (1916). He believed that, in the anterior region of Meckel's cartilage, ingrowing cells effected a "dissecting dissolution" of the interterritorial matrix, thereby leaving some chondrocytes in their more resistant capsules to be enclosed in new bone. Schaffer remarked that Renaut and Dubreuil (1910) had described this Morcellement resorptiv in the development of the sheep's turbinals. Although some mixing of cartilage cells with bone matrix may happen in this manner, at certain prominences with an established periosteum, cartilage-like cells seem to be developing along with osteoblasts and osteocytes (my Figure 7).

REPORTS WITH LONG-BONE CHONDROID BONE ABSENT

Lubosch (1928), in an article mostly concerned with the possibility of osteoblasts themselves transforming into matrix, included chondroid bone as one of the tissues participating in peripheral growth near the ends of non-mammalian bones. At first glimpse, his work seems to support Zawisch-Ossenitz, because he reported chondroid bone as a subperiosteal cell-rich tissue at the end of a phalangeal diaphysis of a 15-day chick, just under the ossification groove (Ranvier's encoche d'ossification).
In the frog tadpole's femur a similar tissue was present within the ossification groove, which is distinctively formed in Anura because of the overhang of the epiphyseal cartilage. Close study of his text and figures indicates that Lubosch was giving chondroid bone precisely Schaffer's (1888) meaning of large-celled bone. Like Schaffer, Lubosch started his report with a description of ossification in the dentary of farm animals, and explicitly equated his avian and anuran chondroid bone with what he and Schaffer saw in the mammalian fetal mandible.

On the other hand, the site of Lubosch's chondroid bone in tadpole and chick is close to Zawisch-Ossenitz's location of chondroid bone in mammalian long bones. While Lubosch made no mention of the presence of cartilage-like cells in the tissue, this remains as a possibility to be explored.

Descriptions of the tissues at the end of the young diaphysis conflict. Some anatomists such as Zawisch-Ossenitz, and Knese and Biermann, speak of chondroid bone in the sense of a true intermediate tissue, while others, in particular Shapiro, Holtrop, and Glimcher (1977) find nothing like it. The latter authors employed electron microscopy, radioautographs of the fate of 3H-glucosamine, and histochemical staining.
Nevertheless, at the ossification groove on many bones of the rabbit, they found only a clearcut distinction between osteoblasts and chondroblasts, and bone and cartilage. Their bones included the femur which was the bone used by Zawisch-Ossenitz. How may the discrepancy have come about?

  • The length of the bone and the duration of development may play a role. A zone of transition would be more extensive the longer the periosteum, which, if allowed to form cells for a greater period, should result in a region of intermediate tissue large enough to be noticed. Shapiro, Holtrop, and Glimcher (1977), although using many long bones, seem, from their figures, to have concentrated their attention on the shorter metatarsal. In the long bones they may not have sampled enough locations around the circumference of the epiphysis at sufficiently frequent intervals to catch the tissue.
  • The periosteum of their animal, the rabbit, may not form as much chondroid bone as other species seem to, although Bidder (1906) saw cartilage on the rabbit's tibia.
  • The chondroid bone reported on long bones may be close to insertions, sites perhaps deliberately avoided by other investigators.
  • Although Altmann (1964) referred the tissue to Ranvier's encoche, the region he had in mind may have lain a little further away from the epiphysis into Zawisch-Ossenitz's telodiaphysarer zone.

    CIRCUMSCRIPT PERIOSTEAL ATTACHMENTS

    Zawisch-Ossenitz's and Knese and Biermann's observations depict a situation due to recur at many young skeletal sites. The germinal tissues of "perichondrium" and "periosteum" lay down tissues already ambiguous in their nature, which may transform into or through other tissues also hard to characterize. Later in development, other events and tissues come to dominate the picture, but often with no clear indication whether the tissues are the earlier ones after metamorphosis or rather their replacements.

    Thus, some chondroid bone (CB I) is deposited directly by the "perichondroosteum," but chondroid bone of a similar appearance may be the consequence of a metaplasia of cartilage or chondroid to bone. A third source of a chondroid bone (CB II) is the mineralization of a kind of cartilaginous tissue which itself arose by the metaplasia of tendon cells. These events are to be seen in fuller expression at more circumscript periosteal attachments and the apophyses.

    Biermann (1957) described the attachments along the shafts and surfaces of bones, located away from the apophyses at their ends. These periosteal diaphyseal insertions fell into two categories: muscular attachments to periosteum (extensive type), and tendinous to bony crests, edges and prominences (circuniscript type) -- a division equivalent to Dolgo-Saburoff's (1929) "periosteal" and "tendinous."
    As an example of the former, Biermann illustrated (his Figure 16) the insertion of the human M. vastus intermedius into periosteum, from which collagen fibers leave to enter the bone obliquely. At the surface of the bone is a very cellular kind of bone crossed by prominent fibers. Biermann thought that the best name for this kind of bone, based on the pattern and external origin of its coarse fibers, was Petersen's (1930) Einstrahlungsknochen.
    From Biermann's figure and his text description of a similar superficial layer of bone on the femur with broad lacunae and round young osteocytes, it is likely that the tissue was Zawisch-Ossenitz's coarse-fibered or beta chondroid bone, which is not cartilaginous enough to justify the adjective chondroid.

    Biermann acknowledged that the periosteum could itself be considered to be an insertion-structure, and in some places it was hard to judge whether it was a tendinous tissue or a periosteum that was present. One site of periosteum, modified to act as a circumscript region of firmer attachment, is the linea aspera. Enlow's (1962a) Figure 20 illustrated chondroid bone at the linea aspera of a young rat. He indicated that it was not fibrocartilage, but was "similar to or identical with the characteristic tissue located on growing alveolar crests surrounding teeth."
    Of the fate of the tissue he wrote, "The cartilage-like cells appear to undergo direct conversion into osteocytes and become included in seemingly typical nonlamellar bone, although this supposition is not certain." From Enlow's Figure 20 the tissue has more than a hint of cartilage about the cells present, as evidenced in his naming it chondroid bone. Its brief life in that condition may be one reason why it has not been reported more often at diaphyseal sites of insertion, but, of course, another explanation could be that it is very rarely present.

    I found no paper on the histology of the older rat's linea aspera, but Gebhardt (1901a) described it in man after early development as bearing an insertion structure of the same kind as those of major ligaments and tendons in maturity.

    TIBIO-FIBULAR FUSION IN RODENTS

    The fusion of the tibia with the fibula in some rodents' normal skeletal development has the periosteal cells veering from their expected inclination to the extent of forming frank cartilage (Murray, 1954). These cartilaginous formations are examples of secondary cartilage and are themselves accompanied by small amounts of chondroid bone.
    According to Murray (1954), the fusion of the rat's tibia and fibula in their distal parts is achieved in the first two weeks of life. At between three and five days of age, cambial cells of each periosteum form a layer of cartilage on the pre-existing bone. The cartilage extends into the fibrous tissue separating the bone, apparently by a transformation of the matrix into cartilage, with the cells experiencing a partial metamorphosis to chondrocytes by becoming vacuolated, "though perhaps never as much so as the cells derived from the cambial layers." Finally, at six days, cartilaginous fusion takes place. Over the next few days, while the bridging pad of cartilage enlarges, it is being rapidly replaced by endochondral bone, bringing the cartilage to an early end.

    Murray first used guinea pigs for a study of the healing of broken fibulae, but on noticing the existence of cartilage pads on tibia and fibula, where these were at their closest, he examined five unoperated guinea pigs. Of the five, he found two to have cartilage pads; of another he was less certain. Murray concluded that the cartilage pads were an event of normal development, which was not absolutely regular in its occurrence and, when present, displayed some variation.
    In the more usual case, the chondrification spread from the cambium outwards through the fibrous periosteum to build a pad common to both bones. More rarely, the two pads remained separate and "form a structure suggestive of a nearthrosis (his Plate 2, Figure 12)." In older animals, resorption and replacing bone encroached into the pads, both from their substratum of bone and their perimeter.

    Murray (1954) perceived several matters of interest in these chondrifications in the rat and guinea pig. The tibial-fibular cartilages as periosteal derivatives are undoubtedly secondary and were so categorized. In the rat, Murray commented on the presence of a "swift but gradual transition" of tibial and fibular cartilages into bone, illustrated in his Figures 21 and 24. This tissue of transition at the periphery of the cartilage is chondroid bone I.

    Murray attempted to relate the chondrifications to Roux's (1895) general hypothesis that mechanical stress promotes chondrogenesis. Murray acknowledged that presumptive embryonic cartilage cells can go on to form cartilage in vitro independently of mechanical stimulation and that non-skeletal connective tissues could be induced to chondrogenesis in circumstances, e.g., injections of alcohol, where humoral rather than mechanical factors seemed to play the key role.
    There was, in Murray's view, a third category - skeletal cells which, while not normally chondrogenic, would produce cartilage if they were subjected to mechanical conditions involving "pressure and probably shear." The tibio-ribular cartilages fell into this last class, because: 1) they appeared where the two bones were close together; and 2) in the rat, the picture gave the impression that "the fibula tried to push into the tibia," and vice versa, allowing for the possibility of "mutual injury" by the bones.

    Murray himself questioned this line of reasoning on the basis of the unions between the tibia and fibula, and radius and ulna, of the frog, and the chick's tarso-metatarsal fusion. All three bridgings were achieved solely by bone, although the periostea of these species are known to produce cartilage in the course of healing fractures. Murray tentatively suggested that there might be less movement between the bones in frog and in chick than at the mammalian tibio-fibular sites, but in the same sentence he expressed doubt about the truth of this suggestion,

    Moss (1977a) has lately described and discussed the fusion of the tibia and fibula in the rat and mouse. Moss confirmed the transitory nature of the secondary cartilage, told of the similarity of its nature and fate in rat and mouse, and offered a figure (his Figure 7) illustrating the presence of chondroid bone where cartilage merges with the bony shafts in the mouse.
    Moss put forward comparisons of the relation between the tibia and fibula in other species and classes and in paleontological specimens, to bolster the idea that biomechanical factors extrinsic to the bones bring about the fusions. Such mechanical stimuli would have their origin in the musculature and soft tissue of the limb, which thus could be viewed as providing a "functional matrix" (Moss and Salentijn, 1969) for the skeletal elements.
    Perhaps the most significant finding in Moss's (1977a) study was the demonstration that intra-cerebral grafts of the newborn rat's "tibia-fibula complex," with attached but presumably ineffective muscles, did not exhibit osseous fusion, although longitudinal growth had taken place.

    Murray was fairly certain that in the guinea pig the fibrous layers of the periosteum also were transformed into cartilage, although this process in the rat was less complete. The fate of the fibrous tissue thus is similar to that of the perichondria at the symphysis of the mandible (Trevisan and Scapino, 1976b), and can be regarded as an example of fibrous-to-cartilaginous metaplasia.

    The tibio-fibular fusion thus involves two phenomena: the de novo formation of chondroid bone by certain periosteal cells on long bones, and the transformation of an established connective tissue (periosteum) to a kind of cartilage. The latter event appears to be widespread in tendons and ligaments, for example, where they insert. In turning to Schaffer (1902 a,b, 1930) for the most systematic account of tendinous chondrifications, we meet two difficulties.
    First, his treatment sought to fit these entities into the entire spectrum of cartilage-like tissues in vertebrates and invertebrates, resulting in a special nomenclature.
    Second, although he granted that many expressions of his "chondroid" tissue could well be regarded as secondary cartilages, he left unclear which of them originated by a Zellmetamorphose (he forbore to use metaplasia) and which directly from early blastemal cells.

    SCHAFFER'S CHONDROID TISSUES

    Schaffer's chief criterion for distinguishing chondroid from cartilaginous tissues was that live chondroid cells could not be stimulated chemically or electrically to retract. As one consequence, in tissue fixed and sectioned by the standards of the time, chondroid cells, unlike most chondrocytes, filled their lacunae. Other differences were the chondroid cells' weaker basophilia and lack of a pericellular capsule.

    Schaffer distinguished four varieties of chondroid supporting tissue:

    1. Blasige, as in the radula of some molluscs and as a basal support in featherduster worms (see Person and Philpott, 1967, and Cowden, 1967, who discuss chondroid in light of recent histochemistry);
    2. Hyalinzellige, as in the frog's Achilles tendon and the cranial skeleton of some teleosts;
    3. Grundsubstanzreiche, mucoide mit verastelten Zellen, as in the tongue-papilla of snails; and
    4. Grundsubstanzreiche, mucoide mit hyalinen Zellen.
      Kinds 2 and 4 were widely distributed in the tendons, ligaments, tendon sheaths, menisci, sesamoids, cardiac skeleton, and elsewhere in Anura, Urodela, Reptilia, Aves and Mammalia.
      Schaffer's (1930) Figure 154 shows where chondroid or vesicular tissue is present in the squirrel's foot.
    In mammals, the cells are dispersed by the considerable admixture of collagen, are basophil and more readily taken for cartilage cells than those in other classes. Schaffer recognized the resemblance of the fibrous kinds of chondroid to fibrocartilage, but insisted on the basis of cellular retractility that the only true fibrocartilages were the pubic symphyseal, intervertebral and avian articular.

    FIBROCARTILAGE-VESICULAR TISSUE DISTINCTION

    Several contempories, Drahn (1922) inter alios, did not acknowledge Schaffer's (1902b, 1930) placement of the tendinous and other cartilages in a special category of "vesicular supporting tissue of a chondroid type," but held them to be variants of fibrocartilage. The distinction as revealed by light microscopy is discussed by Weidenreich (1923 a,b), Petersen (1930), who employed "tendon cartilage tissue," Knese and Biermann (1958), Schneider (1956) with his "hyaline cartilage run through by unmasked tendon fibers", Haines and Mohuiddin (1968), and Knese (1978b).

    The main criterion for assigning a cartilaginous character to a tissue was cellular shape and size; for example, at the insertions on the Cristae tuberculi of the humerus (his Figure 14) Biermann (1957) described the cells as "sometimes vesicularly enlarged."
    The chondrocytic nature of tendon cells was further inferred from the extents of their pericellular basophil capsules (McLean and Bloom, 1940) and metachromasia. However, a large size is insufficient sign of a tendon cell's being chondrocytic, because an enlargement of tendon cells can, at other sites, herald their becoming osteoblasts, e.g., Biermann (1957), and at the rat's anterior tibial tuberosity (McLean and Bloom, 1940).
    Next, when examined by TEM by Muna et al. (1978) the chondrocytic (Schaffer's chondroid or vesicular cells) of the dog's cardiac skeleton definitely differed from chondrocytes. Can the identification of cartilage-specific macromolecules justify a distinction between fibrovesicular tissue and fibrocartilage?

    As with tendons, the histological nature of menisci and disks, e.g., at the knee, has always been hard to categorize, because of the presence of both fibroblasts and chondroblasts (Barnett, 1954; Silva, 1969; Van Sickle and Kincaid, 1978), and the chondroid-cartilage distinction of the Germans and Austrians. With the help of electron microscopy, Ghadially et al. (1978) concluded that the rabbit's semilunar cartilages are best regarded as fibrocartilage, populated by cells with more affinity with chondrocytes than fibroblasts in their shapes, territories, and extracellular proteoglycan particles.

    On the other hand, Eyre and Muir (1977) found man's semilunar meniscus, "which is classified as fibrocartilage," to contain 95% of its collagen as type I - that typical of non-cartilaginous connective tissues - whereas type II was preponderant in the lumbar disk's annulus fibrosus (50-65%). In assessing the significance of this biochemical difference between a customarily accepted fibrocartilage - the spinal disk's - and a sometimes disputed one - the meniscus - it should be noted that the intervertebral disks of kangaroo and pig also fibrocartilaginous, have more collagen of type I than type II. Accordingly, although the biochemists and bioengineers may be as well placed as microscopists to come up with a biologically sound subclassification of the fibrocartilaginous tissues, one has yet to be formulated.

    Since Schaffer himself assigned many of the vertebrate chondroid structures to the class of secondary cartilage, and much of the "retraction" of cells in his fixed preparations was probably artifact, I shall follow the practice of Dolgo-Saburoff (1929), Cooper and Misol (1970), Becker (1971) and others in regarding Schaffer's fibrovesicular chondroid tissue in tendons and elsewhere as fibrocartilage, recognizing that the fibers are not arranged as in, say, intervertebral disks, nor may the cells depart from the linear order and spacing typical of tendon fibroblasts.

    CARTILAGE IN TENDONS AND LIGAMENTS

    One may imprecisely separate tendon chondrifications into the macroscopic, involving large nodules of firmer tissue, e.g., some sesamoids, and the microscopic, where the tissue's cartilaginous or chondroid nature is expressed in the cells dispersed amongst the fibers. Some of the major sesamoids consistently present develop early from blastemal cells as a hyaline cartilage that experiences endochondral ossification to form a sesamoid bone, e.g., the mammalian patella. (In addition, cartilage-like cells are evident on microscopy within the fibrous insertions into the patella.)
    Other sesamoid bones, e.g., the Os peronaeum (Leutert, 1958), have a mixed origin, and are only partly derived from hyaline cartilage.

    Other tendinous and ligamentous chondrifications are identified by Schaffer (1902a,b; 1930) and Knese (1978b) as:
    1) among the fibrous bundles at insertions;
    2) hyaline cartilaginous nodules acting as intermediary between the extensor tendon and the lizard's distal phalanx (Schaffer, 1902b);
    3) in the interior of tendons, principally where these experience pressure - Varaldi (1901) and Drahn (1922) gave many examples in domestic animals;
    4) at the surface of tendons, extending inwards to varying depth;
    5) sometimes in the sheath of a tendon, for example, in the avian foot, where under the perching bird's weight a wash-board surface on the sheath engages chondroid ridges on the tendon, apparently to lock the tendon in flexion and secure the bird's grasp on its twig (Schaffer, 1902a); and
    6) by the periosteum of grooves in which tendons ride.
    Only in a few of these specializations is there evidence that the tissue formed first with fibroblasts, which then became chondrocytic.

    CARTILAGINOUS METAPLASIA IN TENDONS

    Schaffer (1930) was emphatic that at tendon insertions the chondroid cells had not previously been fibroblasts, but had arisen from indifferent cells when the tendon first formed. While this may be so for some cells, as the insertion structure matures, it is described by Knese and Biermann (1958) and others as extending into the tendon, thus necessitating a continuing transformation of tendon cells to chondrocytes.

    A similar metamorphosis is a prominent part of the ossification in avian tendons distant from insertions (Renaut (1871-72) and Engel and Zerlotti, (1967) inter alios). Johnson(1964) described the process thus, "The fibrocytes without mitosis, enlarge, transform to chondroblasts, and produce a sulfated mucopolysaccharide that permeates the entire tendon and converts it to osteoid. With mineralization, some cells become osteocytes, the rest disappear". (Landois (1866) made an early listing of the sites of tendinous ossification in the hen.)

    As mammalian tendons age, chondrocytes take the place of fibroblasts, presumably by a metaplasia, for example, in the rat's Achilles tendon (Barfred, 1971), the equine deep digital flexor tendon (Webbon, 1978), and the human nuchal ligament (Scapinelli 1963). In calcifying tendinitis, mineralization is preceded by a pathological chondrocytic conversion (Sarkar and Uhthoff, 1978). But the strongest support for a tendinous cartilaginous metaplasia comes from the experiments of Ploetz (1937) (see Chapter 4, Pressure-elicited Chondrogenesis on Endochondral Bones).

    OTHER PERI-SKELETAL METAPLASIAS

    The cells of the temporo-mandibular meniscus become more cartilaginous with increasing age (Karakasis and Tsaknakis, 1976), and in the older intervertebral disk an increase in alcian blue-positive material around the inner annular cells is characterized by Pritzker (1977) as a cartilaginous metaplasia. In the normal development of the intervertebral disk, part of the annular fibrocartilage derives from a tissue earlier holding fibroblasts (Prader, 1947; Knese, 1978a).
    The chondrocytic metaplasia thus brings into being secondary cartilages. These in their turn may be subject to a further transformation - calcification - which by custom rather than logic is not granted the status of a metaplasia, and is discussed in the next chapter.

    The calcification of the cartilage derived from fibrous tissue might in certain instances lead to bone. However, by the insertions of the patellar ligament, fibroblasts are reported to be a direct source of osteoblasts. McLean and Bloom (1940) described the process in the rat: "The fibers are transformed into bone matrix, and calcification of the matrix occurs either as it is formed or shortly thereafter ... the tendon cells are seen to undergo all the stages of transition to osteoblasts ... Moreover, in the ossified tendon, many osteocytes are seen to have the lineal arrangement of the tendon cells, together with the stellate form and pale staining area (probably the negative Golgi net) characteristic of osteocytes and osteoblasts."

    Bidder (1906) was skeptical of a fibroblastic origin for the bone cells. He thought that ossification proceeded into the tendon in a manner similar to primary endochondral osteogenesis and accordingly named the process in the rabbit endotendinale Ossification. Later authors (Badi (1972a) inter alios) have confirmed the metamorphosis, but classify its site as the fibrous growth plate rather than with the ligament. Badi (1978) has shown by radioautography that it is the fibroblastic cells that become the osteoblasts of the bundle bone.
    A similar incorporation of already formed collagen fibers into bone matrix was described by Knese and Biermann (1958) at the child's tibial tuberosity (their Figure 18) as "periosteal ossification with inclusion of ready-made fibers."

    Knese and Biermann (1958) also implied that at insertions of the fibrocartilaginous ends of ligaments into hyaline cartilaginous apophyses, a hyalinization can encroach into the fibrous zone, in effect, an alteration of fibro- to hyaline cartilage.


    Chapter 10 CHONDROID BONE II

    Mineralization versus ossification
    Mineralized fibrocartilage at insertions
    Other mineralized fibrocartilages
    Pathological chondroid bone II (fibro)
    Articular chondroid bone II
    Chondroid bone II (hyaline)
    Chondroid bone II (fibro)
    The Nature of chondroid bone II (hyaline)
    Chondroid bone II (hyaline) in epiphyseal plates
    Chondroid bone II (hyaline) in other permanent cartilages
    Cellular viability and chondroid bone
    Occurrence of bone
    Stimuli for degeneration, calcification, and ossification
    Chondroid bone II (elastic)
    Metaplastic interpretations of CB II
    Non-mammalian chondroid bone II

    The patella and cartilage canals
    Tracheal cartilages
    Sites of insertion
    Conclusions
    Confusions between chondroid bone I and II
    The cells and mineralization
    Functions of CB II
    . Growth
    . Mechanical roles
    . Non-mechanical roles
    Secondary cartilages and CB II

    MINERALIZATION VERSUS OSSIFICATION

    Any kind of cartilage, primary or secondary, skeletal or extraskeletal, can experience mineralization. Much hard cartilage may then suffer erosion and replacement by bone and marrow. However, in many places the erosion eventually desists, leaving a small amount of calcified cartilage. When this enduring tissue includes living cells, many anatomists have looked on it as a form of bone. This is what I am calling CB II.

    The origin of the tissue in cartilage is clear, but there is variation in how close the final tissue comes to bone. In mature articular cartilage and tendon insertions the cells keep their chondrocytic form, but in avian tendons the cells become smaller and more like osteocytes, enabling Lieberkuhn (1860, 1863) to use the events to bolster his general metaplastic theory of osteogenesis. The calcification undoubtedly occurs, but is the hard tissue bone? And how and why does calcification take place? The first issue has lapsed into the usual wrangle over nomenclature (Weidenreich, 1930).

    Some time after mineralization, parts of the tendon are resorbed and replaced by lamellar true bone (Landois, 1866), allowing the misconception that mineralization had led directly to the formation of lamellar bone. (In the last century, for a while, it was held that lamellar bone had to be of osteoblastic (non-metaplastic) origin, whereas non-lamellar bone might have arisen by metaplasia.) Taking specimens at closely spaced intervals and from other animals established that the early process was one of mineralization of an established connective tissue and that it was widespread in tendons and ligaments (Gebhardt, 1901a), even if only in particular mammalian species such as the kangaroo (Koch, 1926) was it as prominent as that in birds.

    Gebhardt (1901b) conceived of the initial mineralized tissue as true bone, but one that "yet still possessed many properties of the tendon." McLean and Bloom (1940) also wrote of calcified dense connective tissue, "although it retains microscopic characteristics which distinguish it from bone formed in the usual way, this tissue is probably best classified as bone."
    The term sclerosed connective tissue had currency for a period, until Weidenreich (1923b) deliberately abandoned it in favor of ossified connective tissue. He conceived of the process as an ossification rather than "merely" a calcification, because he believed that an active synthesis of Kittsubstanz (cement or ground substance) accompanied, and was the critical event for, mineralization - a theory still of concern (Hohling et al., 1976; de Bernard et al., 1977).

    Weidenreich (1923c) extended his scheme of classifying ossifications to include mineralized cartilages. His concept of bone thus was very broad and took in ossifications named for the nature of the tissue before mineralization. Thus, tendons and ligaments became ossified connective tissue - one form of Faserknochen.
    Another form recognized that many tendons and ligaments in places had large cells, closer to chondrocytes than to fibroblasts. Schaffer (1902a,b) had drawn attention to such vesicular cells in dense connective tissues. The second type of Faserknochen accordingly was ossified vesicular fibrous tissue.

    The mineralized cartilage lying under articular surfaces Weidenreich grouped separately as Knorpelknochen. In these ways, he recognized two kinds of chondroid bone: calcified subarticular cartilage or Knorpelknochen, equivalent here to chondroid bone II (hyaline or occasionally fibro); and ossified vesicular fibrous tissue, i.e., chondroid bone II (fibro), at insertions and sesamoids.
    He justified the name bone for these materials because mineralization made them a "permanent and morphological and functionally essential part of the hard skeleton," and although the term bone thereby lost some of its precision, the fault lay with the organism.

    Recognition of any chondroid bone must broaden and blur the concept of bone, but calling a tissue chondroid bone may be less troublesome than other names, as is illustrated by Haines and Mohuiddin's terminology (1968). They looked at ground and stained decalcified sections of subarticular cartilage, and the insertions of tendons and ligaments, to reach conclusions similar to Weidenreich's: these tissues mineralized to become part of the skeleton; the process was a direct metaplasia and resulted in a "metaplastic tissue," to which they gave the general name "metaplastic bone." This was to be specified further by its various origins as articular, tendon, or ligament bone.
    Within the context of their paper the nomenclature suggested was useful and consistent. However, their terminology obscures Weidenreich's distinction between the two kinds of chondroid bone II, and between these and mineralized but non-cartilaginous connective tissue.

    Within the wider context of metaplasia, "metaplastic tissue" is too vague, and a "metaplastic bone" achieved by the calcification of cartilage or tendon needs to be separated from ectopic true bone formed by the osteoblastic metaplasia of fibroblasts.
    In addition, "tendon bone" begs to be confused with a sesamoid, and "articular bone" with the subchondral bony plate.

    Fig.,, 8. Radial t.b (Fig. 176 Knese, 1978b)

    MINERALIZED FIBROCARTILAGE AT INSERTIONS

    At both typical tendon insertions and the circumscript periosteal attachments, Biermann (1957) and many others report that, as the fibrous tissue approaches the bone, its cells enlarge and appear more chondrocytic (Figure 8). Routine staining of decalcified sites reveals the cartilaginous region of the tendon comprises two zones separated by a distinct line of demarcation (Grenzlinie or tidemark, Zawisch-Ossenitz, 1929b).

    The stains - e.g., azan (Bierman, 1957) or hematoxylin-eosin-azure II (McLean and Bloom 1940) - bringing the tidemark to view are not generally thought to be staining calcium salts, though some calcium survives routine decalcification (Hough et al., 1974), but other evidence indicates that the tendon on the bony side of the line is mineralized and on the muscular side is not.

    Granting that the tissue of apophyseal and circumscript periosteal insertions is a form of fibrocartilage, then the mineralization within it that yields CB II (fibro) is widespread. The very many locations of this material are listed with a reference to the source of the information. The site of insertion is human unless otherwise stated.

    1. The collateral ligament on phalanges (Petersen, 1930).
    2. The patellar ligament on the tibia (Gebhardt, 1901a; Petersen, 1930; Knese and Biermann, 1958; Ogden, Hempton, and Southwick, 1975), and on the rat's tibia (McLean and Bloom, 1940; Badi, 1972b).
    3. The interosseous ligament into the radius and ulna (Gebhardt, 1901a).
    4. The Peroneus brevis tendon (Petersen, 1930).
    5. The Achilles tendon at the Tuber calcanei (Gebhardt, 1901a; Petersen, 1930; Weidenreich, 1923b; Schneider, 1956; Becker, 1971) and in the panther (Gebhardt, 1901a).
    6. The quadriceps tendon into the patella (Gebhardt, 1901a; Weidenreich, 1923c) and in the elephant's patella (Gebhardt, 1901 a).
    7. Origin of Extensor digit. comm. on the radial epicondyle (Schneider; 1956).
    8. Insertion of M. gluteus maximus on the major trochanter (Schneider, 1956).
    9. Insertion of M. supraspinatus (Schneider, 1956).
    10. Insertions of the biceps muscle (Cap. long.) on the supraglenoid tubercle, and on the Tuberositas radii (Knese and Biermann, 1958) and in the rat (Knese, 1978b, Figure 189).
    11. Insertion of the cruciate ligament on the femur and, in man and the cat, of the Ligamentum teres (Gebhardt, 1901a; Benninghoff, 1925; Weidenreich, 1930).
    12. Insertion of the iliopsoas on the minor trochanter (Becker, 197 1).
    13. In several tendinous insertions of reptiles (Haines, 1969).
    14. Insertions of the triceps on the olecranon of the dog (Haines and Mohuiddin, 1968).
    15. Insertions on the styloid process (Gebhardt, 1901a).
    16. Insertions in the linea 'aspera of the femur (Gebbardt, 1901 a).
    This already lengthy list of sites of mineralized fibrocartilage at insertions does not exhaust those reported in the literature, and other potential locations have not yet been closely examined, e.g., the cartilage ensheathing the handle of the malleolus (Graham, Reams, and Perkins, 1978).

    OTHER MINERALIZED FIBROCARTILAGES

    Fibrocartilage is found in two other kinds of situation: as an articulating tissue between bones (see Chondroid Bone II,p. 171) or between hyaline cartilages attached to bones; and participating in certain firm structures separate from the skeleton, although sometimes these extraskeletal and secondary fibrocartilages are attached to it by collagenous tissue, e.g., for menisci.

    Many species have cartilaginous or chondroid cells among the fibrous tissue of the cardiac skeleton. In some, such as the sheep and cow (Weidenreich, 1930), the tissue regularly calcifies, producing a chondroid bone II (fibro). Other mammals, but not ungulates, have cartilaginous and bony bodies in the penis. The rat has a penile bone and a separate anterior process of fibrocartilage (Wiesner, 1934). As the rat ages, this fibrocartilaginous mass usually experiences a partial calcification followed by a limited replacement by very irregular endochondral bone (Beresford, 1970a).

    Do these observations have a more general significance? Yes, in that any other mineralized fibrocartilage after a while may be partially eroded and replaced by bone, for example, at some of the tendinous insertions described by Knese and Biermann (1958); and no, in the sense that while eventual calcification may be the rule in one fibrocartilage, it is not so in many others despite their larger size (Barnett, 1954), and variations by species must be heeded.
    For example, bone is a normal occurrence in the rat's semilunar cartilage, as in several species (Barnett, 1954). Another example of ossification in the fibrocartilages of the rat, one might think; but, no, the osteogenesis takes place in hyaline cartilage (Pedersen, 1949) (see Knese (1978b) Figure 201A), or according to Schaffer (1930), chondroid tissue mixed with hyaline cartilage.
    In the more fibrocartilaginous menisci of the human knee, calcification and ossification are pathological and rare. Harris (1934) and Pedersen (1949) gave some examples, and there are more recent instances, inter alios Symeonides and loannides (1972).

    PATHOLOGICAL CHONDROID BONE II (FIBRO)

    These uncommon human cases, together with rare mineralizations of such skeletal fibrocartilages as the intervertebral disk's annulus (Amprino and Bairati, 1934; Dussault and Kaye, 1977; Weinberger and Myers, 1978) and pubic symphysis (Amprino and Bairati, 1934), comprise a class of pathological CB II. In this class belongs the calcification preceding ossification that occurs in fibrocartilage uniting the slowly healing tibial fractures in man (e.g., Urist, Mazet, and McLean, 1954).
    Other instances of pathological CB II (fibro) arise when tendons, e.g., of the supraspinatus (Wrede, 1912), and ligaments, e.g., Ligamentum nuchae (Scapinelli, 1963; Lewinnek and Peterson, 1978), chondrify and then mineralize. Uhthoff (1975) gives an account of the cellular hypertrophy, increasing metachromasia and elaboration of alkaline phosphatase in calcifying tendinitis.

    ARTICULAR CHONDROID BONE II

    At the apophyses and subperiosteal locations chondroid bone I is also sometimes present, and a variety of seemingly metaplastic events unfolds. The same may be true of certain joints such as the temporomandibular, where a mandibular secondary cartilage participates in the normal development. However, in maturity, at these joints, and the more typical diarthrosis and synchondrosis, only chondroid bone II is to be found. Since some of these articulations have fibrocartilage, both CB II (hyaline) and CB II (fibro) occur.

    Chondroid Bone II (Hyaline)

    1. Under long-bone articular cartilages, (Pittard, 1852; Muller, 1858; Kolliker, 1889; Gebhardt, 1903; Weidenreich, 1923c, 1930; Benninghoff, 1925; Petersen, 1930; Haines and Mohuiddin, 1968; Hough et al., 1974; Muller-Gerbl et al., 1987).
    2. In the patella (Green, Martin, Eanes, and Sokoloff, 1970).
    3. In the malleus and incus (Weidenreich, 1923c, 1930).
    4. In the ribs (Muller, 1858); the sternal ribs of carnivores and ungulates, and the synchondrosis between the vertebral rib and the intermediate piece in the horse are noteworthy (Lubosch, 1924).
    5. At the vertebral epiphyses or end-plates (MUller, 1858; Schmorl, 1928; Schmorl and Junghanns, 1932; Frangois and Dhem, 1974).
    6. At the Dens epistrophei of the atlas (Petersen, 1930).
    7. At the persisting cartilage of the vertebral spinous process in horse (Stoss, 1918a).
    8. As a widespread calcification of the 'match-head' epiphyseal cartilage of frog (Parsons, 1905; Haines, 1938a).

    Chondroid Bone II (Fibro)

    1. Pubic symphysis (Muller 1858).
    2. Mandibular symphysis of hamster (Trevisan and Scapino, 1976b) and prosimians (Beecher, 1977).
    3. Sterno-clavicular joint (Benninghoff, 1925; Petersen, 1930).
    4. Intervertebral disk (Amprino and Bairati, 1934; Francois, 1965; Frangois and Dhem, 1974).
    5. Collagenous peripheral regions of hyaline articular cartilages (Benninghoff, 1925).
    6. Mandibular articulation of teleost fishes (Haines, 1938b).
    In at least one location there is some dispute over the nature of the tissue that mineralizes. Petersen (1930) listed the human jaw as a site of calcified fibrocartilage, and one may presume that he had in mind the mandibular condyle, whereas Schaffer (1930) regarded the condyle as hyaline cartilage that becomes fibrous only towards its surface, as did Green et al. (1970).
    For the deep mineralized band, the question of the starting tissue may not be as important as at first sight, since, when definite hyaline cartilage is in the deep subarticular position, it loses proteoglycans and appears more eosinophilic and fibrous. In other words, aside from being mineralized, it changes somewhat in the direction of fibrocartilage; but whether it gains collagen is another matter. Therefore, whichever the initial tissue, the end results would be closer in nature.
    In the synchondroses of the spine, both hyaline cartilage of the endplates and fibrocartilage of the annulus at its insertions can be mineralized (Francois and Dhem, 1974), with the calcification being either diffuse or distributed in a honeycomb fashion.

    THE NATURE OF CHONDROID BONE II (HYALINE)

    A critical question for chondroid bone II now must be heard. What is the character of the tissue, in terms of its cells and matrix? The cells are large and vesicular enough to took like chondrocytes, but unlike those of the calcified cartilage participating in typical endochondral ossification they are not hypertrophic (Haines and Mohuiddin, 1968; Kemp and Westrin, 1979); although there is a dearth of measurements to give precise meaning to 'chondrocytically enlarged' and 'hypertrophic'.

    The matrix is eosinophil and can be shown by special impregnations with silver salts (Weidenreich, 1930) to be fibrillar. Maceration (Haines and Mohuiddin, 1968), X-rays and dissection reveal the matrix to be mineralized, but identification of this condition in the stained decalcified section relies heavily on the presence of a basophil line at its border. Zawisch-Ossenitz (1929b) reviewed the significance of such tidemarks. It is generally accepted that in cartilage, tendons, and other connective tissues, a single basophil line marks off mineralized from un-mineralized regions.

    In chondroid bone II, to what degree is the cartilage mineralized? This point would seem to be of great import for any chondroid bone, because if the cells remain chondrocytic without processes ramifying in canaliculi (Haines and Mohuiddin, 1968), some level of calcification would be expected to obstruct the diffusion needed to keep the cells alive, although Gebhardt (1903) mentioned occasional lacunae with canaticuli in calcified articular cartilage.

    Some individuals have thought that chondrocytes cannot survive any calcification of their matrix, but this has long been known to be erroneous from persisting mineralized cartilage in fishes (J. Muller, 1834; Schaffer, 1930; Wurmbach, 1932; Moss, 1977b), amphibians, birds, and mammals (Muller, 1858; Tretjakoff, 1929; Weidenreich, 1930). The viability of the cells and the degree of calcification of the matrix are two issues requiring separate attention and investigation.

    Regardless of what hypothesis predicts, if the cells are alive, however calcified the matrix, they have been able to cope with its condition. For example, Moss (1977b) describes the chondrocytes of the calcified cartilage in the vertebrae and jaws of sharks as appearing to be vital, although the mineralization is "essentially equivalent to that found in vertebrate bone." Kemp and Westrin (1979, Figures 20 and 2 1) confirm the living appearance of the cells by TEM (my Figures 9 and IO). Dispersed in reports on various sites of chondroid bone II are pertinent data derived by recent techniques.

    Lemperg (1971a) treated nine-month-old rabbits with tetracyline and found the intra-cartilaginous tidemark across much of the femoral head to fluoresce, indicating an active mineralization. Duplicate labeling of tidemarks, when a second injection was given, showed that mineralization progressed slowly towards the articular surface, as Fawns and Landells (1952) had suspected from histochemical staining. Lemperg's (1971b, Figures 3 and 4) microradiography shows the matrix to be mineralized, but not as densely as the subarticular bone.
    When Lemperg injured the overlying non-mineralized articular cartilage, a microradiogram revealed a loss of mineral from the calcified cartilage, then at two weeks after injury the fluorescent tetracyline-binding tidemark became wider and more prominent. Chondrocytes of the mineralized zone later displayed a high uptake of 35sulfate, so that not only are they alive, but they can make a metabolic response to the injury above them.

    Haines and Mohuiddin (1968) noted that the subarticular chondrocytes usually stain weakly, although their capsules react well with hematoxylin and toluidine blue. Their von Kossa-staining showed "successive layers of more or less dense calcification" in the matrix and heavily calcified capsules around the chondrocytes. Lemperg's (1971a) Figure 6 of strong tetracycline-labeling around the chondrocytes of the mineralized zone indicates an active calcification close to the cells, and perhaps mediated by them, but Lemperg cautioned that there is some autofluorescence on the part of the chondrocytes.
    [Lemperg's (1971b) experiment was unlike Meachim's (1972) complete experimental penetration of the articular cartilage and underlying bony plate at the rabbit's knee. The early reparative events described by Meachim did not differ from those in holes drilled laterally in a para-articular position, and followed the sequence typical of fracture repair. The new tissues were the typical ones - bone, cartilage, and interposed chondroid bone (type I) - as the subchondral bone reacted to harm.]

    Electron microscopy and histochemistry have helped establish the vitality of chondrocytes in CB II. For example, at canine tendinous and ligamentous insertions, Cooper and Misol's (1970) electron microscopy revealed chondrocytes looking well, despite being completely surrounded by mineralized matrix. Becker (1971) also saw chondrocytes surrounded by mineral, and believed at least some of the cells to be degenerating, but the nucleus in the chondrocyte of his Figure 12 is not pyknotic. Badi (1972b) observed viable small chondrocytes still reactive for alkaline phosphatase in old calcified cartilage of the rat's patellar ligament. In the course of following other examples of chondroid bone II, the state of the cells will be indicated, if the authors have revealed it.

    The extent to which CB II can become mineralized is uncertain. While Lemperg's work (1971a) suggested that it was less calcified than bone, the matter is less clear in the mandibular condyle. In later stages of its human development, Wright and Moffett (1974) describe the innermost zone of cartilage as exhibiting "a basophilic staining reaction which indicates mineralization of the matrix." This layer is wide in their Figure 18 of a 19 year-old condyle, but narrow in a 23-year-old. The layer shows up as "highly mineralized" in the microradiogram of a 191/2-year-old condyle (Ingervall,Carlsson, and Thilander, 1976).

    In one of the very few papers devoted to the calcified cartilage layer, Green et al. (1970) reported on the microradiography and histology of the tissue. In the patella, linear radio-opacities occurred over, but wider than, the tide-mark, and at the osteochondral junction. Elsewhere the cartilage was only as radiodense as the bone. However, in the adult human intervertebral disk and at the toe phalanx of an old Rhea americana, the calcified cartilage was distinctly denser, i.e., more mineralized, than the bone. These authors also found the human layer to be PAS-positive, but to lose its alcianophilia after the age of 25. With increasing age the number of viable chondrocytes fell drastically, but the same was true for the osteocytes in the neighboring bone.

    CHONDROID BONE II (HYALINE) IN EPIPHYSEAL PLATES

    Ossification in the secondary center of the epiphysis spares the articular cartilage, bringing about a longstanding junction of bone with calcified cartilage. On the other side of the secondary center, at the growth plate, bone and cartilage again lie side-by-side for long periods. Can CB II arise in this similar situation?

    Dawson (1929) extended his earlier report (1925) on the failure to close of several epiphyseal plates in the rat. Rats over 90 days old still had cartilaginous growth disks. The cartilage closest to the two confining layers of bone appeared to be experiencing a metaplasia into bone: "The final obliteration of the cartilage, when it does occur in old rats, also appears to be the result of such a metaplasia rather than destruction succeeded by substitution ... In the zone immediately adjoining the limiting sheets of bone these modified cells assume the form and staining reactions of osteoblasts and in many places appear to be directly incorporated into the bone."
    In cases where the cartilage was obliterated, the chondrocytes disappeared and a line of basophilic substance separated the limiting plates of bone. Then, when the plate was no longer cartilage, vascular penetration across the epiphyseal line could at last take place.

    Dawson's pictures do not show a clear tidemark, but it seems reasonable to conclude that the cartilage adjacent to the bone, especially the basophil remnant when the gap between the bony plates was narrow, is mineralized, enduring, hyaline cartilage (CB II, hyaline). Dawson also mentioned that areas of this tissue became fibrillar before its direct transformation "into a kind of bone (osteoid) tissue," but "this evidence for chondroosseous metaplasia is admittedly not completely conclusive."

    Haines (1975) has lately suggested that his "metaplastic tissue" in the form of calcified cartilage (chondroid bone II) participates in epiphyseal closure, in general, as the hard tissue first uniting the ossification centers and arresting growth.

    CHONDROID BONE II (HYALINE) IN OTHER PERMANENT CARTILAGES

    While the cartilages of the ribs and airway generally last throughout life, they undergo many changes, among which often are calcification and ossification. Schaffer (1930) and Hintzsche (1931) reviewed such regressive and kataplastische alterations in hyaline cartilage in some detail. Schaffer cited Donders (1846) as the first to liken the coarse fibers in older cartilage to asbestos fibers, and Rheiner (1852) as accurately describing the changes with age in laryngeal cartilages.
    Does the calcification of these hyaline cartilages convert them to chondroid bone? When bone forms, is chondroid bone involved? What causes the cartilage to calcify, ossify, or be replaced by bone? Is there a metaplasia of cartilage or chondroid bone into bone?

    Cellular Viability and Chondroid Bone
    For a tissue to be chondroid bone II, as distinct from non-vital calcified matrix, it should have living cells in a matrix of collagen and mineral. The calcification of aged costal and airway cartilage is seen in areas of matrix where the cells are mostly fading or absent. When the cells are present, are they alive enough to make the tissue chondroid bone?
    The implication of the three cellular degenerations described by Schaffer (1930) - Verdammerung, chondromucoid transformation, and fatty degeneration - is that sooner or later the cell is lost. Although the tissue of the resulting cell-free zones is not chondroid bone (though it may become well mineralized), does niineralization occur around chondrocytes that have a prospect of staying alive?

    Few authors have paid serious attention to this question: most have commented in more general terms on the great variability in the appearance of the cells and matrix within any one zone of an old cartilage. (Rheiner (1852) first noted that in a cross-section of a cartilage the subperichondral region differs from the central zone, and both these zones from an intermediate or transitional zone lying between them.) Nevertheless, for the older costal cartilage, Hass (1943) remarked, "in many fibrillary, densely calcified fields the cells often are well preserved."

    Nevinny (1927), and many before him, noted calcification to start in the vicinity of the chondrocytes as fine granules. The long-held notion of an active cellular participation in the initiation of calcification was reinforced by the sighting in electron microscopy of clusters of mineral crystals within electron-dense bodies - matrix vesicles - membrane-bound, and hence of cellular origin (Bonucci, Cuicchio, and Dearden, 1974). They applied histochemistry and electron microscopy to costal and tracheal cartilages of rats up to two-years-old. Calcification had started by 30 days of age and was extensive in the central zone of six-month- and two-year-old rats. Calcification completely surrounded some cells, which remained alive but laden with large lipid droplets. While the relevance of these observations for man is limited by the differences in the span of life and the sizes of the cartilages, it is possible that the calcified regions of costal and airway cartilages in man (and several domestic animals: Sussdorf, cited by Nevinny, 1927) retain some long-lived cells, and could justly be classed as chondroid bone II.

    Another change in the cartilage that might bring it closer to bone is that interterritorial regions of the central zone, the zone most subject to calcification, also display an increasing eosinophilia and prominence of the collagen fibrils. In its extreme form this tendency leads to the large parallel fibers of the asbestotic transformation (Schaffer, 1930; Hough, Mottram, and Sokoloff, 1973).
    Is the matrix of aging hyaline cartilage increasing its content of collagen fibrils? There is no definite answer. A loss of the proteoglycans that mask the fibrils has long been suspected (Bohmig, 1929; Hass, 1943; Linzbach, 1944) and confirmed histochemically (Quintarelli and Dellovo, 1966), but this event does not reveal whether the chondrocytes synthesize additional collagen. Bonucci et al. (1974) saw unusually thick fibrils by some older chondrocytes, but offered two alternative explanations for them: abnormal chondrocytic synthesis, or an accretion from soluble collagen no longer bound to proteoglycan. Dawson and Spark (1928) had earlier suggested that the fibrous transformation in aged rats' costal cartilage resulted from a dissolution of the original fibrils, followed by a new precipitation with another orientation determined by mechanical conditions.

    Occurrence of Bone
    Oppel (1905) dated the acquisition of knowledge that the laryngeal and tracheal cartilages ossify from Realdus Columbus (1572) and P. Dionis (1696), respectively. Oppel cited several authors reporting laryngeal bone formation, but gave most space to Chievitz's (1882) thorough account. Chievitz (1882), Hart (1928), Schaffer (1930), Amprino and Bairati (1933a), Hass (1943), Linzbach (1944), Keen and Wainwright (1958), and many others contributed these observations regarding aging costal and airway cartilage:

    1. The abnormal formations of cartilage and bone in the tracheal soft tissues - tracheopathia osteochondroplastica - are unrelated to normal aging within the permanent cartilages.
    2. More cartilages exhibit calcification than ossification.
    3. Calcification can exist without ossification, but the converse is not seen (of course, ignoring the calcium salts within the bone).
    4. Bone can be deposited on uncalcified cartilage (Pascher, 1923; Bohmig, 1929).
    5. The first rib is early and regularly affected, the other ribs later and less consistently (Werner, 1978).
    6. The laryngeal cartilages, aside from the elastic ones, are more involved than the tracheal rings.
    7. Within the trachea, the gradient towards lesser involvement runs from the larynx downwards, with bronchial cartilages least calcified and rarely the seat of bone.
    8. The position of the bone varies with the particular cartilage. In the ribs, it tends to lie as a sheath near but not at the periphery (Bohmig, 1929), although Hass (1943) gave it more a central position in the cross-section.
      In the trachea, bone forms near the outer perichondrium at the anterior part of the ring. The place of onset and the manner of ossification are disputed. For example, Nevinny (1927) thought that tracheal bone formation progressed from the perichondrium, inwards; Linzbach's (1944) contention was from the interior, endochondrally, outwards.
    9. Calcification and ossification in the human nasal septum are very rare (Alverdes, 1933).
    10. In man, the mineralization's time of appearance, extent and position within the cartilages differ between the sexes.
    The bone that forms within and remains a part of a permanent cartilage has a cartilaginous location, but with the exception of what may be present in birds, and Linzbach's suspect osteoid (see Tracheal Cartilages, p. 187), the bone is real bone, even displaying occasional osteons in the trachea (Nevinny, 1927), larynx (Pascher 1923), and rib (Bbhmig, 1929), and may play host to metastases from a nearby carcinoma (Carter and Tanner, 1979).

    Stimuli for Degeneration, Calcification, and Ossification
    Why do costal, laryngeal, and tracheal cartilages calcify and experience a partial replacement by bone? These events accompany many others with varying degrees of regularity. The apparently haphazard nature of the time of onset, and the kind and extent of the changes in the tissue, have kept explanations to generalities, unable to account for specific changes at individual cells with certainty.

    Linzbach (1944) and Beneke et al. (1966) maintained that the continuing growth of tracheal cartilages makes them prone to degeneration because the cells outstrip their perichondral nutrition, which, as a factor of area, must lag behind tissue requirements rising as a function of volume. Hass (1943) and Rahlf (1972) drew the same conclusion from measuring the areas of normal and altered regions in cross-sections of aging ribs. Lack of knowledge about the distribution or extent of the vessels within the young healthy cartilage, and how these vessels change with growth and aging, limits the validity of measurements of perichondral area vis-a-vis cartilage volume to a generality for the cartilage as a whole, and can throw no light on individual cells and their relations with vessels.

    Impoverished nutrition of the chondrocytes is believed to lead relatively early to their inability to synthesize enough proteoglycans to keep pace with the the degradation or dissolution. Later, some cells die and, according to Linzbach (1944), may release materials attracting vessels to break down the cartilage matrix and invade it. The penetrating vessels bring with them cells ableto form bone.

    The unknowns concerning this calcification and ossification are many. Electron microscopy suggests that the cells play an early and active part in initiating calcification (Bonucci et al., 1974), but in man calcification is extensive in areas now free of cells or with verdammert cells. Were the cells alive and responsible when calcification started? Are the changes in proteoglycan amount and chemistry of importance for calcification or resorption? Stagni et al. (1977) postulated two factors as responsible for calcification's not occurring in nasal cartilage: macromolecules inhibiting the precipitation of calcium phosphate, and only a low affinity for ionic calcium on the part of its mineral-binding glycoproteins. What is the role of collagen "bared" by a loss of proteoglycans? Cartilages in the ribs, larynx, and avian trachea that consistently calcify and experience some ossification may help answer some of these questions.

    CHONDROID BONE II (ELASTIC)

    Infrequently, calcification affects elastic cartilages such as those of the epiglottis and the Eustachian tube (Meyer, 1849a). Elastic cartilages of the ear also have a limited involvement. Weidenreich (1930) designated the tissue elasticher Kalkknorpel, i.e., CB II (elastic), and gave two examples of calcified elastic cartilage; but Schaffer's (1930) treatment was more thorough, with cited examples (Baecker, 1928) of a regular mineralization of the external auditory cartilage in the mole and the guinea pig (Schaffer's Figure 272), the pinna of dog, and pathologically in the human auricle and epiglottis.
    Baecker (1928) noted that cartilage of the meatus in the mole, although calcified, had very few elastic fibers. That of the guinea pig was more elastic and not only calcified but also was extensively replaced by bone.
    Incidentally, very small mammals have a matrix-poor, cellular auricular cartilage, giving some histological support to Schaffer's (1930) classification of elastic cartilages as secondary.

    In man, calcification of elastic cartilage is rare: Amprino and Bairati (1933b) found none in over 80 examples of the epiglottis and auricle. Dreyfuss (1916) found minor calcification in the older epiglottis, while Adran (1965) reported extensive mineralization in a 68-year-old man. For the elastic cartilage of the auricle, Siebenmann (1977) has drawn 130 cases of calcification from the literature. Of these, his own patient and one other had histologically proven ossification, and in his patient the epiglottis also was partly replaced by bone. He suggested that endocrine disturbances, in particular the treatment of Addison's disease with deoxycorticosterone, were a likely cause of some auricular calcification. The role of excess corticoid hormone is disputed by Zillessen, Gless, and Baldauf (1978) on the basis of another case of auricular ossification. Chadwick and Downham (1978) list several local and systemic conditions in which auricular calcification happens.

    Calcified elastic cartilage has not been studied from the standpoint of the viability of its cells and the possibility of metaplasia. For the general resistance to calcification of elastic cartilage in old age, one wonders if the cartilage's high content of hyaluronic acid (Wusteman and Gillard, 1977) may be significant. Since some elastic fibers are present in the cartilage of fracture callus (Murakami and Emery, 1967), and other "non-elastic" cartilages (Keith et al., 1977), one may also ask whether elastin can be a minor component of chondroid bones other than CB II (elastic).

    METAPLASTIC INTERPRETATIONS OF CB II

    Non-Mammalian Chondroid Bone II
    Non-mammalian species also have chondroid bone II at tendinous, ligamentous, articular, and epiphyseal sites. There are four reasons for treating them separately from the mammal's.
    First, there is a long-standing tradition of divided discussion, for example, by Muller (1858) for mammals, amphibia, birds, and fishes, and by Heidsieck (1928), who gave a synopsis of the several reports on reptilian bone
    Second, chondroid bone II is more abundant in non-mammals, because of two factors: the resorption in endochondral ossification spares more calcified cartilage (Muller, 1858; Lubosch, 1924) and may proceed slowly; and birds and reptiles have more ossifications in their tendons and more sesamoids, e.g., Schaffer (1902b), and Haines (1940).
    Third, reptilian CB II has so consistently been taken as evidence of chondro-osseous metaplasia that an overview of it can also act to introduce metaplasia as the first of several general issues raised by CB II.
    Fourth, comparisons of endochondral ossification can throw light on the phylogenetic and biomechanical significance of differences in the sites, pace, and architecture of cartilaginous bone formation (Parsons, 1905; Haines, 1938a).

    In the enchondral bone formation of chick, frog, and salamander, more cartilage matrix calcifies, and more of this matrix is left unresorbed, than in mammals (Muller, 1858), as confirmed by Katschenko (1881), Eggeling (1911), Haines (1938a) and others. Thus, calcified cartilage with intact cells is left not only in a sub-articular position, but cartilaginous islands remain for long periods in the diaphysis and in secondary ossification centers (if formed); see, for example, Figure 29 of a reptile's proximal ulna (Haines, 1969), and Figure 5 of Haines (1940).

    The amount of residual mineralized cartilage, already appreciable in reptiles, can be increased experimentally. Belanger, Dimond, and Copp (1973) injected turtles (Chelydra serpentina serpentina) with calcitonin, with or without parathyroid extract, and confirmed with microradiography that the hypertrophic cartilage plate and an abnormal epiphyseal cartilaginous appendage were mineralized.
    In normal hens cartilage cells remain in the wide epiphyseal spicules (Lubosch, 1924), and their number increases in cockerels given estradiol (Salomon et al., 1947), and chicks made magnesium-deficient (Chou et al., 1979). Among the agents causing the retention of chondrocytes in mammalian metaphyseal trabeculae are fluoride (Figure 13 of Kameyama, 1974) and dichloromethylene diphosphonate (Schenk et al., 1973).

    Does such enduring mineralized cartilage experience a metaplasia into a tissue closer to bone? Muller thought that, although the lacunae became smaller and uneven, the cells did not become true bone cells, but he did not absolutely rule out a very minor metaplastic contribution to the total osteogenesis of a bone.

    Katschenko (1881) claimed two modes of ossification in Anura: endochondral new bone formation and a metaplastic ossification of calcified cartilage. More metaplastic bone developed in the pelvis and coracoid bone than in long bones. In the metaplasia which followed the calcification and partial resorption of the cartilage matrix, the chondrocytic capsules stained more with carmine than hematoxylin, then the carminophilia spread out further into the matrix. The lacunae became uneven, angular, and later stellate and, he believed, developed communicating processes.
    However, his Figure 12 shows the "bone" formed in this metaplastic manner as distinct from that laid down by osteoblasts. The process involved the margins of the cartilage trabeculae before their interior regions, and could start with indivdual cells in a circumscript way or involve an extensive seam in a more diffuse kind of metaplasia.

    On the other hand, Klintz (1911), in salamander, and Tretjakoff (1929), in frog, found no evidence for such a metaplasia. Tretjakoff noted that femoral ossification took place from perichondral or medullary connective tissue at the expense of uncalcified cartilage. Only later was the extensively calcified cartilage of the epiphyses resorbed partially and replaced by bone. This occurred both in the epiphysis proper and where the cartilage extends into the bony tube (Tretjakoff, 1929, Figure 7). In both sites, the bony trabeculae held substantial spicules of cartilage. The latter and the persisting subarticular calcified shell are CB II (hyaline).

    Schauinsland (1900) described a process in the developing vertebra of the reptile, Sphenodon, as a Kontaktmetamorphose. An ossification or, as he preferred, a calcification, spread into the cartilage from the bony plate that had established itself ventrally. He viewed the event as more than a simple deposition of calcium salts, because the matrix lost its affinity for hematoxylin in favor of indigocarmine and eosin.
    This occurred elsewhere where bone ensheaths cartilage, as in the ribs, skull and extremities, and, to a lesser extent, in the carpals and tarsals. He maintained that if one can judge by the staining, the cartilage does not merely calcify but becomes like bone. Except in the vertebrae, this metaplastic bone was itself later resorbed.

    "Sclerosed cartilage" was a new name for the metaplastic bone in Urodela, introduced by Eggeling (1911). In his extensive descriptions of ossification in reptilian extremities, Heidsieck (1928) continued the use of his mentor's Knorpelsclerose for the apparent conversion of cartilage to bone. His fullest account was for the gekko, Platydactylus guttatus, in which he found sclerotic cartilage in the humerus, radius, ulnar patella, and carpals. At the boundary between hyaline cartilage and endochondral bone, under the humeral epiphysis and the apophyseal center of the major tubercle, the cells had the appearance of either shrunken or still large chondrocytes, but lay in a matrix staining like bone.
    This configuration he also saw more peripherally between endochondral bone and bone laid down by the periosteum (Heidsieck's Figure 10). Following Zawisch-Ossenitz (1927), at first he attributed this peripheral chondroid bone to an earlier activity of the periosteum, but he decided against calling the tissue ossified. He argued that the genesis and fate of the tissue were special, and Zawisch-Ossenitz had denied such cells a true chondrocytic nature, calling them instead "pseudocartilage" cells. But from his eventual choice of Eggeling's Knorpelsklerose, it appears that Heidsieck's thinking on the origin of the peripheral tissue changed against periosteal deposition and in favor of a mineralization of cartilage.

    Thus, he divided the "sclerosis" into two categories: an inner sclerosis, involving hyaline cartilage, i.e., CB II (hyaline), and an outer sclerosis, whose initial tissue is either hyaline cartilage or fibrocartilage (leading to CB II fibro), as at the insertions of ligaments and joint capsules, and at the sides of tendons abutting the epiphyses. For instance, the radius had more outer sclerotic cartilage than the humerus.
    Remnants of outer sclerotic cartilage could lie both deep in the periosteal bone and at the surface under the periosteum. A basophilic tidemark marked off subperiosteal sclerotic cartilage from the overlying connective tissues. Some cells in the sclerotic regions appeared to be degenerating; others, more towards the middle of the diaphysis, looked osteocytic. The sclerotic cartilage (chondroid bone II) in places merged indistinctly into periosteal and endochondral true bone.

    Haines (1969) reviewed his own (1941) and others' work on reptilian osteogenesis. Among his points these are relevant here.

    1. Whether or not a secondary ossification center forms (Haines, 1938a), accompanying the slow-paced ossification in reptiles such as the Crocodilia and Testudines are large calcified hyaline cartilage remnants that appear to become bony (his Figure 9), with cells transitional between chondrocytes and osteocytes. Such a metaplastic change also occurs in the axial skeleton, and in Ophidia.
    2. Where secondary epiphyseal osseous centers develop, e.g., in the lizard Agama agama, the union of the epiphysis with the shaft is by calcified cartilage, which may constitute the last phase of growth and not be succeeded by bony closure. This would be an exaggeration of the use of CB II (hyaline) to first unite epiphyses generally (Haines, 1940, 1975).
    3. Sesamoids are common in lizards, and some may incorporate mineralized fibrovesicular tissue (1969: his Figure 37), i.e., CB II (Fibro), as do tendons' attachments to bone (his Figure 15).
    4. As he had earlier (Haines and Mohuiddin, 1968), he employed the names metaplastic tissue and metaplastic bone, which obscure the differing origins of fibrous and hyaline chondroid bone II.
    5. His conclusion on chondroid bone deserves quotation: "Metaplastic tissue forms much of the surface of mature mammalian bones, but is thin, being restricted by erosion and replacement. In lizards it forms a larger part of the skeleton, especially of the epiphyses and sesamoid bones, and its abundance would allow critical study by modern methods."
    There are many situations where the cartilage abutting another tissue takes on an appearance which, in combination with its intermediate position, has encouraged an interpretation of metaplasia. Examples pertinent to CB II are when endochondral resorption is incomplete: in the anuran and reptilian bone just discussed; in rickets (Chapter 23); in the rat's persisting epiphyseal plates (Dawson, 1929); in the epiphysis of the chick, where in one sentence Fell (1925) suggested that some calcified cartilage is transformed directly into bone; and in the permanent cartilages such as the tracheal rings. Other situations are at insertions and around vascular canals in various cartilages.

    The Patella and Cartilage Canals
    Not only is the patella a site of insertion, but the manner of its internal ossification is of interest, since this has commonly been held up as an example of a metaplasia of hyaline cartilage to bone. By inference, this makes the patella a site of chondroid bone while the transformation is underway. Close scrutiny of the paper concerned (Carey and Zeit, 1927) is therefore in order. Carey brought to this study his belief that he (1922) had seen metaplasia in the cartilage underlying the bony collar of the femoral shaft.

    The authors observed that the dog's patella develops as a hyaline cartilage penetrated by numerous vessels. At 61/2 weeks of age, the vessels around the ossification center are enclosed in a broad rim of eosinophil matrix, the cells of which have the typical morphology and structural arrangement of cartilage cells. Noting that the matrix stains similarly to osteoid matrix, Carey and Zeit believed that cartilage was experiencing a metaplasia into osteoid that would later calcify to become bone. (In other regions they saw hypertrophic chondrocytes, basophil calcified cartilage and the other events of a typical endochondral ossification.)
    Judging from their figures and description, it appears that Carey and Zeit mistook the usual eosinophilia of the matrix around cartilage canals (my Figure 11) (e.g., Stoss, 1918b; Lubosch, 1924; Stump, 1925) for a sign of osseous metaplasia. That their bibliography included no sources on cartilage canals, e.g., Langer (1876), Bidder (1906), Eckert-Mobius (1924), strengthens the suspicion that theirs were not true examples of chondroid bone and metaplasia.
    Fig. 11

    Such sources would have informed them of another controversy involving cartilage canals. Hintzsche (1928), one of their contemporaries, interpreted the eosinophilia and fibrillar nature of the matrix in the vicinity of canals as a sign that cartilage was turning into collagenous connective tissue. This early stage in a process of vascular invasion supposedly involved a dissolution of cartilaginous ground substances (Stump, 1925). On the other hand, Pascher (1923), Eckert-Mobius (1924) and Haines (1933, 1937, 1974) rejected the idea of a chondrolytic penetration by vessels, and regarded the deficiency of chondromucin common to both around the canals and the matrix just under the perichondrium as evidence for a process of inclusion, whereby perichondral vessels became enclosed within canals as the cartilage grew appositionally. However, Kugler et al.'s (1978, 1979) electron microscopy gives an impression that chondroclasts erode the canals; the pattern of mammalian canals cannot be accounted for solely by a process of passive inclusion (Levene, 1964); and in the well canalized avian epiphysis there are signs that "vascularized mesenchyme" (Lufti, 1970) erodes cartilage in the construction of the canals.
    Labelling with tritiated thymidine to show dividing cells, Shapiro (1998) finds that there are extensive mitoses throughout the rabbit epiphyseal canal tissue, but few in the surrounding cartilage, so demonstrating that an active vascular invasion is taking place.

    Zawisch-Ossenitz (1929b) appears to have been misled by Carey and Zeit's (1927), and Hintzsche's (1928) reports on this special peri-canalar cartilage matrix. She considered the acidophil tissue that borders cartilage canals where bone is absent, as seen by Hintzsche (1928) and others, to be chondroid bone. But this eosinophil matrix has no apparent close relation with bone; thus, although collagenous, it has not been shown to be calcified. Haines (1975) and others familiar with canals regard it as proteoglycan-poor hyaline cartilage, not as chondroid bone.

    The circumstances become more involved when new tissues develop from the canal's cells. Bone is common, but Zawisch-Ossenitz (1929b, Figure 50) saw a canal penetrating to the femoral marrow cavity to be lined by chondroid bone. This tissue was of the same type as she had seen under the "telodiaphyseal" periosteum (see Chapter 9, this volume), and it included cells ranging from degenerate Pseudoknorpelzellen to "true young chondrocytes." Since new cartilage can be deposited within canals in addition to or in place of bone (Hintzsche, 1931), she viewed the chondroid bone as another example of the ability of the germinal cells of canals to form intermediates as well as pure tissues.
    Hintzsche and Schmid (1933), in commenting on her observations and their own in canals, took the same line, and cautioned that this formation of an in-between tissue did not constitute a metaplasia. Gray and Gardner's (1969, Figure 8) example of bone forming in a cartilage canal of the human fetal humerus is a formation distinct from the cartilage lining the canal. The nodule actually appears in large part to be made up of chondroid bone 1, with vesicular cells in a sometimes dark matrix.

    Another solution to how to introduce vessels into epiphyseal cartilage is solved for echidna by a single large "endochondrium"-lined cavity of the momotreme echidna (Thorp and Dixon, 1991).

    Tracheal Cartilages
    Occasional observers of the bone in airway cartilages have suggested that some or all of it forms by a direct metaplasia of cartilage, as in the bird's trachea. Schaffer (1888) cited Gegenbaur as a proponent of metaplasia there, and in antlers and horns, although Gegenbaur assigned his osteoblasts the task of forming all other bone. Mjassojedoff's (1919) report, at a meeting of the Russian Pathological Society, of a metaplasia of cartilage to bone in the hen's trachea appeared as a German abstract. However, Nevinny's (1927) own observations in man, and the theoretical improbability of Mjassojedoff's claim that chondrocytes could extend their processes out to the point of anastomosis, led Nevinny to reject metaplasia as a source of tracheal bone. On the other hand, birds share with other non-mammalian vertebrates a mode of long-bone endochondral ossification that leaves more calcified cartilage unresorbed (Miiller, 1858). The bone in the avian trachea may form endochondrally, but the resorption might spare sizable remnants of cartilage to transform to a more bone-like state.

    In human tracheal cartilage the moderate frequency of ossification was recognized much later (Amprino and Bairati, 1933a; Linzbach, 1944) than for birds. Linzbach's paper probably was the first report of metaplastic ossification within human tracheal cartilage. He saw not only that the vessels within aging tracheal cartilage increase in number, but also that they were occasionally present before degeneration had set in, for example his Figure 7 shows a cartilage canal in a six-year-old boy. Thus, there is no absolute difference between the costal cartilages which have numerous canals (Bohmig, 1929) and younger tracheal cartilages.

    Around the vessels in healthy and abnormal cartilage, Linzbach observed that the matrix was eosinophil, and either the perivascular connective tissue cells appeared to be becoming chondrocytes or the reverse was taking place. Where the cartilage had degenerated, chondroclasts and vessels destroyed it and bone was laid down in a conventional endochondral way.
    Bone was also deposited on healthy cartilage. Linzbach interpreted the loose cellular tissue within cartilage canals and the surrounding eosinophil matrix as signs of a vascular destruction of normal cartilage that freed chondrocytes to become osteoblasts and form the new bone, what he termed "a pure osseous metamorphosis of healthy cartilage."
    With respect not to the cells, but the matrix, he considered the idea that the vessels also acted to transform the adjacent cartilage matrix into osteoid, but admitted that the preparations were inadequate evidence. Furthermore, if eosinophilic cartilage matrix and osteoid were the same, he admitted having no explanation why the normal peripheral (sub-perichondral) zone of a cartilage, also eosinophilic, did not mineralize and become bone.

    Linzbach's tentative conclusions seem to be another example of misinterpretation of the nature of the tissues in and around cartilage canals, along with a failure to distinguish canals from resorption cavities in calcified or degenerated cartilage. The proximity of normal hyaline cartilage, eosinophilic cartilage, immature connective tissue, osteoid (if present), and bone need not mean that any or all of these are participants in a sequential transformation.

    Sites of Insertion
    The same caveat - a spatial succession need not reflect a temporal one - applies here. Nevertheless, there are factors making the tissue picture at insertions more convincing of a metaplasia: the regularity of the sequence in which the tissues lie; the slow pace of transformation; and the restricted role of endochondral ossification.
    The scene is complicated by the great variety of tissues and processes participating. Thus, tendons can insert into bone and cartilage, be calcified and non-calcified, chondrocytic and fibroblastic. Chondroid, hyaline cartilage and fibrocartilage may mineralize, andperhaps turn into bone; but other bone forms from osteoblasts endochondrally, with an accompanying destruction of cartilage.

    One paper attempting to chart the many events is Knese and Biermann's (1958), and several of their observations have received confirmation in the reports of other anatomists.

    Knese and Biermann described four kinds of chondroid bone at insertions and drew metaplastic conclusions from all of them. Around young insertions a cellular blastemal tissue either formed a CB 1, made directly in that condition, or formed a "subperiosteal" chondroid which transformed into bone, implying an intermediate phase as chondroid bone. These forms and the transformation of tendon into fibrocartilage are mentioned in Chapter 9 as expressions of secondary chondrogenesis.

    After the neonatal period, as the insertion structure matures, relationships and tissues change and some of the early tissues, such as chondroid and palisading cartilage, disappear, to be replaced by other tissues. For example, where the patellar ligament inserts into the tibial tuberosity in the 25-year-old man (Knese and Biermann, 1958, Figure 19) the ligament with its large cells is crossed by a tidemark, on the bony side of which is an area of CB II (fibro).
    Knese and Biermann claim in the figure legend that in this tissue there is a transformation of the matrix to a bony condition (Umbildung der Interzellularsubstanz in den knochernen Zustand). The same mineralized region of the tendon inserting into the radial tuberosity is designated in their Figure 26 (my Figure 8) as Einstrahlungsknochen mit knorpelahnlichen Zellen (in-streaming bone with cartilage-like cells), elsewhere as parallel faseriger chondroider Einstrahlungsknochen - both clearly a characterization of CB II (fibro).

    At some other sites they made a better case for metaplasia, but a classification of the accompanying chondroid bone with either CB I or CB II is difficult. In their figures the CB is not demarcated from cartilage by a clear tidemark. A moderately cellular zone, acting as the source of the tissue, forms cartilage, from which the chondroid bone then derives. Thus, although its degree of mineralization is unclear, this CB seems to be altered cartilage on its way to bone rather than a direct blastemal deposit.
    They distinguished it from CB II above by calling it metaplastisch gebildete chondroide Knochen. It appears in their Figures 24 and 25 (my Figure 12) of one- and five-year-old children's radial tuberosities, in Figure 29 of the five-year-old's lateral femoral condyle (where it is called Knorpel-Knochen durch Metaplasie gebildet), and in reduced amount in Figures 30 (my Figure 13) and 31 of the 25-year-old's lateral femoral condyle, where the cartilage cells "slowly lose the hallmarks of chondrocytes and gain those of osteocytes; they undergo metamorphosis."
    Fig.12

    In their conclusion Knese and Biermann brought together the results of all their observations favoring a metaplasia:

    1. There is continuity between tendon or ligament, cartilage (fibro- or hyaline), and bone.
    2. The character of the matrix changes, but in the absence of any spaces in which osteoblasts could have made the bone. (Resorption cavities lie deeper or are well spaced along the transforming front, as in Figures 28 and 29, Knese and Biermann, 1958.)
    3. The cells lie in the same patterns in the cartilage and in the bordering bone, and in certain locations the deeper cells appear to have become more osteocytic.
    The combined effect of the metaplastic change and the endochondral ossification spaced out at intervals across the front was to extend the bone-cartilage boundary further out into the ligament or tendon,

    In their discussion, Knese and Biermann wavered between regarding the ostensibly metaplastic change as: a metaplasia of an established tissue - cartilage; a final differentiation of an unfulfilled intermediary or provisional tissue; or a sequential expression of the pluripotentiality of the skeletal cells within the insertion, i.e., a modulation.

    Amprino is another microscopist who thought CB II undergoes a metaplasia to bone. Thus, in their study of mineralized permanent cartilages, Amprino and Bairati (1934) suspected one pubic fibrocartilage was experiencing a direct conversion to bone, and Amprino and Catteneos (1936) described tendons as ossifying both by substitution and by a direct metaplasia via calcified cartilage. Ogden, Hempton, and Southwick (1975) believed a transformation of fibrocartilage to bone occurs at the human infant's tibial tuberosity, but they named the process "intramembranous ossification."

    Conclusions
    A couple of reports of a transformation of CB II to bone have been dismissed as misinterpretations of the nature of the cartilage around canals, but many other instances of suspected metaplasia remain. The latter include the unresorbed calcified epiphyseal cartilage of Reptilia and Anura, the late-closing epiphyseal plates of rats, and the slowly advancing bone-mineralized cartilage boundary of insertion structures, and maybe synchondroses.
    In these situations the suspicion of a metaplasia is based mostly on changes in the hardness and staining reactions of the matrix. Although the cells are sometimes described as becoming small and dark, they lack processes, and investigators such as Matschinsky (1892), Weidenreich (1923b) and Haines and Mohuiddin (1968) have hesitated to call them osteocytes.

    The chemical implications of a metaplasia of tendon to cartilage, and of its mineralized derivative, CB II, to bone are many. For example, did the tendon cells as they became chondrocytic modify their synthesis to produce cartilage-specific proteoglycans and type II collagen? There are reports of the manufacture of a fine-fibered collagen in the pre-mineralizing region of tendons (e.g., Becker, 1971).
    When and if the deeper aspect of this zone of CB II transforms to bone, do the cells then revert to bone-typical synthetic products? How far away from the chondrocytic cells can their products spread and take effect? And are these effects on mineralization, on collagen, or on both? And with what mechanical consequences? How do the cells keep the width of the mineralized zone in articular cartilage and insertion-structures limited? And in what way does this control fail in such diseases as ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis (Resnick, 1977), where mineralization spreads into ligaments, tendons, and synchondroses. (The hazard of a general mineralization throughout one's soft connective tissues would seem to call for very elaborate chemical safeguards comparable to those involved in preventing a disastrous intravascular clotting of the blood.) What is the nature of the mineral? Mohr et al. (1979) believe that in the intervertebral disk it is calcium pyrophosphate.

    For chondroid bone II arising in unequivocal cartilage, e.g., costal or articular, there are three issues to be kept apart.
    What happens in a cartilage when it mineralizes to become CB II?
    What occurs if mineralized cartilage transforms into bone?
    Do either of these changes (or both together) justify the name metaplasia?
    The few biochemical questions posed above are as yet unanswered. Until more details of the changes in CB II are known, labeling them as a metaplasia is more a matter of cloaking ignorance than of precise designation. Of course, if one follows Weidenreich (1923c) and Haines and Mohuiddin (1968) and calls mineralized cartilage 'bone', then ipso facto cartilage has become bone and metaplasia has taken place.

    CONFUSIONS BETWEEN CHONDROID BONE I AND II

    The first significant misunderstanding arose with Schaffer's (1888) use of chondroider Knochen for young ovine mandibular bone with large osteoblasts and basophil lacunae. Zawisch-Ossenitz (1929a,b) continued the practice for fetal long bones, but also applied it to particular regions where some cells were more chondrocytic, her Pseudoknorpelzellen. In such places a true kind of chondroid bone existed: her fiber-free to fine-fibered variety.

    The third entity that she named chondroid bone, at second-hand, was the acidophilic cartilage around canals (Hintzsche, 1928). These three kinds of CB, two of them "false," associated with Zawisch-Ossenitz make for confusion when authors equate other tissues with her "chondroid bone." Weidenreich (1930), for instance, held the reptilian sclerotic cartilage (CB II) of Heidsieck, Eggeling, and others to be similar to the "chondroid bone" described by Schaffer in the mandible and Zawisch in the guinea pig's long bones.

    This kind of confusion still persists, but is based additionally on Orban's (1944) illustration of chondroid bone I at an alveolar process. Haines and Mohuiddin (1968) referred to Orban in their paper on articular and tendinous "metaplastic bone" (CB II). Of the trans-septal part of the kitten's periodontal ligament, they wrote, "the deeper part of this ligament is progressively mineralized to form a peculiar, metaplastic tissue, the 'chondroid bone' of Orban (1957), whose capsules and coarse fibers can be stained with silver," (Haines and Mohuiddin, 1968, Figure 16).
    Were Haines and Mohuiddin correct in thinking that the crest grows by the mineralization of a ligament as, say, the spread of calcification into a ligament at the knee? In Chapter 11 (see p. 209, Cartilage on Alveolar Processes) I argue that the growth of the alveolar crest is by the action of a chondrogenic zone like that of the mandibular condyle, but quite unlike that of older ligamentous insertions. Hence, the early alveolar crest is a site of CB I, not CB II.

    Bones may be the site of both kinds of chondroid bone, although probably not at the same time. This development has been observed in the long bones and seems to occur in the mandible. The deepest part of the articular cartilage of the older mandible is well known to be mineralized, e.g., Weinmann and Sicher (1964) and Irving and Durkin (1972), and is CB II. Earlier in its development, the mandible is the seat of several secondary cartilages, each of which may be merged with bone through a greater or lesser amount of chondroid bone I.
    One site in particular needs more attention. Baume (1962b) briefly reported secondary cartilages appearing at the site of attachment of the aponeuroses of the masticatory muscles. Chondroid bone I would be expected to join these cartilages to the mandibular bone, but since the sites are insertion structures, the later development of CB II (fibro) is likely when the aponeuroses become denser and more heavily loaded.

    THE CELLS AND MINERALIZATION

    Chondroid bone II (fibro) may constitute an artifical distinction within the mineralized collagenous connective tissues, since seemingly non-cartilaginous tissue experiences calcification in some tendons and the fibrous growth plate (Smith, 1962b; Badi, 1972a), and during conventional osteogenesis. The observation that mineralization does not require the tendinous cells to be chondrocytic, if true, leads to the question: Are the cells of CB II unimportant for mineralization, as Weidenreich (1923b) implied? The form of the cells may well be less important than what they produce. For example, Moss (1969) found "no evidence for participation by any variety of cartilaginous tissue" in the dermal sclerifications of reptiles, but did note histochemical signs of a probable contribution of sulfated mucopolysaccharides to the calcification.

    Chondroid bone II has non-hypertrophic chondrocytes, e.g., in subarticular cartilage (Haines and Mohuiddin, 1968) or in "small-celled" fibrocartilage (McLean and Bloom, 1940; Badi, 1972b), but their lack of gross enlargement may not keep them from behaving like epiphyseal mineralizing-zone cartilage cells. Thus, Yamada (1976) applied electron histochemistry to the insertion of the Achilles tendon into the rat's calcaneus, and showed that matrix vesicles bearing alkaline phosphatase are present in the mineralizing fibrocartilage and seem to be derived from non-hypertrophic chondrocytes. This finding suggests an active cellular control of mineralization.

    Some observations raise doubts of an exclusively cellular initiation of mineralization. First, matrix vesicles are present around the chondrocytes of elastic cartilage that is uncalcified, and unlikely to calcify (Cox and Peacock, 1977), but these vesicles lack alkaline phosphatase and ATPase (Nielsen, 1978). Second, in mineralizing avian tendon, the mineral is concentrated in the gap regions of the staggered collagen molecules (White et al., 1977), denoting some influence by the collagen on the deposition of crystals. With electron microscopy, Cooper and Misol (1970) saw a pattern to the crystals on the collagen of tendon insertions of dogs; Becker (1971) made similar observations in rats.
    Sela and Boyde (1977), from the pattern of mineralization in osteosarcomas, also emphasize the role that collagen plays in mineralization. In aging rat cartilage, Bonucci and Dearden (1976) saw mineral within numerous matrix vesicles, perhaps derived from verdammert cells, but could not directly relate the vesicles to the matrix calcification.

    Materials made by the cells and released will interact according to physicochemical laws with each other and blood-borne substances, but the matrix can hardly be said to take on a life of its own. How alive connective tissue matrices are was a question asked from time to time in the German literature, e.g., Weidenreich (1923d). The idea persists in Knese and Biermann's (1958) conception of a Weiterdifferenzierung der Interzellularsubstanzen involved in the mineralization and suspected changes in the collagen at apophyseal insertions.

    The spread of mineralization from existing bone into a tendon or articular cartilage may give an impression that the softer tissue - cells and matrix - is being overwhelmed by an encroaching neighbor beyond its control, as expressed by Schauinsland's (1900) Kontaktmetamorphose. However, the extent of the spread is normally limited, although in avian tendons, extensive. Mineralization also is found independently of the skeleton within ligaments and tendons (Weidenreich, 1930; Engel and Zerlotti, 1967) where an invasive spreading of calcification could not have initiated the process. Moreover, Cooper and Misol (1970) proposed that the calcification of insertions might even serve to block the diffusion of mineral into the tendon or ligament.

    The limit on calcification is evident in articular cartilage. Green et al. (1970) found the calcified cartilage layer to have a very consistent width of 134 =/- 7 um (SE) in the patellae of individuals varying greatly in age, 4-74 years; and Benninghoff (1925) drew attention to the relatively constant width of the calcified layer regardless of the thickness of the non-calcified articular cartilage above it. Without citing Benninghoff, Muller-Gerbl et al. (1987) came to the opposite conclusion from measurements on the thickness of the calcified layer and of the directly overlying unmineralized cartilage in human femoral heads. They noted a close correlation between the two thicknesses, and argued that mechanical factors influenced both values.
    Another way of looking at the calcified articular cartilage is as one hard part of the subchondral plate (Milz et al., 1995). In the patella there is only a low correlation between the thickness of the plate - subchondral bone + calcified cartilage - and the unmineralized cartilage above.

    The width of the mineralized zone can be altered by experiment and is subject to genetic influence. As an example of experimental alteration, Tarsoly and Mateescu (1972) gave frequent high doses of testosterone to male rats. The femoral articular cartilage narrowed drastically, and at the same time proportionately more of the cartilage reacted positively for alkaline phosphatase, and calcified.
    As for genetic influence, in Figures 5, 6 and 7 (Silberberg, 1973) of the vertebrae of aging dwarf mice (strain dwdw) each cartilaginous vertebral endplate appears to have two separate zones of calcification, one by the bone, the other facing the abnormal disk. Of course, the width of the calcified layer reflects the rate of resorption as well as the extent of calcification.

    There is much to be learned about the role of cells and agents in the mineralization and endurance of matrix, and study will need the application of such techniques as tetracycline labeling and microradiography (Lemperg, 1971a), microchemistry (Pita and Howell, 1978), and elemental analyses by microprobe (Ali et al., 1977) to these and other sites of CB II.

    FUNCTIONS OF CB II

    Several purposes to be served by the mineralized regions within cartilages have been put forward.

    Growth
    The calcification of articular facets, tendons, and ligaments (whether chondroid or not) may add permanently, albeit slightly, to the hard skeleton (Weidenreich 1930; Petersen, 1930; Biermann, 1957; and Haines and Mohuiddin, 1968). The calcification is therefore a supplementary mode of bone growth. However, does the mineralized epiphyseal cartilage contribute to longitudinal growth? Dawson (1929) thought that if proliferation preceded the apparent transformation to bone in the older growth plate, some growth in length might occur.
    A similar proposal is Johnson's (1964) for the deep calcified layer of articular cartilage. Johnson followed Ogston (1876, 1878) in suggesting that articular cartilage regenerates continually by a shedding of superficial tissue, a proliferation of deeper chondrocytes, and a transformation of its calcified cartilage to bone, thus very slowly adding to the bone's length. But what Ogston actually proposed, for non-arthritic cartilage, was that the calcified cartilage matrix dissolved, and it was only its cells that became first marrow cells, then the osteoblasts which, he supposed, formed most of the trabecular bone in the epiphysis.

    Lane, Villacin, and Bullough (1977) measured the vascularity and extent of resorption in the articular calcified cartilage of human femoral and humeral heads. In both sites, resorption decreased from adolescence until the sixth decade of life, whereupon it increased. The authors endorsed Ogston's idea that this destruction is a part of a process of slow endochondral growth, but one serving, in their view, more a remodeling or reshaping of the cartilage and its subjacent tissues; perhaps having the effect of increasing joint congruence to the detriment of nutrition, lubrication, and the distribution of loads.

    There are really two separate issues concerning the significance of resorption for growth. First, the eroded spaces in the subchondral bone and calcified cartilage may be filled in with bone to some extent, thus constituting a local but internal growth. Second, the erosion might be tied in some way to a) an encroachment of calcification further into the cartilage, and b) proliferation and growth in the non-calcified part of the cartilage, but such a connection has not been shown conclusively for man after adolescence.

    The reduplication of the tidemark has been taken as indicating that "continued abortive growth activity goes on in the osteochondral region in the adult years" (Green et al., 1970), but the duplication was not evident in all adult patellas. The authors did not keep the above two issues distinct, for they wrote of "the possibility that insidious growth in length of the bones may also come about at the expense of the cartilage, through the advancing endochondral calcification and ossification." But, if the process is at the expense of the cartilage, this view implies that it is not itself growing. If the non-mineralized cartilage is not growing, the bones will not lengthen, insidiously or overtly.
    In the older (nine-month) rabbit, Lemperg's (1971a) tetracycline labeling indicated a slow advance of calcification into the cartilage, but Mankin (1963) and Havdrup (1979) found no sign of proliferation within the articular cartilage after six months of age, when tritiated thymidine was injected into the knee. Taken together, these findings suggest that there is no growth in the more superficial cartilage, to which the process of calcification of the cartilage could be linked.

    Mechanical Roles
    The bonds between soft and calcified zones in articular cartilage, and between subarticular bone and the calcified cartilage, are both powerful. Considerable shearing stress detaches the cartilage at the tidemark (Sokoloff, 1973) rather than at the chondro-osseous junction. The only known weakness of the osteochondrat boundary is in the humerus of some large dogs (Knecht et al., 1977). Fast-growing animals such as pigs are prone to degenerative cracks and cysts in the calcified cartilage layer (Reiland, 1978).

    Gebhardt (1903) and Petersen (1930) thought that mineralization across the subarticular bone-cartilage boundary kept the junction from being unstable. But while the fibrils run across the tidemark in the cartilage, with light microscopy there appears to be a discontinuity at the osteo-chondral junction (Benninghoff, 1925). According to him the strength and resistance to shear lie in fine interlocking projections and the mineralization of both tissues. While this may be true on the macroscopic scale, scanning and TEM revealed to Hough et al. (1974) an interweaving across the osteochondral boundary of fibrils originating separately in bone and cartilage.
    Within the boundary, the fibrils in mature individuals (man, squirrel monkey, and dog) splayed out their component microfibrils and became more disorderly, perhaps accounting for the absence of birefringence. The junction in mouse, guinea pig, rabbit, and child was only sparsely fibrillar. Furthermore, within the calcified cartilage, the murine fibrils were fewer and thinner than those in bone and lacked a periodic cross-banding, whereas the human fibrils were thick, plentiful, and clearly cross-banded (Hough et al., 1974, Figure 3).

    The tidemark at the superficial boundary of the articular calcified cartilage (CB II) is unlike the osteo-chondral line of juncture in its reaction with Bodian's protargol-silver, collagen birefringence, and higher content of sulfur (Hough et al.). The tidemark reacts for lipid and the enzymes alkaline phosphatase and ATPase (Dmitrovsky, Lane, and Bullough, 1978). At the tidemark, the collagen fibrils form a distinctive network visible in scanning electron microscopy (Hough et al., 1974; Redler et al., 1975). The latter authors concur with the idea of Greene et al. (1970) and others that the tidemark region has the task of tethering the uncalcificd collagen, so as to prevent shearing at the mineral boundary. (Minns and Steven (1977) have also viewed fractured calcified articular cartilage by SEM.)

    The radial orientation of the fibrils of the calcified layer troubled Gebhardt (1903), because they seemed to be oriented parallel with the direction of pressure on the articular surfaces. He suggested that as the fibrils were forming, and before mineralization, the pressure on the deformable cartilage produced a secondary sideways pressure in the cartilage where this abutted the hard, less yielding bone. It was this sideways pressure that gave the fibrils of the mineralized layer their orientation. This notion was an early attempt to reconcile the fibrillar architecture of skeletal tissues with Roux's ideas of the roles of pressure and tension in skeletal histogenesis and morphogenesis (Chapter 4).

    One special site of enduring calcified cartilage lies centrally in the epiphyses of certain lizards, such as Phyllodactylus porphyreus (Haines, 1941). The secondary center never ossifies, so that CB II (hyaline) there takes on whatever mechanical role secondary ossification centers might perform (Parsons, 1905). Haines (1938a) conjectured that the separation of articular and growth functions in an epiphysis makes the cartilage so wide that it needs internal reinforcement.

    Gebhardt (1901b) proposed that the bone-like transformation of tendons and ligaments at their regions of insertion achieves a special intimacy of attachment, analogous perhaps to the use of an intervening third layer of metal to fasten two dissimilar metals. (The layer of calcified cartilage between an articular surface and subchondral bone was to serve a like function.) Furthermore, the rigidity of the mineralized section of the tendon was significant mechanically in another way, by altering the angle between the tendon and the bone and thereby partly directing the tensional loading. Redler et al.'s (1975) mooted reduction of shearing forces echoes Schneider's (1956) conception of the role of apophyseal tendon insertions. He believed that a direct fastening of a tendon into bone would be especially vulnerable to shearing and bending forces.
    But Knese and Biermann (1958) thought of the cartilaginous region of the tendon as a Dehnungsbremse - a stretching brake - acting in the following way. As the tendon is tensed it tries to narrow in width, but this narrowing is resisted by the cartilage cells between the fibers, thereby reducing the amount by which the tendon in the vicinity of the bone can be stretched. They did not mention how the subsequent mineralization of the fibrocartilage would affect this action.

    Knese and Biermann (1958) distinguished zugfest or tension-resistant cartilages from druckfest or pressure-resistant ones: a dichotomy which, they suggested, corresponds approximately with the secondary-primary division of Schaffer, although not all secondary cartilages were zugfest. The cartilage of insertion structures was to be secondary and zugelastisch.
    But is one justified in linking a particular tissue to just one kind of mechanical load? Knese and Biermann (1958) themselves answered this by noting that hyaline cartilage, at those apophyses which have it, resists tension, but in other sites hyaline cartilage is considered to be more an adaptation to pressure.
    However, to take the matter a step further, is it even possible to separate tension and pressure or are the two, in practice, inseparable? A difficulty arises here because the discussion (and any measurement, e.g., Tipton, Matthes, and Martin, 1978) starts with macroscopically large pieces of tissue, but there is a temptation to extrapolate down to the cellular level. For instance, in writing of chondroid bone at tubercles, Enlow (1962a) postulated: "Although the fibrous matrix of bone tissue in the tubercle is subject to tensile forces, the individual chondroid cells themselves do not receive direct tensile stress. To the contrary, they must be resistant to the pressure exerted on them by the surrounding fibrous matrix which is under direct tension" - in essence, the proposition of Knese and Biermann (1958).
    What is the evidence that even at macroscopically definable points on bones only tension or only pressure acts? And, can data obtained for structures of this size accurately indicate what is the actual mechanical situation of, say, a chondroid bone cell?

    An answer to the first of the last two questions can be approached by a theoretical examination of the normal dynamic use of the skeletal part. Oxnard (1971) tested the few musculo-skeletal situations where, when he had considered the full cycle of normal movement, the net influence on a skeletal region was probably tensile. He applied the deduced major forces to a two-dimensional model of each bone in photo-elastic material, of the kind used by Smith (1962a,b) to show that the fibrous parts of certain epiphyseal plates were those under tension. Oxnard concluded "that in rare cases where precise anatomical architecture is such that net tension may be present, then bone is not formed; such regions consist of collagenous structures." Where collagenous structures such as tendons came under compressive forces, these forces were, he believed, resisted by sesamoids.

    When Oxnard followed tensed collagenous structures to their attachments, he was intrigued by the regions where one knows chondroid bone II to be present. Thus he wrote, "For instance, in the localized region at the attachment of tendons and ligaments (e.g., the ossifying flexor tendons in the legs of some birds), surely net tensile forces exist. Here the answer may lie in the particular microscopic anatomy of these small bony regions, the concomitant differences in the physical properties of the contained elements and the complex microscopic stress effects produced within them during function. The physical arrangement may be such that, although tension may exist in the tendinous architecture, compression may nevertheless dominate the bony elements."
    He took this last phrase beyond the context of bony insertions to include other regions of bone in his general conclusion, "that net tension rarely exists in significantly large regions of bones during normal function." In other words, bone, and at insertions, chondroid bone, serve more to resist compression than tension, though not exclusively.
    If this is so, the tissues adapted to resisting compression are several: cartilage, calcified hypertrophic cartilage, chondroid bone (II and I), and bone. One is reminded of the remark of Gebhardt (1901a), to which Biermann (1957) drew attention, that the actual bony structure present represents one solution for the prevailing mechanical requirements, but not necessarily the only one. For instance, in the lizard's jaw even bone may be exposed directly to pressure in mastication (Throckmorton, 1979).

    Non-mechanical Roles
    While the skeletal cells and their products in manifold combinations are meeting mechanical demands, they are perhaps accommodating other constraints, for example:
    in the order in which skeletal tissues can succeed each other in time, while maintaining continuity in performing their mechanical duties;
    in how closely vessels can approach; and
    in controlling the extent of resorption, and the site's service as a mineral store.
    Concerning the second two points there is evidence indicating that chondrocytes make a factor inhibiting vascular invasion (Kaminski et a]., 1977), and that they may inhibit bone resorption (Horton, Wezeman, and Kuettner, 1978). A role of the chondrocytes of subarticular cartilage and tendinous insertions might be to prevent or delay erosion and remodeling, and possible weakening, of the tissue when it is mineralized.
    For example, when monkeys were immobilized in body casts (Noyes et al., 1974), cortical bone below the insertion of the anterior cruciate ligament was extensively resorbed, but resorption only occasionally extended into the zone of mineralized fibrocartilage. Heavy loads tore through the bone or the ligament, but the insertion itself did not fail, suggesting to the authors that the fibrocartilaginous zone was "protective" in its resistance to erosion.

    Brookes (1971) reviewed several papers on the diffusion of intravascularly injected materials that imply a non-mechanical role for mineralized sub-articular cartilage, namely as a pathway for nutrients from the subchondral vessels to the overlying soft cartilage.
    But recent evidence based on a hydrogen-washout method suggests that this route is blocked when the cartilage mineralizes (Ogata, Whiteside, and Lesker, 1978).

    Although the cartilaginous change in tendons and other fibrous tissues appears to be in response to certain mechanical demands, it may be wrong to read a mechanical purpose into any subsequent mineralization and bony substitution. Hintzsche (1931) noted the contradiction that while Erdheim (1931) suggested that stress promoted costal calcification (also Beneke et al. (1966) for tracheal mineralization), Pascher (1923) had thought that stresses from use kept calcification out of the region of the cricoid cartilage by the cricoarytenoid joint.
    In avian tendons, Amprino (1948) could see the mineralization only as weakening the tissue: a process made worse when osteonal bone replaces the hard tendon, if the bone's cement lines are a site of weakness. Thus, it is conceivable that the initial chondrocytic transformation meets a mechanical requirement, but any subsequent mineralization and osseous replacement of the fibrocartilage are triggered, inappropriately for the site, by the chemical nature of the tissue.
    Amprino (1948) proposed that the osseous substitution in the sequence is an expression of the widely used means of making the mineral in a hard tissue accessible for the general metabolism and may have no local mechanical significance. Calcification must alter the static and dynamic physical properties of cartilages and attachments, but an effect on stress-resistance is not a cause of the calcification which changed the resistance.

    SECONDARY CARTILAGES AND CB II

    Schaffer's (1930) secondary cartilages embraced those of the mandibular condyle and its articular tubercle, cartilaginous or fibrovesicular sesamoids, chondroid or vesicular tissue in the insertions of tendons and ligaments, and in the hearts and penises of certain species, and elastic cartilages - all of which have instances of mineralization and conversion to CB II. Is all CB II then derived from secondary cartilages? No. Subarticular, appendicular, spinal, pelvic, and costal calcified cartilage, that in non-mammalian endochondral ossification centers and any principal reptilian skeletal bones mistaken for sesamoids (Haines, 1969) are of primary origin.
    This twofold origin of CB II serves to reinforce Knese and Biermann's (1958) point, that the mechanical roles that CB II may play cannot be tied directly to the separate question of whether mechanical factors evoked secondary cartilage. Moreover, that a tissue may be cartilaginous for other than mechanical reasons cautions against concluding that because cartilage can withstand pressure, and is found at sites of compression, pressure brought it into existence by determining the differentiation of tendinous or other precursor cells. Knese and Biermann (1958) commented, "From the manifold possibilities of histogenetic differentiation, the relatively simple interpretation of a tissue's developing because of mechanical factors must be doubted ... among the correlative determinants mechanical ones could belong."

    Chapter 11 MAMMALIAN SKULL

    Introduction
    The mandible
    . The number of chondrogenic sites
    . Angular and coronoid cartilages
    . Anterior mandibular cartilages
    . Cartilage on alveolar processes
    . Mandibular condyle
    Introduction
    Uebergangsgewebe: Chondroid Bone I?
    Condylar cartilaginous tissues: a stable basis for comparisons?
    Persisting cartilage cells
    Periosteal bone: living cartilage boundary
    The disk of the mandibular joint
    Squamosal fossa and tubercle
    Fuchs' comparative studies
    Human articular tubercular covering
    Chondroid bone in rodent fossa
    Temporal chondroid bone: reactions and remodeling
    Maxilla and pterygoid
    Pterygoid
    Maxilla and palatine bone
    Cranial vault sutures: two kinds of cartilage?

    INTRODUCTION

    The primary-secondary dichotomy concerning the skeleton was introduced to cope with a problem arising in mammalian cranial development. Bones of the vault and face form "in membrane" rather than in cartilage as is usual for almost all other bones. Kolliker (1849), and others classified endochondral bones as primary (including those of the chondrocranium), to be distinguished from secondary bones of membranous origin. Kolliker regarded the mode of histogenesis of particular bones to be a reliable guide to homology between the bones of different species and classes. This hypothesis was soon refuted by Muller (1858), and was undermined more deeply and criticized further many times thereafter, for example, by Gegenbaur (1870), Gaupp (1903), and de Beer (1937).

    Kolliker noted that among the bones he had categorized as secondary, the mandible had cartilage at the condyle and angular process. These mandibular cartilages were not, in his view, part of the primordial cartilaginous skeleton but appeared on the developing mandible to assist in its functions. In this way the cartilages were accessory to the bone, and accessory is the name under which they, as a class, were discussed for the next fifty or so years (Low, 1909).
    While secondary as applied to bone was going out of use, since it had no basis in a bone's histogenesis, secondary gradually came to be the term of choice for accessory cartilages. Restricted to cartilage, secondary meant, in the first place, that the cartilage came into being after the first appearance of the primary cartilages of the axial and appendicular skeleton. This criterion is met by all of Schaffer's (1930) secondary cartilages, except, as Knese and Biermann (1958) point out, the clavicle's.

    Adherence to this temporal criterion resulted in Schaffer's (1930) including in the secondary category instances of cartilage, e.g., in fracture callus, and extra-skeletal and elastic cartilages, that are not always accessory to membrane-bones or, in other instances, to any bone at all.

    Any cartilage on a bone believed to form intramembranously, i.e., without cartilage, inevitably caused controversy over what proportions of, say, the mandible, developed through cartilage and by a more direct noncartilaginous ossification. Not only were the relative membranous and endochondral contributions to ossification in dispute, but how the mandibular cartilage actually ossified was, as well. Schaffer's (1888) scrupulous work stilled the contention that the condylar cartilage ossifies directly, but, for the mandible as a whole, the issues are by no means settled even today.
    Only recently has the meagerness of the contribution of the condylar cartilage to the over-all growth of the mandible been accepted (Meikle (1973b) and Sprinz (1979) inter alios). At the mental symphysis, Trevisan and Scapino's (1976) papers make it clear that there is still work to be done on the participations by the secondary and primary (Meckel's) cartilages, and the degree and nature of any metaplasia in the secondaries.

    The primary-secondary division among cartilages comes into especially sharp focus in places such as the maxilla and mandible, where in many species, primary and secondary cartilages are very close to each other, on occasion may fuse with one another (Trevisan and Scapino, 1976b), and both experience ossification. In this situation: 1) frequent histological samples of the site are needed to establish which tissue precedes another; and 2) one has to be able to distinguish cartilage from bone, to determine the mode of ossification.

    As for the first requirement, the time at which samples are taken has caused trouble in determining the exact number of cartilages accessory to the mandible of a given species. Some cartilages, such as those of the coronoid and angular processes, can be small and short-lived and hence quite easily escape notice. Timing matters in another way. Although a secondary cartilage is secondary because it appears after the primaries, these primary cartilages are mostly situated at a distance, and this temporal consideration pales beside the more pressing local one, of whether ossification preceded chondrogenesis at a particular site on the bone of interest.
    In other words, is the cartilage also secondary in its immediate context because it appears after bone has been established locally? An answer is important, since for many secondary cartilages, the cartilage very soon experiences endochondral ossification. Hence, if cartilage appeared first at the site, it would be acting as a typical primary precursor of bone. In fact, the margin by which the cartilage is secondary in this sense is very close; bone is first off the mark only by one or two days, as is seen at the rat's mandibular condyle (Duterloo and Jansen, 1969) and penile bone (Ruth, 1934; Beresford, 1975c).

    Next, distinguishing bone from cartilage in the developing skull presents several problems:

    1. Young bone has something about it of the nature of cartilage, e.g., large cells and stained pericellular capsules, and accordingly was named chondroid bone by Schaffer (1888), and in places perhaps mistaken for cartilage by Gaupp (1907) and Meyer (1849a).
    2. The cranial secondary cartilage is not exactly like primordial hyaline cartilage though it resembles primordial hyaline cartilage; for example, its matrix is meager and stains feebly, and hence has been termed unreif (Schaffer, 1888), "embryonic" (Durkin, 1972), or given the name chondroid by Zawisch (1953) and Knese and Biermann (1958). Thus, the question arises of how like and how different from hyaline cartilage is the secondary cartilage.
    3. The resolution of this last issue is hindered by the apparent existence of two varieties of secondary cartilage on membrane bones, but it can be argued that one is merely a smaller, earlier version of the other, so that the subdivision is redundant.
    4. Related to this point is secondary cartilage's development from progenitor cells whose nearby neighbors have formed bone, resulting often in a transitional tissue lying between the bone and the cartilage. When only a small nodule of secondary cartilage forms, none of it is very far from bone, and the transitional zone constitutes a greater proportion of secondary cartilage and attracts more notice than does that in the larger cartilages, such as the mandibular condyle's seen well after its establishment.
    5. While the larger secondaries are known to experience an endochondral ossification that departs in various aspects from that in epiphyseal plates (Brock, 1876), there are reports that the smaller cartilages undergo a direct transformation to bone and fibrocartilage. Chondroid bone I therefore may arise both in the course of a switch from osteo- to chondrogenesis, and during any osseous metaplasia of cartilage.
    6. Dividing the mandible and hard palate of certain animals is a cartilaginous symphysis. For example, the mandibular halves of cattle are joined by fibrocartilage and fibrous tissue which in the view of Rigier and Mensek (1968) constitute a synchondrodesmosis. There is a strong likelihood that the part of this fibrocartilage closest to the bone is mineralized, as it is in prosimians (Beecher, 1977). Such calcified persisting cartilage is chondroid bone type II. In most instances it is more easily distinguished from bone than its other names, such as metaplastic bone, might suggest.
    Since the mandible was the source of the concept of accessory cartilages, this structure is discussed first, followed by the maxilla, (another bone complicated by the proximity of primary cartilage); then the bones of the cranial vault are dealt with.

    THE MANDIBLE

    The Number of Chondrogenic Sites
    In his series of fetal pigs Reichert (1837) observed that the mandible formed as two bony halves. Each half had two posterior bony processes, of which the more posterior "covered itself with cartilage" and formed a joint with the temporal bone. He was the first to give the ramal bone temporal precedence over the condylar cartilage and to note the independence from Meckel's cartilage of mandibular ossification.
    Reichert anticipated but never found a bony locking piece (Schlussstuck) analogous to a premaxilla, to join the mandibular halves. This anterior site is of concern because the bridging of the two parts of the mandible is generally by cartilage, with a participation in some species by Meckel's cartilage, which is, of course, a primary one. Thus, when later authors referred to a cartilaginous nucleus anteriorly on the alveolar process, unless they specifically excluded Meckel's cartilage, it is uncertain whether Meckel's or the anterior secondary cartilages was meant.

    Another source of misunderstanding is the term, alveolar process. The early histologists working on the lower jaw studied fetal domestic and farm animals without chins, whose mandibles curve up to end around the incisors. In these animals the anterior part of the alveolar process is the anteriormost region of the mandible above the symphysis, where a secondary cartilage occurs in several species (Baumuller, 1879; Pensa, 1913; Trevisan and Scapino, 1976a).
    Another location where cartilage-like tissues develop, but with less regularity, is what Masquelin (1878) and Baumuller (1879) referred to as the upper edge of the alveolar walls, and what now would be termed the crest of the alveolar process. From hereon, alveolar process will be used in this, the modern sense, and the site of the other secondary cartilage by the symphysis will be referred to as the anterior mandible.

    In the posterior mandible, the absence of a histogenetic relation between the bone and Meckel's cartilage soon became clear. The issues here were: how many secondary or accessory cartilage nuclei are there, and how do these participate in osteogenesis? Since Reichert (1837), there has been no doubt that a condylar cartilage exists. Cartilage has also been reported on the coronoid process for the cow (Bruch, 1852), the pig (Streizoff, 1873a; Schaffer, 1888), the sheep (Schaffer, 1888), man (Masquelin, 1878; Henneberg 1894, cited by Schaffer, 1897; Pensa, 1912; Hanau cited by Koller, 1896; Low, 1905; Momigliano-Levi, 1930b,d), the deer and the mole (Low, 1905), the shrew (Momigliano-Levi, 1930b,c) and the rat (Bhaskar, 1953; Youssef, 1969).
    On the angular process, a cartilage nucleus is present in the cat, the mouse, and the rabbit (Stieda, 1875), the pig (Stieda, 1875; Brock, 1876; Low, 1905), the deer and the mole (Low, 1905), the cow (Kolliker, 1850; Bruch, 1852); man, the bat, the shrew, and the opossum (Momigliano-Levi, 1930b,d), and the rat (Bhaskar, 1953; Youssef, 1969). Schaffer (1888) suggested that, for the coronoid process, the size and life of the cartilage nucleus varied with the ultimate prominence of the coronoid process, so that in species where the process is insignificant the small and short-lived coronoid cartilage may easily be overlooked.
    While this proposal has not been systematically investigated, if true, it would be expected to hold for the angular process, but not enough fetuses have been studied to establish for sure whether, as Low (1905) thought, some species lack a coronoid cartilage, perhaps the mouse (Rohlich, 1933), and others an angular nucleus.

    Another complication is the description in some animals of one posterior cartilaginous mass in the place of condylar and angular cartilages, which only later acquire separate identities, e.g., in the pig (Parker, 1874; Brock, 1876; and Low, 1905), the bear (Fuchs, 1906), and the rat, on the 17th day postinsemination (Bhaskar, 1953). In a fetus of Halicore dugong, Matthes (1921) noted a marked development of the accessory dentary cartilages, including a distinct coronoid one, and a large cartilaginous complex extending from the condyle into the angulus, and anteriorly.

    These observations provide a perspective for examining the report of Gaupp's (1907) demonstration to the German Anatomical Society of the sectioned fetal head of a flying lemur (Galeopithecus volans). He described a posterior mass of cartilage occupying the region of the angular, condylar, and coronoid processes, and extending far forward. A similar cartilage-like tissue was to be found in the whole palatine process of the maxilla, within the maxilla and in the parasphenoid (pterygoid) and squamosal bones. Other preparations revealed the same tissue in the rabbit's maxilia and the hedgehog's pterygoid (parasphenoid). Could some of this extensively distributed tissue be bone mistaken for cartilage, because of the similarity between young bone and cartilage (Schaffer, 1888)?
    Fuchs (1909a), having examined Gaupp's slides at the meeting, pronounced Gaupp correct in identifying the tissue as cartilage in all instances, except in the vomer, zygomatic, and frontal bones. From his own experience with alcoholic fixation, Fuchs believed Gaupp's tissue in these latter three elements to be badly shrunken bone. Schaffer (1930) questioned Fuchs' competence in the matter and believed Gaupp's account to be accurate. (Another report of secondary cartilage in the zygomatic bone was Toldt's (1905) for the rat.)

    Thus, one may conclude that:
    1) the developing mandible can have several secondary cartilages - in some species, one at each protruding part, including, to a variable and distinctly limited degree, the crests of the alveolar bone,
    2) some of these cartilages can attain a considerable extent; (this is not grounds for viewing them either as elements homologous with the separate bones of the dentary of lower vertebrates (Bardeleben, 1905), or as phylogenetic derivatives of a primordial cartilaginous skeleton (Fuchs, 1909a);
    3) there is interspecies variation in the size and duration of the secondary cartilages; and
    4) the significance of the number of cartilage nuclei is limited by the existence of single masses serving two or more eventual processes.
    What needs to be considered now is the nature of the tissue reported at each mandibular site, how it is related to chondroid bone, and whether there are signs of a metaplasia.

    Angular and Coronoid Cartilages
    In the pig, the single large cartilage that forms at the posterior and inferior margin of the mandible undergoes growth and ossification, which eventually lead to two separate cartilage-capped angular and condylar processes (Brock, 1876). The cartilage of the angular process disappears at some time before birth, but earlier in fetal life the cartilage is larger in the angular region than in the condylar. In fact, the condyle was not to be identified until the embryo had reached a length of 6.5 cm.

    The cartilage of the posterior mandible was quite distinct from Meckel's cartilage, but although hyaline, the angular-condylar cartilage displayed some unusual relations with the bone. First, it formed inside the periosteum as a derivative of periosteal cells. Starting in its early development, the cartilage was linked with the nearby bone by a transitional Uebergangsgewebe, i.e., chondroid bone, where the calcification increased, and it was hard to decide whether the cells were chondrocytic or osteocytic.
    Brock understood this intermediate tissue to be cartilage experiencing a direct metaplasia into bone, noting this event particularly near the surface of the cartilage. In addition, he noted that osteoblasts could lay down trabecular bone upon such transformed cartilage. Lastly, with the establishment of the condyle, a third mode of ossification eventually came to dominate the picture, as the metaplastic osteogenesis waned, namely, a modified endochondral variety.

    Schaffer (1888) was to describe in more detail these variations from the endochondral pattern seen in growth plates, but Strelzoff (1873a) and Steudener (1875) had already drawn notice to the lack of chondrocytic columns, irregularities in the line of ossification and depth of erosion, and the persistence of remnants of cartilage in the trabeculae.

    Brock saw both osteoclasts and capillaries involved in the resorption of cartilage and extensive destruction of some of the bone. His Figure 12 (Brock, 1876) illustrates a canal extending into a zone of chondroid bone. Since there is so much destruction of tissues, it cannot be assumed that the chondroid bone will survive long enough to turn into bone, and Brock remarked that some chondroid bone was destined for early dissolution. In view of its superficial position, some of Brock's Uebergangsgewebe or chondroid bone could have arisen directly from the periosteal cells that gave rise to bone and cartilage, and need not therefore be interpreted as proof that cartilage is turning into bone, although such a change remains a possibility.

    For Brock's Uebergangsgewebe in the interior of the cartilage, it is a different story. In the interior of the cartilage, the matrix around the cells calcified, but the cells seemed to hesitate and did not alter and take on an osteocytic form. While he wrote that here an Uebergangsgewebe accumulated that either underwent resorption or was converted to bone by an endochondral route, his later text suggests that Brock was observing typical calcified cartilage, but was misled into thinking that metaplasia was underway because of the similarity with the irregular pattern of erosion seen in rachitic cartilage. Brock perceived that, as the ossification line became more regular in the later fetal condyles, the larger residual islands of spared calcified cartilage disappeared, and an endochondral non-metaplastic ossification took over.

    Brock's contemporary Stieda (1875), from investigation in only a few and varied animals, maintained that no metaplasia was to be found in the mandibular cartilages. Low (1905) made no mention of metaplasia in the angular cartilages.

    Of investigators who observed cartilage in the coronoid process, Strelzoff (1872, 1873a,b, 1876) was of the metaplastic school. His views were not given much attention since he had reached the conclusion, by starting with rather late pig embryos, that the whole mandible is preformed in cartilage, which ossifies solely by metaplasia. Aside from Strelzoff, only Momigliano-Levi (1930c,d) suggested that the coronoid process is a site of direct metaplasia, but it was minor compared to the typical osteoblastic ossification.

    Anterior Mandibular Cartilages
    According to Baumuller (1879), Kolliker (1861) and Strelzoff (1873a) had made fleeting reference to cartilaginous nuclei at the anterior of the mandible. Their identity was first made clear by Stieda (1875) in a 13-cm-long fetal rabbit, which had two small round cartilage nuclei lying symmetrically on either side of the midline above the symphysis, along with the two Meckel's cartilages.

    Stieda grouped the former with the angular and condylar cartilages as accessory. Baumuller (1879) observed similar anterior nuclei in older fetal pigs and newborn cats, and in the pig claimed that the anterior cartilage experienced in part a direct metaplastic ossification (his Figure 6 shows chondroid bone), likened by him to what Gegenbaur (1867) had reported in horns and rachitic bones. Pensa (1913) identified the anterior cartilage in man as the noyau incisif, Schaffer (1916) by the term, Symphysenknorpel (see Knese's (1978b) Figure 193).

    The relations between the anterior accessory cartilages, Meckel's cartilages, and the mandible are the subject of two very interesting recent articles (Trevisan and Scapino, 1976a,b). These authors give a comprehensive review of the participation of cartilage in the development of the mandible's rostral end, which makes it unnecessary to do more than call attention to certain points emerging from their histological work and the literature since Baumuller.

    1. Accessory being a synonym for secondary, to call cartilage at the symphysis of the mandible the "symphyseal accessory cartilage," as Bernick and Patek (1969) did for the rat, was injudicious, when they examined rats no younger than one day and hence were unable to say whether the cartilage was derived from Meckel's (primary) or secondaries, or from both by fusion.
      However, Youssef (1969) expressly distinguished an anterior secondary cartilage from the anterior ends of Meckel's cartilages in the rat.
    2. Trevisan and Scapino (1976a) saw such a fusion of the anterior secondary cartilages with Meckel's cartilage in the hamster and, from an examination of figures published for rats and mice, held that in at least three species of rodent the symphyseal cartilage is a composite structure formed by a unique fusion of primary and secondary cartilages.
    3. In the kitten, two anterior secondary cartilages develop astride the midline, undergo partial endochondral ossification, with the remainder forming an enduring symphyseal fibrocartilage, to which Meckel's cartilage does not contribute, despite its presence, until the second post-natal week (Schachter, Furstman, and Bernick, 1969). Trevisan and Scapino (1976a) cite Spatz (1967) as having found only a participation by secondary cartilage in the tree-shrew's symphysis, and refer to several studies implying the existence of anterior secondary cartilages in other species, including man.
    4. For species where the mandible remains as separate halves joined by cartilage, in the weeks right after birth, the hyaline cartilage transforms into fibrocartilage, e.g., in the cat (Schachter, Furstman, and Bernick, 1969), the rat (Bernick and Patek, 1969) and the hamster (Trevisan and Scapino, 1976b).
    5. The symphyseal fibrocartilage appears to undergo mineralization of its regions abutting mandibular bone, as evidenced by the tidemarks on the adult hamster's fibrocartilage in Figure 10a of Trevisan and Scapino (1976b). They interpret the tidemark as a site where cartilage is turning into bone by the same metaplastic process as occurs in the infant's secondary cartilages.
      However, the picture is more reminiscent of the calcification of fibrocartilage common at apophyses and other synchondroses, that was discussed earlier as yielding a separate type of chondroid bone, CB II. Their Figure 10a clearly depicts a three-fold layering: bone to the left, CB II next, and unmineralized fibrocartilage in the center. While the chondroid bone region is, as they note, part of the symphyseal plate, it is not typical bone.

      In Figure 11 of Schachter, Furstman, and Bernick (1969), at the upper right, in the 60-day-old kitten's symphysis a distinct tidemark runs through the cartilage near to the bone. These writers identified only a process of endochondral ossification in the cat's secondary cartilages, which ceased before completion and sealed off the remaining cartilage by bone. Their figures suggest that CB II participates in this "seating off."
      Last, Beecher's (1977) examination of the partially fused symphyses of the prosimians, Lemur macoco and L. fulvus, suggests that the linking ligaments chondrify before they calcify.

    6. It will be recalled that Baumuller (1879) suspected a metaplasia within the anterior secondaries of the pig. According to Trevisan and Scapino (1976a), in areas of the hamster's secondary cartilage adjacent to the bony symphyseal plates, "individual and/or clusters of cells appear to be surrounded by bone.... Examination of serial sections discloses that bone is being deposited between cells of the cartilage at some distance from the nearest capillaries, thus eliminating the possibility that vessels brought in osteogenic cells."
      Thus, what they describe and show in their Figure 11 is chondroid bone I appearing to turn into bone. Their second paper (1976b) does not give much more detail of the process, but discusses it at some length, and has more illustrations of CB 1, thus their Figures 4b, 5a, 6a. (Their Figure 10a in this series depicts, I believe, the different process of mineralization of fibrocartilage to form CB II (fibro).)
      That the other Figures are of CB I is reinforced by such comments in the discussion as "material that stains as bone between chondrocytic cells." The distance of this tissue from 'clasts and capillaries led Trevisan and Scapino to infer a metaplasia, for which they preferred Urist, Wallace and Adam's (1965) term "chondroidal ossification."
    7. Elsewhere at the hamster's symphyseal cartilage, the endochondral ossification resembled that in the mandibular condyle, rather than that in long bones. Trevisan and Scapino's attempt to specifically designate this "quasi-endochondral type" as "replacement ossification" is infelicitous, because of the long-standing use of Ersatzknochen (e.g., Gaupp, 1903) for typical endochondrally formed bones.

    Cartilage on Alveolar Processes
    The several editions of Orban's (1944) textbook of oral histology are the one place where the general reader finds chondroid bone mentioned and illustrated. His example at the alveolar crest has come to be regarded as a definitive instance of the tissue, cited for example by Enlow (1962a). The cartilaginous tissues of the alveolar margin are, however, too variable in their microscopic appearance and attendance to serve reliably in this capacity.
    As in the similarly shaped sutural margins (see Cranial Vault Sutures: Two Kinds of Cartilage? p. 228), the one germinal envelope may produce bone, chondroid bone, or secondary cartilage merging through chondroid bone into bone.

    Orban certainly was not the first to see a cartilage-like tissue at alveolar crests. Schaffer (1930) gave Steudener (1875) priority, but Masquelin (1878) may have been the first to distinguish two types of material there. In the ossifying condyle, Masquelin identified three components: hyaline cartilage; a tissue dominated by connective tissue fibrils (osteoid or bone, most likely); and an intermediate, seemingly a combination of the other two, that Masquelin called fibro-cartilage. From its supposedly direct ossification to bone this last should be chondroid bone 1. The 95-mm CRL human embryo had cartilage along the alveolar margins, while one of 170 mm had "directly ossifying" fibro-cartilage in the same regions.

    Fig. 14 Alveolar crest

    Low's (1909) report is too brief to identify the tissue, but most subsequent descriptions allow one to recognize either secondary cartilage or chondroid bone. Indeed, Schaffer (1930) chose, as an example for his section on secondary cartilages, a picture (his Figure 265) of the alveolar process in a 4-month human fetus (compare my Figure 14). Since Orban's (1944) example is regarded as an exemplar for chondroid bone, it is instructive to compare his figure of the crest with Schaffer's.

    Schaffer illustrated a cellular perichondrium enclosing a cap of cartilage merging with a large-celled trabecular bone. The intent of the figure was to show (by a leader) the cartilage and its resemblance to the main accessory cartilages of the mandible. His leaders to chondroider Knochen lie more on the large-celled bone than on the tissue of transition, where cartilage graded into bone, i.e. the true CB I present. But, in addition, the bone itself has some cells that exceed the size expected in newly formed membrane bone. Therefore, Schaffer's (1888) earlier application of "chondroid bone" to new mandibular membrane bone in general here has some justification.

    By contrast, Orban's Figure 8-3 (8th ed., 1976) gives no age, but the density of the nearby connective tissue suggests a specimen older than Schaffer's. Cartilage is absent between the mandibular bone and the surrounding proliferative tissue. The inner part of the bone, where the leaders to chondroid bone lie, consists of a tissue with large, dark, apparently basophil regions obscuring most of the cells, whose size cannot therefore be made out.
    The fate of the cartilage and chondroid bone of the crest is uncertain, despite the belief of Masquelin (1878) and others that these experienced a metaplasia, because the new tissues are, with little delay, subjected to resorption. As the tooth grows it is unlikely that this particular region will be replaced by bone, as Orban proposed, or be allowed to turn directly into bone.

    Cartilage and chondroid bone also grow in the crests of maxillary alveolar bone. Miles (1950) wrote of the human maxilla: "Areas of tissue having an appearance strongly suggestive of cartilage of which the matrix has calcified are sometimes found within the substance of the developing jaws. Unlike 'secondary cartilage' it does not appear to be distributed according to any pattern. Lehner and Plenk (1936) call this tissue 'chondroid bone' and state that it tends to develop in situations of particularly rapid growth. Bone of this nature is shown in Figure 4."
    His (Miles') illustration, from a 60-mm embryo in the region of the deciduous canine tooth germ, has a tissue rich in cells like large osteocytes or small chondrocytes.

    On the other hand, Dixon (1953) gave the outer alveolar wall of the human maxilia as a site of secondary cartilage. Next, the tissue shown in Mohammed's (1957) Figure 39, at the anterior alveolar crest of the first maxillary molar of a four-days post-natal rat, is called by him, and looks like, cartilage. Hall's (1971) Figure 8 depicts a nodule of maxillary cartilage in the mouse, characterized as "ectopic," which is justified to the degree that its occurrence is haphazard.

    The fullest description of alveolar chondroid bone is Lehner and Plenk's (1936). They distinguished chondroider Knochen, a Mischform between bone and cartilage, from bone, bone with basophil islands (Zawisch-Ossenitz's (1929) Inselknochen), and secondary cartilage, noting that the symphysis, condylar, and coronoid processes had both cartilage and Mischgeweben.
    Their Figures 112 and 113 depict chondroid bone adjacent to mandibular incisors in a 390-mm human fetus (compare my Figure 15). The cells varied between typical osteoblasts and ones without processes, but enclosed in cartilage-like capsules. The authors remarked that the CB came not from an osteoblastic seam but from a cellular tissue resembling a perichondrium.

    This last observation throws more doubt on Haines and Mohuiddin's (1968) proposal that alveolar chondroid bone grows as a typical chondroid bone II (their "metaplastic bone") by the mineralization of the transseptal part of the periodontal ligament. The cellular germinal zone capping the crest not only forms the chondroid bone, but separates it from the developing connective tissue. Moreover, at this time in fetal life, the tooth has no root and hence the introduction of periodontal fiber groups to explain the origin of the CB is premature.
    Lastly, the CB is promptly eroded by osteoclasts (see Figure 15, p. 212), the rapid pace of reshaping of the crest making unlikely any progressive mineralization of a chondrifying ligament, as described by Haines and Mohuiddin at the many bona fide sites of CB II.
    This is not to say that tendinous insertions are not themselves subject to relocation along bony surfaces (Hoyte and Enlow, 1966; Videman, 1970), but this event, the maintenance of the periodontal ligament's fastening to the labile alveolar bone, and the early formation of the alveolar crest are not one and the same phenomenon.

    Fig. 15

    MANDIBULAR CONDYLE

    Introduction
    Schaffer's (1888) analysis of ossification in the sheep's jaw acknowledged, both in the text and summary, that the histological events in the condyle and coronoid process almost begged for a metaplastic interpretation. To cite the last part of his summary, the factors enticing one to believe in a direct transformation were these:
    "By the direct deposition of cartilage-like bone on bone-like cartilage, two very similar tissues, which neither morphologically nor in their staining have a sharp demarcation, come into a most intimate spatial relationship. By the circumstance that young bone reacts to staining more like cartilage than mature bone. Because of the rich vascularity and highly irregular penetration of the marrow cavities into the condylar process. By the calcified cartilage's (in the 'perichondral' phase of ossification) being resorbed by osteociasts after the manner of bone; and the formation of globuli ossei, which are commonly so cut by the section that they appear to be surrounded by cartilage matrix. Finally, by the persistence of intact cartilage capsules or groups of these within the area of ossification".

    The secondary nature of the condylar and coronoid cartilages had been known since Reichert's and Kolliker's reports at the middle of the century. But Schaffer's investigation provided proof that bone preceded cartilage, and the cartilage formed from erstwhile periosteum, and was unconnected with primary cartilages such as Meckel's; although Fuchs (1906) later tried to dispute the whole concept of secondary cartilages and those of the mandible in particular.

    The mandibular condylar cartilage is the most studied of the secondary cartilages (see Chapter 3, this volume), but in certain aspects knowledge has not advanced much beyond Schaffer's findings. It is with these special topics relating to chondroid bone that the remainder of this section deals.

    Uebergangsgewebe: Chondroid Bone I?
    The bone first established at the condyle had, Schaffer thought, enough large encapsulated osteoblasts in it to deserve the name chondroider Knochen, but it is typical membrane bone. Then, at its end appeared a large-celled, matrix-poor, almost cartilage-like tissue, an Uebergangsgewebe, just before the cartilage proper was to form.
    The Uebergangsgewebe observed on the condylar process of the 51/2-cm-long sheep had large, ovoid, encapsulated pale cells, with a fibrous eosinophilic matrix, and served, Schaffer thought, as a tissue of transition to the more hyaline cartilage due to appear shortly on the condylar process. It is probable that this tissue was a variety of chondroid bone formed by cells from the stock that had just been the source of osteoblasts. These cells could change towards chondroblasts in their morphology, but continue in the tradition of forming a bone-like fibrous matrix, and thus lead to the dual-natured tissue described by Schaffer. Schaffer did not say if the Uebergangsgewebe turned into anything else, but Momigliano-Levi (1930c) thought that the tessuto di transizione underwent metaplasia into bone, as Pensa also thought (1913).

    The condylar intermediate tissue was superseded by a distinctive cartilage, which, once present, stained so weakly with hematoxylin in comparison with Meckel's cartilage that Schaffer viewed it as unreif (unripe). Furthermore, as the cartilage grew, the cells very soon enlarged to leave so little matrix between them that the cartilage came to resemble the very cellular variety in fishes described by Kolliker as Zellenknorpel, and by Rollett as Parenchymknorpel.
    The most impressive aspect of the secondary cartilages that are accessory to membrane bones is the rapid development of large cells, likened by many to the hypertrophic cells of epiphyseal cartilage at the ossification line. Thus, the condylar Uebergangsgewebe was intermediate to a bone that looked somewhat cartilaginous and a cartilage that differed in its weak staining reaction and high cellularity from the general run of hyaline cartilages.

    Condylar Cartilaginous Tissues: A Stable Basis for Comparisons?
    One factor supporting the existence of chondroid bone I and secondary cartilages as true entities is the similarity between tissues at various skeletal sites, to which histologists every so often have drawn attention. For example, Schaffer (1888) remarked on the resemblance of the tissue at bony margins of the crania to the condylar Uebergangsgewebe; while Knese and Biermann (1958) equated a cranial marginal tissue to the hyalinzelliges Chondroidgewebe at the mandibular condyle, clavicle, and elsewhere.
    Both sets of comparisons are legitimate, but they refer to tissues differing in age and appearance, although originating in the same kind of cell. Thus, likening materials to those of the developing mandibular condyle did not tie the cranial and clavicular tissues to a precise and accepted entity, but threw them into the already blurred spectrum of supporting tissues described above.

    A major obstacle to discussing the various instances of accessory cartilage has been the variability of the tissues involved. Aside from mostly minor differences based upon species, variation occurs along two dimensions:
    histogenetic, in that the germinal cells form different kinds of bone, chondroid bone, and cartilage; and
    temporal, because each tissue may rapidly change its character as it matures, as noted by Momigliano-Levi (1930c,d).
    These variations combine to introduce more types of tissue, with very subtle differences, than can be accommodated by the few names generally agreed upon on. Attempts at more refined schemes of classification, e.g., by Zawisch-Ossenitz (1929b), have not really helped. The term, chondroid, has probably clarified least.

    For example, Knese and Biermann (1958), noting Zawisch's (1953) description of the cellular cartilage of the young clavicle as a Pseudoknorpel, included it in the category of hyalinzelliges Chondroidgewebe. They proposed that the special form of hyalinzelliges Chondroidgewebe in the clavicle, mandible, and, from their own observation, the trochanteric region of the femur and some locations in the skull, be considered, from its morphology and prospects, as a form intermediate between the usual skeletal cartilage and the Ansatzknorpel - the cartilage at tendinous and ligamentous insertions, with which their article was chiefly concerned. Thus, the tissue at the edges of cranial bones and the very early condyle was defined not in direct terms in its own right, but by reference to other tissues to which it is apparently related as an intermediate - tissues which themselves cannot serve as sufficiently stable points of reference for such an exercise.

    Knese and Biermann (1958) mentioned this last factor in their sensible attempt at a resolution of the status of the chondroid tissue:
    "These special expressions of this form of cartilage, determined apparently by site and time during development, scarcely justify distinguishing it as a 'Pseudocartilage,' differing fundamentally from skeletal cartilage. The morphological differences between the two forms are hardly greater than the variations of skeletal cartilage itself.... The insertion-cartilages - probably all so-called secondary cartilages - must be seen as a true hyaline cartilage, since the collagen fibers are extensively masked by the ground substances."

    The two "identifiable" varieties of cartilage in the condyle are: the first tissue coming after initial osteogenesis, which is cartilage still with a bony tinge (Schaffer's Uebergangsgewebe or CB I); then the much more extensive cellular hyaline cartilage, which acquires layers and experiences endochondral ossification. Similar entities are found bordering the cranial sutures (see Cranial Vault Sutures, p. 228).

    Persisting Cartilage Cells
    Schaffer (1888) found that the irregularity of the erosion of the mandibular condylar cartilage left some intact chondrocytes behind. Their existence is not of itself, evidence for a metaplasia of cartilage into bone. If anything, their temporary persistence shows that cartilage cells do not become osteocytes. The spared cartilage cells do not last indefinitely but disappear with the extensive reshaping of the mandible. Chondrocytes enclosed in matrix survive erosion in other sites of endochondral ossification; so they are not a peculiarity of secondary cartilage (see Chapter 10, Metaplastic interpretations of CB II).
    This phenomenon differs from the apparent survival of chondrocytes after erosion has opened their lacunae, a form of survival of which the mandibular condyle has long been suspected (Rohlich, 1933; Silbermann and Frommer, 1972). These latter authors based their conclusion, in part, on evidence from contemporary tissue culture experiments on other cartilages, e.g., Holtrop (1967).

    Periosteal Bone: Living Cartilage Boundary
    Schaffer (1888) emphasized what Muller (1858) had already observed, that in endochondral ossification bone is laid down not only on calcified, hypertrophic and perhaps moribund cartilage, but also on living cartilage which may have quite modestly sized chondrocytes. This event is evident from the cross-sections of fetal human long bones studied by Knese (1956, 1957), with Figures 1 and 2 of the second paper illustrating the phenomenon well.
    Knese noted that along the boundary between cartilage and bone the cells are not all in the same relation to the two matrices. Some cells are enclosed in cartilage matrix, others are to a greater or lesser degree encompassed by bone matrix. From Muller's time onwards, there have been questions about the precise nature of this first seam of subperiosteal bone and its relations with soft connective tissue, cartilage, and older bone.
    Schaffer (1888) and Knese (1956) reviewed the history of these issues, but more recent electron microscopy makes it clear that the tissue is a form of bone, or, at the least, osteoid. For example, Crissman and Low (1974) described the osteoid deposited on both uncalcified and calcified cartilage in the developing vertebra of the chick.

    In the endochondral ossification of both primary and secondary cartilages, when a germinal layer has been engaged in making cartilage, there is a layer of this tissue, clad in a perichondrium, the innermost layer of which is populated by germinal cells. If some of these cells become osteoblasts, bone matrix can be laid down upon living cartilage, usually with a quite sharp boundary visible between osseous and cartilaginous matrices. Where the last chondrocytes to be formed lie in relation to this line of demarcation depends on whether they had time to enclose themselves totally in cartilage matrix, before the germinal cells switched to osteogenesis and started synthesis.
    If the last cartilage cells have deposited matrix on only their deeper aspect, their superficial aspect could receive a covering of bone matrix from the osteoblasts now differentiating from the germinal layer, in the same way that osteoblasts may be covered over not so much from their own efforts as by those of the next generation of cells above them (Cameron, 1963). These relatively small, living cartilage cells with cartilage matrix on one side, bone on the other, were misconstrued by authors such as Carey (1922) as evidence for a metaplasia.

    Since Schaffer's paper, such cells have been mentioned every so often, for example, by Murray (1963) and Murray and Smiles (1965), as situated where avian bone and adventitious cartilage abut one another, and by Knese (1956) as under the bone first deposited upon the midshaft of the fetal human tibia.

    Knese suggested that the superficial cells of the cartilage might themselves add more bone to the inner surface of the midshaft collar, but this activity has not been substantiated for bones of the limb. Rather, by staining for bone with Da Fano's silver method and cartilage with methylene blue, Fell (1925) was able to show in the chick's leg bones that a thin layer of cartilage always intervened between the peripheral chondrocytes and the bone, but "in ordinary preparations the peripheral cells seem to be directly applied to the surface of bone."

    Thus, along the junction of subperiosteal bone and living cartilage there is a "tissue" comprising cells, in some places enclosed in bone, elsewhere in cartilage matrix, and perhaps by different matrices on the two sides. The "tissue" is little more than one cell wide and its extent is easily overlooked, unless, in the long bone, one uses the transverse sections favored by Muller (1858) and Knese (1956, 1957). In other developing bones the junction temporarily extends for greater and more noticeable distances. Bidder's (1906) Figure 3 shows the extent of the sleeve on the rib. His name for the bony extension over the cartilage was begrenzende perichondrale Ossificationslamelle, an extension of the primare Periostlamelle on the embryonic diaphysis. The leading edge of the sleeve of ossification, at the point where periosteum takes over from perichondrium, in a way constitutes a scaled-down and ungrooved version of the encoche d'ossification of Ranvier on developing long-bones; and the sleeve of bone can be equated with the "bone bark" encircling the epiphyseal growth cartilage (Shapiro, Holtrop, and Glimcher, 1977).

    The kind of chondroid bone just described is an almost inevitable accompaniment of a switch to osteogenesis by previously chondrogenic germinal cells; however, it is not typical CB I because it can come about without any cells being programmed to form a mixed or hybrid tissue, when the changeover is effected rapidly. Although the changeover permits some slight variation in the relation of cells to matrix along the bone-cartilage line, the boundary zone essentially has area but no real depth and does not merit the status of a separate tissue.
    On the other hand, if the germinal cells take their time about changing their synthetic and matrix-controlling programs, but nevertheless there is a stimulus for them to divide and do something, the something done could be the making of chondroid bone I. In these circumstances, with both old and new instructions in effect at the same time, the cells would produce a tissue having cartilaginous and osseous characteristics, and if enough cells divide and perform synthesis, the tissue could pick up the appreciable widths seen in many sites of chondroid bone I.
    Whether the actual stimuli and controls of gene expression work in such a manner is unknown. What certainly needs explanation is why, since the cells can switch abruptly and leave a fairly precise line of demarcation between bone and cartilage, the cells react slowly and gradually at other sites, particularly in accessory secondary cartilages.

    Schaffer called attention to the elongated pyramidal shape of the young mandibular condylar cartilage: a shape described also as wedge- or carrot-like. In both the penile bone and the mandibular condyle, the phase of an elongated growth cartilage lasts no more than a few days, before the cartilage grows and is reshaped to a more crescentic convex cap (see Figure 1, Chapter 1). Much of the sides of the early pyramid or wedge is enclosed in a sleeve of bone, which lies for the most part on living cartilage. The superficial cells of this cartilage themselves therefore already participate in a very tenuous sheath of a dubious kind of chondroid bone.

    At the most proximal extent of the bony collar, that is, where it reaches highest up the carrot of cartilage, the nature of the boundary is more ambiguous, because there chondrocytes may be surrounded by bone matrix (compare Figure 19, Chapter 14). This CB I, reported by Schaffer (1888), Rohlich (1933) and others, appears to be produced by circumstances - the switching of a perichondrium to osteogenesis - which are the converse of those that first established the secondary cartilage on the bone, and also briefly gave rise to a CB I.Br> Rohlich rightly held that Masquelin's (1878), Pensa's (1913) and Momigliano-Levi's (1930c) placing a metaplastic construction on the tissue was unwarranted. But his own idea that the bone-like matrix is a product not of the cartilage cells but of nearby osteoblasts is not "much more probable." Because of the appreciable depth of the tissue, the chondrocytes bear a prima facie responsibility for the material enclosing them.

    The Disk of the Mandibular Joint
    Adjacent to the condyle is the disk: another secondary cartilage. Karakasis and Tsaknakis (1976) and Kopp (1976) list more recent authors than Schaffer (1930), who have written on the cartilaginous nature of the mandibular articular disk in man, guinea pig and other species. The greater number of chondrocytes in older disks, whether viewed as pathological or as a phenomenon of aging, is usually accounted for by a metaplasia of some of the fibroblasts of the younger disk. Since capillaries are scarce in the central region, the hypothetical perivascular mesenchymal cell is even less likely than in other connective tissues to be the source of the cartilage cells.
    An experiment illuminating another metaplastic potential of the disk involved its transplantation into the brain of young rats (Ronning and Koski, 1969), where the disk sometimes formed cartilage, and later bone with marrow.

    SQUAMOSAL FOSSA AND TUBERCLE

    Kolliker (1889) was, according to Schaffer (1897, 1930), the first to describe cartilage on the cranial bone with which the mandibular condyle articulates. Several microscopists have confirmed the presence of a cartilage-like tissue in the mammalian glenoid fossa but report it as varying in its extent and time of disappearance. In some rodents, controversy exists as to whether the tissue is present at all.

    Fuchs's Comparative Studies
    The widest ranging comparative study was by Fuchs (1906) in a paper more concerned with homologies of the auditory ossicles and dentary between mammals and reptiles.
    In human fetal development hyaline cartilage formed early in the glenoid fossa, but by the third and fourth month had already been partly destroyed. His Figure 27 shows cartilage extending across the face of a four month-old joint. The cartilage was partly calcified and in consequence, here, was rather less easy to distinguish from the bone. Endochondral bone replaced the cartilage during development, so that the cartilage never achieved a conspicuous size. As a rule, in adults, only traces of it remained.

    Fuchs, from examinations of fetal mice and rabbits, believed that in rodents it was very probable that no cartilage formed on the cranial face of the joints. Fuchs found that other species had a significant squamosal cartilage. He saw a moderate covering of cartilage in ermine and zorilla (an African mustelid) and a greater amount in the hedgehog. (In the hedgehog, hyaline cartilage was also present in the meniscus of the temporo-mandibular joint, another example of a secondary cartilage, except that for Fuchs all secondary cartilages represented primary cartilaginous skeletal elements.)
    Fuchs saw cartilage in its most luxuriant growth on the squamosal bone of the newborn badger and the prosimian, Lemur mongoz. In the badger, cartilage covered the whole surface of the joint and was itself clad in a thin layer of spindle-shaped chondrogenic cells. The deeper cartilage was calcified and undergoing a modified endochondral ossification - modified in that bone enclosed islands of spared calcified cartilage, but Fuchs was unable to study the fate of the tissues in a mature badger.

    In the fetal cat, the squamosal cartilage was not as thick as in the badger but was extensive, with a wide sheet of calcified cartilage capped by a small region of uncalcified cartilage as shown in his Figures 36 and 37. Fuchs's calcified cartilage in Figure 36 is trabecular and, while it may be eaten-out calcified cartilage, its appearance suggests it might be bone. However, the uncalcified cartilage certainly looks cartilaginous. A fetus of the bear (Nasua socialis) had an extensive cartilage similar to the cat's, except that calcified cartilage was mostly absent.

    Last, in two species of opossum Fuchs detected a peculiar cartilage that lay within the connective tissue on the joint's surface, but was not fused with the bone and was not a part of the meniscus (see his Figure 40). He was unable to find cartilage in this site in a grown Didelphysis virginiana. The cartilage he saw would constitute yet another secondary cartilage associated with the squamosal-mandibular joint.

    Fuchs's only insectivore was the hedgehog, but the shrew has a very large triangular condyle with separate bearing surfaces (Fearnhead, Shute, and Bellairs, 1955). Two separate glenoid shelves accommodate the two condylar facets. From their text and Figure D, the adult glenoid surfaces remain as cartilaginous as the condylar ones. A fibrocartilaginous disk is interposed in the superior articulation. Momigliano-Levi (1930d) described cartilage joined by a transitional zone to the underlying temporal bone in the long-winged bat, but the opossum had only "groups of small cartilaginous elements surrounded by very thin basophil capsules, dispersed in the bone matrix." His table gave the sites of craniofacial secondary cartilage in four species.

    Human Articular Tubercular Covering
    The human mandibular joint, unlike many animals', has a prominent articular tubercle against which the condyle presses. It is on this tubercle that a kind of cartilage was seen by Kolliker and later authors, e.g., Momigliano-Levi (1930b,c). In the 90-mm long fetus, Symons (1952) observed, "the bone of this region, being of open texture and with such large cell-spaces, has almost the appearance of an area of secondary cartilage," and by the 150-mm stage, "in the temporal region an area of secondary cartilage has appeared," but by full term "all trace of active growth of secondary cartilage had disappeared from the temporal region of the joint."
    Symons interpreted this cartilage as a development paralleling the condylar element but starting later, being smaller, and "apparently associated with a quite local and transient necessity for rapid growth."

    Baume's (1962a) Figure 8 from a 72-mm fetus illustrates the tissue and again identifies it as a secondary cartilage. He mentioned a Spanish and two Italian authors who had used the presence of the temporal cartilage to argue that the condyle and the glenoid elements came from the same blastema. Baume's paper was directed at refuting this conclusion and the temporal cartilage was mentioned only once. Recent studies of the joint pay the tubercular covering more notice.

    Wright and Moffett (1974) thought the osteogenesis on the neonatal human tubercle odd: "Tissue being deposited there can be called immature or chondroid bone. It shows a gradual transition from overlying articular tissue in that the cells become more rounded, enlarged, and proportionately closer together but without the presence of mitotic figures and isogenous groups of cells. At the same time, the collagenous matrix gradually becomes basophilic giving a total picture of metaplastic conversion to bone analogous to that seen in the mineralization of tendons and in sutural and periodontal ligaments."
    The "immature bone" persists in the tubercle in the months after birth, but its formation diminishes between six months and two and one-half years of age. An incidental observation was that the temporo-sphenoidal suture media] to the joint has chondroid bone along its bony surfaces, and deposition of this tissue is still under way at two and a half years.

    Thilander, Carlsson, and Ingervall (1976) reported a "transitory cartilage" on the newborn infant's tubercle that soon decreased in width, but which until 17-18 years kept a cellular proliferative zone that increased in thickness at puberty. After 20 years, "the superior and anterior parts of the condyle and the postero-inferior parts of the articular tubercle still contain cartilage with only a few cells. This has been considered to constitute the basis for remodeling processes which occur later in life in response to various stimuli."
    Their Figure 3 shows the "cartilage" on the tubercle to be chondrocytes in a bone-like matrix, and hence a chondroid bone. Kopp (1978) finds more metachromasia in the "chondroid cells" placed laterally on the temporal eminence than those located medially.

    Chondroid Bone in Rodent Fossa
    Mammalian species differ widely in the shape of the joint. The rat has a squamosomandibular joint in which the antero-posteriorly oriented fossa, at five days of age, is lined by a "cartilage-like mass of tissue" (Collins, Becks, Simpson, and Evans, 1946):
    "With increasing age, this modified tissue expands and continues to conform to the changing shape of the condyle........Calcification of the cartilage-like tissue occurs in older animals, but even in old age, 465 days, the tissue can still be distinguished. The persistence of this tissue is probably related to the continued capacity of the fossa to be remodeled to parallel any changes in the shape of the condyle arising from unusual stresses either physiologic or pathologic."
    Fig 16 Fossa
    The tissue in question is shown in my Figure 16, where alcian blue has stained the large cells but chlorantine red has colored the matrix like bone. Aside from Collins et al. (1946) and Jolly's (1961) observations of a cartilage-like tissue lining the fossa, Bhaskar's (1953) Figure 48 clearly illustrates something lining but differing from the squamosal bone, although he did not refer to the squamosal bone in the text.

    The presence of cartilage-like tissue in the fossa is not undisputed. Furstman (1965) was unable to find any cartilage in male Holtzman rats aged one day to two years. Among the stains that he used was alcian blue, which should have revealed any cartilage. Thinking that the reason why he could not find the cartilage reported by Collins et al. (1946) lay in their use of only females, Furstman examined some females but still found nothing cartilaginous. Youssef (1969) saw none in fetal rats.

    In the mouse, Levy (1948), Hall (1968a), and Dawson (1962), in a thesis cited by Hall, saw no secondary cartilage on the joint surface of the temporal bone, although Silbermann (1976) has reported the presence of chondrocytes there.

    The lack of agreement regarding the rat and mouse might be explained by the exercise of a rigorous criterion for cartilage on the part of those denying the presence of the tissue. What I have seen has a distinctly bone-like matrix, and only the large size of the cells and their staining with alcian blue suggest cartilage. From this appearance the tissue is better described as chondroid bone than cartilage.
    With obvious cartilage present in the condyle for immediate comparison, some microscopists may have been unwilling to concede that the squamosal lining tissue is in any way cartilaginous. There is a tissue of a cartilaginous cast in the rat, but the amount is slight and the tissue is at least as bony as it is cartilaginous.

    Temporal Chondroid Bone: Reactions and Remodeling
    Collins and others' comments on the fossal tissue of the rat provoke these related queries.
    First, does chondroid bone "expand" or experience interstitial growth?
    Second, how calcified is the tissue? They claimed that "complete calcification of this tissue does not occur" but they were judging calcification only by changes in the reaction to hematoxylin and eosin of decalcified sections.
    Third, is chondroid bone remodeled? And if so, is it by a turnover of matrix materials such as that which occurs in cartilage, or did they mean that it is resorbed and replaced by endochondral bone from its deep, squamosal aspect?
    There is only meager evidence on any of these points, but the tissue does sometimes respond in an experiment aimed at some other structure, of which there are three examples.

    In the first, Jolly (1961) saw "fibrocartilage" between the fibrous surface layer and the bone of the fossa in rats 21/2-6-months old. When the condyle was removed, the bony neck formed a new head, angled to articulate with the cranium outside the fossa. Chondroid bone, like that formed at the stump, sometimes grew on this lateral surface of the squamosal bone. Also, "new bone was frequently laid down on the surface of the old articular fossa," but Jolly did not say what happened then to the chondroid tissue already lining the fossa, and he gave very little space to chondroid bone in his discussion.

    The second experiment was Silbermann's (1976) daily injection of a glucocorticoid hormone into young mice. His Figure 13 shows a marked proliferation of chondrocytes at the articular surface of the temporal bone, after 84 days. Not only are the chondrocytes in obvious nests, but one can also see in his figure that there is a hematoxylinophil tidemark separating the superficial zone with clustered hypertrophic chondrocytes from a deeper lying and wide region, having a darker matrix but still with ovoid, chondroid cells.
    The excess hormone undoubtedly altered the tissues in the squamosal fossa. The changes appeared to include a proliferation of "fibrocartilage" at the surface and the appearance of a tidemark, perhaps the limit of calcification, between superficial cartilage and underlying chondroid bone (see Silbermann's Figure 4a). However, the experiment does not prove that the cells within chondroid bone can divide or that the tissue grows expansively.

    A third experiment in which the squamosal cartilage reacted to a change in its circumstances was Simon's (1977). When he removed the incisors from 10-day-old rats, after 70 days "the squamosal bone is almost devoid of hyaline cartilage, and is covered by a layer of fibrous connective tissue which is thinner than that seen in the control." He did not discuss this finding, being concerned with changes in the condylar cartilage, and so by what route the squamosal cartilage disappeared is unknown.

    The components of the mandibular joint are reshaped during childhood growth and thereafter. The notion that chondroid bone by virtue of its partly cartilaginous nature facilitates the remodeling of the joint crops up often and appears to stem from this line of thought. In enchondral growth, cartilage is partly resorbed to make way for bone and, at the same time, can influence the form of the substituting bone. Remodeling of a bone thus occurs where there is cartilage within it. Hence, if cartilage is present in a bone, it is evidence of both the occurrence and the site of remodeling.
    What is obviously wrong with such reasoning is that bone resorption and replacement also occur at many places remote from cartilage, and bones lacking cartilage are reshaped. Furthermore, some chondroid bone, rather than being more prone to resorption and osseous replacement than normal bone, seems to be less so and likely, instead, to undergo a direct metaplasia into bone, or, in the case of chondroid bone II, for example, under articular surfaces (Haines and Mohuiddin, 1968) simply to persist.
    This is not to say that renewal of bone deep to the fossal chondroid bone does not proceed apace. Indeed, Wright and Moffett (1974) remarked on the extensive remodeling and new osteons under the chondroid bone of the articular tubercle, and Enlow (1962a) noted a marked reconstruction below chondroid bone capping tubercles and the linea aspera on long bones. There is however, some variation in this, and Biermann (1957) called attention to sites where compact bone underlay a prominence.

    MAXILLA AND PTERYGOID

    The maxilla and pterygoid resemble the symphysis of the mandible in having both secondary cartilages and a proximity to primary cartilages, e.g., the nasopalatine, which may confuse the identity and role of the secondaries. A further complication is introduced when homologies are sought between the pterygoid, parasphenoid, maxillary and premaxillary bones of the various classes (Gaupp, 1903; de Beer, 1937).
    More difficulties arise because some facial bones form by the fusion of membranous and endochondral components. Thus, cartilage at the edge of a mostly membranous bone does not have to be a secondary development. To show a cartilage to be secondary requires careful study begun before the emergence of either bone or cartilage.

    Pterygoid
    The foregoing only hints at the complexity of the origins and homologies between facial bones (de Beer, 1929, 1937), illustrated by the mammalian pterygoid, thought by Gaupp (1906) to be homologous with the wings of the reptilian parasphenoid, but by de Beer (1929) to represent the reptilian pterygoid. The controversy arose partly because of peculiarities of the site in Echidna, and partly from the uncertain role of the cartilage associated with the pterygoid. Fuchs (1909b) described in the rabbit how the pterygoid started its development as hyaline cartilage, within the perichondrium of which bone started to form as an independent entity. However, the bone rapidly came to take up a typical "perichondral" position apposed to the cartilage, which was then destroyed and replaced by bone. Fuchs would not call the cartilage secondary because it preceded the bone. He regarded it as a "rest" of an old typical cartilaginous bone, specifically the pterygopalatine process of the reptilian palatoquadrate bone, which, he thought, in mammals fused with a membrane bone to form a mixed entity.

    De Beer (1929) did not think that the pterygoid cartilage represented any primitive cartilaginous structure. Among other factors, he based his conclusion, that the pterygoid cartilage of the shrew was a precocious secondary cartilage, on its histology, which "is identical with that of the nodules of cartilage which are to be found in the angular and coronoid regions of the dentary bone of the mandible in several mammals," and is unlike the undoubted primitive cartilages. This nodule of secondary cartilage on the pterygoid is widespread among mammals, except the Monotremes (Fawcett, 1905; Momigliano-Levi, 1930b,d; de Beer, 1937; Youssef, 1969).

    Another primary-versus-secondary controversy existed for the vomer of the cat. Fuchs (1909b) attempted to refute Zuckerkandl's (1908) claim that periosteal, i.e., secondary, cartilage grows on this bone. Fuchs saw only a fusion of vomeral bone with the paraseptal cartilage, which experienced a partial endochondral osseous substitution.

    MAXILLA AND PALATINE BONE

    The maxilla may have more than one site of secondary cartilage; it has an adjacent primary cartilage - the nasopalatine - and there is a palatine bone, so that the one name, palatine, can become attached to several cartilages. A sound report of any secondary palatine cartilage has to inform as to its origin, site, fate, and relations with bone and other cartilages close by. Few observations are based on enough animals to meet these criteria; Mohammed's (1957) series of rats is an exception.
    Hence, the studies of the palatal suture in mouse and cat by Bernick, Furstman and their colleagues (references in Kurtz, Furstman, and Bernick, 1970) cannot help in establishing the nature of the cartilage, because they deal with only postnatal development, when it is already present. Their work on the rat (Figures I I and 12 of 21-day fetal palate, Hughes, Furstman, and Bernick, 1967; Figure 2 of a newborn rat's palate, Anderson, Furstman, and Bernick, 1967) implies that the cartilage is secondary, but they neither address this point nor use the term.

    In several species, cartilage is a regular occurrence at the edge of each palatine process and serves to achieve palatal fusion across the suture. De Beer (1937) regarded these paired maxillary cartilages as secondary, and in the shrew (de Beer, 1929) he was able to find nasopalatine and palatine cartilages and the secondary cartilage of the palatine process and thus establish the separate identity of the last.

    Several reports on individual embryos testify to the presence of cartilage elsewhere in the maxilla. Gaupp (1907) found an embryo of flying lemur to have the whole palatine process composed of cartilage, which was also present in the body of the maxilla. A fetal rabbit also had cartilage-like tissue in the zygomatic process, and the tissue was present in the parasphenoid (pterygoid) of a hedgehog fetus. Secondary cartilage occurs in the maxilla's zygomatic process in the opossum and man (Momigliano-Levi, 1930b,c,d; Dixon, 1953).

    Fuchs (1909b) observed a rod-shaped nucleus of hyaline cartilage within the maxilla directly caudomedially to the last tooth germ in fetal cats of 8-9 cm length. It underwent endochondral replacement by bone. Fuchs sought to trace the cartilage phylogenetically to primary cartilage of the reptilian palatoquadrate bone, but Gaupp in the discussion on Fuchs's paper held it to be an example of secondary chondrogenesis.

    Fuchs (1909a) also reported cartilage within the fetal rabbit's maxilla in the vicinity of the nasal capsule, but its location is otherwise imprecise. In human embryos of 27 and 47 mm, Miles (1950) illustrated in his Figures 2 and 3 a small mass of cartilage within the maxilla on its lateral aspect, in the region of the malar process and close to the primordial teeth.

    Two cartilages are intimately associated with the developing maxilla of the rat (Mohammed, 1957), and a third - the nasopalatine - is close by. Thus, at the 20th day of fetal life:
    "The maxillary cartilage is a cartilaginous bar at the ventrolateral corner of the body of the maxilla, running from an area caudal to the future premaxilla-maxillary suture to a level anterior to the molar dental lamina. Anteriorly, the cartilage lies ventrolateral to the bone (Figure 21). Posteriorly, it becomes incorporated in the body of the maxilla by the apposition of bone tissue around it (Figure 22)."
    In the first days after birth the cartilage grows anteriorly, but experiences endochondral ossification from its perichondral splint of bone, until at 15 days of age the "entire structure has calcified."
    Mohammed's phrasing that "the cartilage lies ventrolateral to the bone" makes one think that the cartilage and bone might be developing separately, but his Figure 21 indicates that the cartilage is enclosed by and has developed from the periosteum of the bone.
    This is not so evident for the origin of his second maxillary cartilage - the palatine cartilage - forming medially to the palatine process, as in his Figure 19 of a 19-day post-insemination rat. Here, it is not clear that the cartilage and bone share common germinal cells, although the figure certainly does not rule out a shared derivation.

    Fig 17 Palatal suture

    Regardless of their size and position, the fate generally described for the maxillary cartilages is erosion and replacement by bone, if needed for the changing shape of the growing maxilla. Such an endochondral substitution is regularly described for the palatine sutural cartilage (my Figure 17). Mohammed (1957) saw the perichondrium of the cartilage as the source of appositional growth for the transverse extension of the palatine process. Each cartilage thus looks and acts rather like an epiphysis and has been described as "epiphysis-like" by Pritchard, Scott, and Girgis (1956), and Anderson, Furstman, and Bernick (1967), a resemblance which the latter authors pursued in an experiment with growth hormone (Kurtz, Furstman, and Bernick, 1970).

    The fate of the cartilage in rat was followed after birth by Pritchard, Scott, and Girgis (1956) only to the extent that as shown in their Figure 20 palatal cartilaginous fusion "appears to be imminent", and the legend has "cambial layers replaced by cartilage" for this six-day rat. However, Anderson, Furstman, and Bernick (1967) found that cartilaginous fusion was not established until between 25 and 35 days. Cartilage at the suture (called the palatine symphysis by Mohammed) was still present at 200 days, so that it was not as temporary as Pritchard et al. had supposed, at least for the region level with the molar teeth. Moreover, even at 30 days of age, the cartilage cells present are still able to react by proliferation and hypertrophy to excess growth hormone (Kurtz et al., 1970).

    In the rat, Mohammed (1957) could see that nasopalatine and his two palatine (secondary maxillary) cartilages form independently of one another. Although in other species an adequate sample of fetuses is needed to determine the true developmental circumstances, the maxillary cartilages are generally accepted as secondary formations, when their existence is known.
    For example, Kochlar and Johnson (1965) gave high doses of vitamin A to pregnant rats at 9 to 12 days after insemination. Examination of their fetuses at 17-days' gestational age revealed "the occurrence of chondrogenesis in and around the maxillary areas of all treated embryos ..... this heterotopic cartilage partially or wholly replaced the maxillary bone, and sometimes was continuous with mandibular bone thereby producing maxillo-mandibular ankylosis."
    Without a doubt the extent of the chondrogenesis and the ankylosis are abnormal, but the cartilage is not wholly ectopic because small amounts of cartilage are present in the palatine process, at alveolar crests, by the molar teeth in the normal fetal maxilla (Mohammed, 1957), and in the zygomatic process (Youssef, 1969).
    Youssef also saw "a few small nodules of secondary cartilage" in the separate palatine bone, and Momigliano-Levi (1930b,c) referred a nucleus of secondary cartilage to the ascending part of the human palatine bone.

    CRANIAL VAULT SUTURES: TWO KINDS OF CARTILAGE?

    Other cranial sutures than the maxillary bear cartilage or a tissue akin to cartilage (Schaffer, 1888; Sitsen, 1933; de Beer, 1937; Pritchard, Scott and Girgis, 1956; Ragol'skaya, 1959 (cited by Polezhaev, 1972); Aaron, 1973; Markens and Taverne, 1978; and others). That the nature(s) of the tissue is problematic has been acknowledged already in discussing the value of referring skeletal tissues to those of the mandibular condyle.

    Schaffer (1888) referred to Robin and Herrmann's (1882) observation of a tissue at the tip of the growing antler that resembled, but was not proper cartilage. Schaffer commented that a material matching their description was present in microscopically small amounts at the margins of bones of the forming cranial vault. The material was homogeneous, had lacunae separated by thin walls giving it an areolar look, and resembled the tissue occurring at the very young mandibular condyle as the intermediary between bone and about-to-develop cartilage. The nature of the cranial marginal tissue was ambiguous then and has not been satisfactorily resolved since.

    Continental European authors have implied the existence of two skeletal tissues on cranial bones in general: one sometimes called Chondroid in an attempt to pin down its indeterminant nature; followed, in some locations, by one more like hyaline cartilage but by no means identical with primary skeletal cartilage. Certain British and American microscopists seem to have identified the same two tissues on cranial bones, but at the same time have drawn another distinction between them founded on their apparent fate: endochondral versus metaplastic ossification.
    De Beer (1937) may have started this practice, based on work by Momigliano-Levi (1930c). De Beer noted the marked resemblance of the secondary cartilage widespread on membrane bones to the hypertrophic cartilage of embryonic cartilage about to experience endochondral ossification. Fell's (1933) in vitro demonstration that chick femoral cells could form a large-celled cartilage which turned into bone caused de Beer to view secondary cartilage as a "special histological manifestation" of cells normally destined to make bone.
    In his agenda of questions needing study he asked "can secondary cartilage cells be demonstrated in vitro to turn into osteoblasts and secrete phosphatase?" From this it appears that he accepted Levi's (1930c) proposal that the initial regions of secondary cartilages transformed into bone in vivo.

    In their study of fetal and infant sheep, cats, rabbits, rats, and humans, Pritchard, Scott, and Girgis (1956) found cartilage at the sutural margins of several bones:
    "It was most common in the sagittal and midpalatal sutures at the end of the period of rapid growth, and was of two types:
    The first type occurred as irregular islands or areas of large-celled cartilage with scanty matrix, interspersed with, or capping the trabeculae of woven bone at or near the sutural edge. In one specimen such cartilage ran from one parietal to the other across the sagittal suture.
    The second type presented a more orderly appearace. In the palate of the rat, for example, both cambial layers of the suture were temporarily transformed into expanded epiphysis-like masses covering the margins of the bones. Each mass shows a regular gradation from pro-cartilage near the middle of the suture, through definitive hyaline cartilage, to hypertrophic cartilage adjacent to the bone".

    Moss (1958) soon followed with similar observations in the developing frontal suture in the rat. Anteriorly in the suture, at the free margins of the endocranial bones and at the point of fusion,
    "bone morphology was different. Large, irregularly spheroidal lacunae were seen. The endocranial cells were enlarged and pale-staining with eccentric nuclei. These cells were much more numerous and closer together than in the ectocranial bone (Figures 7, 8, 9). The thin eosinophilic matrix about these cells was somewhat retractile, birefringent, and had an interwoven fibrillar appearance. These fibers were continuous with and extended into the sutural tissues."
    This description by Moss of a variant of bone matches Schaffer's (1888) account of the condylar Uebergangsgewebe: for example, "the whole gives the impression of a cartilage-like tissue whose somewhat coarse-fibered intercellular substance still stains intensely with eosin. It merges gradually with the surrounding formative tissue, the character of which we already know, and constitutes a tissue transitional to the hyaline cartilage appearing here in the next stage."

    More anteriorly in the rat's frontal suture Moss found large cells surrounded by a scant, basophilic matrix. Still further forward were processes resembling endochondral ossification,
    "areas containing pale hypertrophic cells and pale-staining basophilic, non-birefringent matrix were seen surrounded by newly-formed bone..... Definite vascular erosion of the earlier hypcrtrophic tissue was seen (Figure 13). .. . No true osteoblasts or chondrocytes were observed. Most anteriorly, at the site of first fusion, these replacement processes had been completed. The tissue forming the endocranial ridge consisted of bone trabeculae alone. The osteocytes were much smaller than the antecedent cell types and highly basophilic."

    Moss (1958) noted the similarity of the fusion of this suture to fracture healing in its rapidity and the fate of the tissues involved, in particular, the "transitory tissue" first uniting the suture. Of this tissue he wrote,
    "The classification of these hypertrophic, pale-staining, cells surrounded by their thin refractile matrix is most difficult. Several alternatives must be considered. This tissue may be immature bone, secondary cartilage, or an intermediate tissue type of a lower order of differentiation".
    Moss ended with all three entities fused in his "intermediate form of secondary cartilage," which by being "transformed directly into bone" is, in effect, an immature form of bone.

    He compared the tissues that he saw in the frontal suture with what Pritchard, Scott, and Girgis (1956) had seen in the palatal and other sutures, and concurred with them that sutural fusion involved two types of secondary cartilage. One was the intermediate form of a "lower order of differentiation," with larger and more irregular cells than in the second, definitive, secondary cartilage.
    Moss wrote: "The term definitive secondary cartilage should be limited to those areas of transitory cartilage which differentiate without reference to the primitive chondral skeleton, and which are replaced by the processes of endochondral bone formation."

    While the first criterion is in agreement with Schaffer's definition of secondary cartilage, the imposition of the second, endochondral replacement, straightaway places certain members in an anomalous position. Moss offered more than one criterion to distinguish definitive from intermediate kinds: not only the subsequent mode of ossification, but also the size of the cells and the regularity of their arrangement. Multiple criteria raise the possibility of a tissue's meeting one measure but failing in another, thus blocking an attempt at a rigorous separation into two categories. Examples of such an obstacle confound the attempt of Pritchard et al. (1956) and Moss (1958) to subdivide cranial secondary cartilage.

    Moss suggested that the palatine cartilage is a site of a definitive secondary cartilage which undergoes endochondral osteogenesis. However, Anderson, Furstman, and Bernick's (1967) Figure 2 shows that this cartilage in the newborn rat has the large irregular cells typical of Moss's intermediate variety before it acquires a more regular layering and endochondral replacement commences.
    The other maxillary secondary cartilage also has large, irregular chondrocytes, as in Mohammed's (1957) Figure 39, but according to him the tissue is replaced by bone rather than experiencing a metaplasia.
    Another site where the criteria for intermediate versus definitive give a confused verdict is in the anterior secondary cartilages on the mandible, which Baumuller (1879) saw undergoing both metaplastic and endochondral modes of ossification.

    The basis for the metaplastic interpretation is the presence of chondroid bone I. As in fracture callus, tumors, and elsewhere, chondroid bone enters into the cranial margins in two ways:
    (1) the skeletal precursor cells may form a tissue intermediate between bone and cartilage, and such a chondroid bone is seen joining the smaller secondary cartilages to bone, e.g., Figure 21 of Pritchard et al. (1956) and Figure 39 of Mohammed (1957); or
    (2) as an obligatory step in the transformation if, as several have believed, cartilage turns into bone, although it is possible that the observers may have construed chondroid bone actually arising from precursor cells, as a token of metaplasia. For example, Young (1959) gave a very dynamic account of metaplasia in the posterior metopic suture of rats made hydrocephalic:
    "Endocranially, hypertrophic cartilage cells appeared connecting the two bones. Intercellular substances increased in amount, while the cells shrank, until they resembled new osteocytes (Figure 27). The matrix then increasingly took on the staining properties of bone."
    While the impression is of events watched in the same cells and tissue, the story is, as usual, based on separate observations at the same site. Common to the observations was the merging of cartilage with bone through a transitional zone, as is seen in Young's Figure 27 with its legend, "Note large amount of secondary cartilage, and evidence of its direct conversion to bone."

    The simplest explanation for the variety of tissues and events reported is one offered before for the mandible, viz., that one is seeing the results of cells switching from osteogenesis to chondrogenesis, but being allowed sometimes more time, sometimes less, to do so. When the periosteal germinal cells first turn into or towards chondroblasts, the resulting tissue is close to bone, spatially and by its nature. Confusion has arisen because it has been classified not as chondroid bone, but as anomalous cartilage: Moss's intermediate cartilage. If the germinal cells soon revert to osteogenesis, the "intermediate cartilage" is enclosed by bone, as Moss (1958) observed, producing circumstances suggestive of a metaplasia.

    If the superficial proliferative cells have time to produce additional chondroblasts, a more cartilaginous tissue becomes established, in which the sequences and layers typical of endochondrally ossifying cartilage (Moss's definitive secondary) appear, as at the palatine suture. Early on these larger, more hyaline cartilages are joined to the pre-existing bone by the CB I that formed as the switch to chondrogenesis began. This linking CB also can give an impression that cartilage is becoming bone directly.

    Should the above account be true, there is no basis for Moss's assignment of a "lower order of differentiation" to the intermediate cartilage. Furthermore, it is unwise to make the questionable metaplastic fate of some of the tissue grounds for a subdivision, but better to take Knese and Biermann's (1958) view that all these post-primary cartilage-like tissues on cranial bones belong in the secondary class.
    If a distinction needs to be made, it could be to recognize the boniness of the first cartilage formed, i.e., its status as CB I. What is more important is to discover what underlies the difference in time allowed for chondrogenesis, and the determinations that endochondral osteogenesis and fusion are to be implemented in one site but not another.

    A high dose of X-irradiation to the 12-day rat fetus can reverse the normally very high ratio of bone to chondroid bone and cartilage in the early cranial vault (Schmahl et al ., 1979). At 18 days post-conception, "abnormal chondrification is manifest by a neurocranial capsule consisting of a mass of cartilaginous tissue instead of the individual ... bones." The authors attribute the cartilage to sutural mesenchymat cells.


    Chapter 12 AVIAN SKULL

    Sites of secondary cartilage
    Avian cranial chondroid bone I
    Reptilian secondary cartilage
    Markers of chondroblastic differentiation

    SITES OF SECONDARY CARTILAGE

    Strasser (1905) referred to a cartilage forming at the inner end of the pigeon's pterygoid, and proposed that stress and strain resulting from movement stimulated the development of such secondary cartilages. Murray (1963), who provided the most detailed account of avian cranial articulations, cited Bock (1959, 1960) as describing fibrocartilage on each articular surface, where the internal process of the mandible meets the skull in the skimmer (Rhynochops nigra).

    In the chick, Murray (1957, 1963) found secondary chondrifications on the bones at many sites, but principally at articulations. In the case of the quadrate-quadratojugal, quadrate-pterygoid, quadrate-squamosal, and pterygoid-cranium, the joint had an articular cavity. A cavity was absent at the squamosal-otic capsule, Meckel's cartilage-surangular and angular, and pterygoid-palatine. In one instance, adventitious cartilage appeared not at a joint but on the squamosal underneath M. depressor mandibulae. While some of the above bones are less mobile than others, Murray observed that for both mobile and less mobile bones the cartilage arose on those surfaces to which the arrangement of the muscles probably directed pressure.

    Hall (1967b) performed a similar study on the eastern rosella, Platycercus eximus (Shaw). He examined nestling and juvenile birds. Secondary cartilage was present "on the first-named component in all the nestlings and fibrocartilage on both components in all the juveniles" at the quadratojugal-quadrate, pterygoid-quadrate, palatine-pterygoid, pterygoidparasphenoid, surangular-Meckel's cartilage, and squamosal-otic process of the quadrate.
    The frontal-maxillary and jugal-maxillary articulations are not fully formed in the nestling. In the juvenile, both surfaces have cartilage and it is adventitious, i.e., more hyaline than fibrocartilage. As in the chick, cartilage formed at both mobile and relatively immobile articulations. The later development of fibrocartilage at articular surfaces is seen elsewhere in the bird, e.g., Fuchs (1909a), and is likely related to the predominance of collagen of type I observed at the avian articular surface by Eyre, Brinkley-Parsons, and Glimcher (1978).

    Not every cranial articulation had secondary cartilage. Its absence where the quadratojugal and jugal overlap and at the articulation of the angular with Meckel's cartilage,
    "where the two elements lie parallel to one another along the whole of their lengths, and where shear and tension are the chief components of the mechanical stress, argues for pressure as the evocator of adventitious cartilage" (Hall, 1967b). Chapter 4 reviewed Murray and Hall's experimental evidence in support of this hypothesis.

    AVIAN CRANIAL CHONDROID BONE I

    Murray (1963) commented of 18-day chick embryos,
    "one could distinguish between obvious cartilage, obvious bone, and a third tissue which seemed, after chlorantine red and alcian blue, to combine characters of each (Plate 18, Figures 4 and 5).... In the intermediate tissue the cells resembled cartilage cells and had capsules like those of adventitious cartilage cells, but were embedded in a matrix dyed more or less intensely red. There seemed to be no sharp topographic boundary between this tissue and either obvious bone or obvious cartilage. Because of the character of the cells, this tissue seemed to be a late stage in the history of the adventitious cartilage; but because the cells did not seem to develop canalicules, it did not seem possible to recognize it as bone."
    This item was under the subheading "Possible direct transformation of cartilage with bone," but Murray deferred his discussion of metaplasia until a later article (Murray and Smiles, 1965).

    He did waver in his estimate of the time of development of the intermediate tissue, thus (Murray, 1963),
    "this specimen throws some doubt on the view expressed in section [as quoted above] that tissue having cartilage-like cells in bone-like matrix is a late stage in the life history of adventitious cartilage, for here the bone-like character was present almost from the first differentiation of the tissues."

    Hall encountered the same intermediate tissue on the parrot's surangular and its articulation with Meckel's cartilage and at the quadratojugal-jugal joint; and his Figure 9 (Hall, 1971) reveals the transitional tissue on the surangular of a one-month-old fowl.
    In the bird, as in mammals, accessory secondary cartilages therefore may be joined to their bones by chondroid bone. Neither Murray nor Hall named it chondroid bone. The secondary cartilage and chondroid bone are both shortlived, as erosion extends into them from the bone, and fresh cambial cells make a new overlying tissue which may or may not be cartilaginous.

    Townsend and Gibson's (1970) description of some of the chick's angular bone suggested a chondroid nature, but it was unaccompanied by secondary cartilage. Thus, from the eighth to 10th days of incubation,
    "Occasionally, patches of more intense alcian blue staining were evident in the area subjacent to the periosteum," (their Figure 16), and "......The cytoplasm of the bone cells also stained with alcian blue."
    The same subperiosteal region had the most intense metachromasia. The authors attributed the bone's staining with alcian blue to a glycoprotein or its precursor, and linked the extracellular metachromasia to a sulfated protein-polysaccharide bound to collagen.

    REPTILIAN SECONDARY CARTILAGE

    Since the snake, a reptile, has as kinetic a skull as the bird, Murray (1963) anticipated the existence of secondary cartilages, but Hall was unable to find any in a nearly full-term fetus of Notechis sculatus.
    In the lizard, Fuchs (1909a) reported cartilage on the pterygoid of Lacerta vivipara. He described the cartilage as merging gradually into the bone, thereby implying an intervening zone of CB. Whether reptiles have secondary cartilage on their membrane bones needs thorough study.

    MARKERS OF CHONDROBLASTIC DIFFERENTIATION

    Making use of the ease with which skeletogenic cells at the avian cranial secondary cartilages can be switched experimentally to osteogenesis or chondrogenesis, Hall (1968d, 1969, 1972a) has attempted to discover how the cellular commitment to forming one particular tissue is first detectably expressed. Pritchard (1952) had earlier essayed this task using histochemistry and light microscopy. Pritchard noted several features held in common by maturing chondroblastic and osteoblastic cells, with little to distinguish them absolutely until their synthetic products appeared. Moreover, in Hall and Storey's (1968) Figure 19 depicting diagramatically the ultrastructural changes in developing avian osteoblasts and chondroblasts, only one cell represents the newly differentiated osteoblasts and chondroblasts. However, these authors listed some subtle differences in the cells and matrix that, they believed, distinguished the germinal cells destined to form secondary cartilage from those which would make bone; for example, more mitochondria in pre-osteoblastic germinal cells.

    Although Murray (1963) had seen chondroid bone at the margins of the cartilage, Hall and Storey observed no cells intermediate between osteoblasts and chondroblasts. This finding could be viewed as confirmation that the cells of chondroid bone at this site are, as Murray described, cartilage-like, and it is the matrix which has a bony cast.

    The quest for an early indication of the determined state has led Coffin and Hall (1974) and Thorogood and Hall (1976) to look not at the cells' macromolecules in the matrix, but at their complement of enzymes. Thorogood and Hall (1976) reported that among the cells of the chick's quadratojugal bone, "determined but cytologically 'undifferentiated' progenitor cells" can be distinguished on the basis of the ratio of lactate dehydrogenase's activity to malic dehydrogenase's (LDH/MDH). Cells determined for chondrogenesis have a ratio greater than one and the converse holds for pre-osteogenic cells. From this, one may predict that the ratio of enzymes would be around one for a cellular population destined in, say, callus or a tumor, to form chondroid bone I.

    However, does the adaptation in enzymes of, say, chondrogenic precursor cells to less aerobic circumstances reflect the environment in which they now find themselves, or is it a first step in anticipation of a future decrease in oxygen tension? If it is the latter, can it not in effect be viewed as an early but actual step in the differentiation of a chondroblast? If so, the cells that have altered their LDH/MDH ratio to a value above one are already chondroblasts and are no longer unexpressed progenitors. They have already expressed themselves by raising their level of LDH.
    Weiss and Amprino's (1940) expectation
    "that the conventional distinction between 'determination', i.e., implicit differentiation ... and 'manifest differentiation' will fade, as our physical and chemical methods of discrimination become more sensitive and refined"
    is only now slowly being realized for the connective tissues.


    Chapter 13 ANTLERS AND HORNS

    Introduction
    Postulated tissues and mechanisms of antlerogenesis
    Cartilage, bone or chondroid bone? Chondroid bone and chondrobsteoid
    Chondroid bone and metaplasia
    Cornugenesis

    INTRODUCTION

    These splendid and fascinating structures have been known since early in the last century to be mostly bone (antler) or to have a bony core (giraffid-ovid-bovid horns). The significance of horns or antlers for the bearer's behavior and in evolution was of interest to Geist (1966). [ More recent comprehensive reviews are in Bubenik and Bubenik (1990).] The annual shedding and regrowth of antlers are initiated by photoperiodicity (Goss, 1977) through fluctuations in the secretion of testosterone (Bubenik et al.,.1974; McMillin et al., 1974), and is associated with an osteoporosis (Hillman, Davis, and Abdelbaki, 1973). The induction of antler development by trauma (Jaczewski and Krzywinska, 1974) may shed light on a possible mechanism of tumorigenesis and some secondary chondrogenesis. Wislocki (1942) presciently accorded the overlying epithelium a place in the initiation of antler formation, and thereby anticipated the interest in epithelio-mesenchymal interactions.

    Osteogenesis and chondrogenesis have lent themselves most readily to examination in deer, which have little commercial value, develop their antlers after birth, except for a brief phase of determination and early differentiation of a pedicle in fetal males (Lincoln, 1973), and regrow their deciduous antlers according to a well-known timetable. By contrast, papers dealing with the histogenesis of horns are few, for instance, Robin and Herrmann (1882), Gadow (1902) and Atzkern (1923).
    Horns are discussed after the antler, and it can be assumed, unless noted, that Europeans looked at antlers of roe, red, or fallow deer, and Americans looked at white-tailed or other American species of deer. The same descriptions, problems, and divisions of opinion arose on both sides of the Atlantic, so that Banks' (1974) suggestion that there may be differences, at the level of tissues, according to species - metaplasia in red, but not in white-tailed - is unlikely, particularly in light of Modell and Noback's (1931) direct comparison, and their conclusion that the processes of ossification were so similar in the Virginia (white-tailed) deer, Wapiti, and European red deer that a description of only one - the Wapiti or elk (Cervus canadensis) - would suffice.

    The history of antler development is thick with controversy over the presence of cartilage and the possibility of metaplastic osteogenesis. Robin and Herrmann (1882) have references to the prehistological studies in the 18th and early 19th centuries on the tissues in the forming antler. Rorig (1900) reviewed the work of the rest of the century. With interest in the structure already aroused, the early microscopists seized upon the antler along with all manner of bones in the hope of solving once and for all whether bone develops by metaplasia or a "neoplasia" by osteoblasts. The antler did not provide them with a simple answer. The rival views of Muller (1858, 1863) on behalf of endochondral ossification and osteoblastic osteogenesis, and Lieberkuhn (1865) and Gegenbaur (1867) for a direct osseous transformation of cartilage are echoed still by Banks (1974) and Goss (1970).

    POSTULATED TISSUES AND MECHANISMS OF ANTLEROGENESIS

    If cartilage does participate in the development of antlers and horns, it would be secondary, both in time - forming after the primary cartilages - and in position, quite removed from them. Almost all investigators have agreed that the antler's first-formed firm tissue is cartilage-like. But, young bone is itself somewhat similar to cartilage, so is this early tissue: 1) typical cartilage; 2) a special cartilage; 3) typical immature bone; 4) a special variety of bone; or 5) an intermediate chondroid bone, i.e., a special kind of bone and cartilage?
    Opinions on the character of the antler's initial firm tissue have been so polarized, stressing either its boniness (3 and 4), or its cartilaginous properties (1 and 2), that the choice, chondroid bone, (5), has not been mooted, although it would give recognition to the validity of each school's observations. Assuming for the present that the first-formed tissue is a chondroid bone 1, but veering towards the cartilaginous, how does such a cartilaginous CB contribute to the formation of the proper bone appearing as the antler continues to grow?
    The answers are varied, and have been offered in different combinations:
    1. The tissue, cells and matrix, is transformed into bone.
      (a) If the initial material is held to be a cartilage, this is classical osseous metaplasia as proposed by Lieberkuhn (1865) and Goss (1970).
      (b) On the other hand, if one regards the starting tissue as an already partly bony precursor of bone - a preosseous substance - its transformation can be regarded as a special kind of metaplasia, the view of Robin and Herrmann (1882), or
      (c) merely as a step, more prominent than usual, in the differentiation of bone, i.e., no metaplasia at all, as Landois (1865a) and Wislocki (1942) would have it.
    2. In the opinion of Modell and Noback (1931), the cells of the tissue degenerate and die, but the existing matrix is adapted to become bone by newly arrived osteoblasts' causing a calcification of the matrix. The cellular atrophy and degeneration are accompanied by shrinkage, so that the initially thick columns lying between the numerous vessels narrow as they are transformed into the trabeculae of the deeper lying spongiosa. Osteoclasts are present, but play a minor role in the narrowing.
    3. Opposing the idea of a self-induced shrinkage held by Modell and Noback (1931) and Wislocki (1942), is the more widely advocated mechanism of chondroclasia or osteoclasia (Muller, 1863; Gadow, 1902; Mollelo et al., 1963; Banks, 1971) depending on how one looks upon the target tissue, with large cells actively resorbing the initial firm tissue.
    4. Opinions on how such a chondroclasia fits in the overall osteogenic sequence have differed. Harking back to an idea common around 1865, Macewen (1920) proposed that a resorption of the matrix liberates chondrocytes, which thereupon become osteoblasts laying down bone on what remains of the cartilage columns. This interpretation is exceptional, for most histologists have had the inter-columnar connective tissue (Landois, 1865a; Wislocki, 1942), or more distant subdermal connective tissue (Modell and Noback, 1931), as the source of the osteoblasts.
      Thus, the cartilage-like first tissue was seen by Muller (1863) and his followers as serving a similar role to that in long bones and other bones formed in hyaline cartilage, namely, to be partially resorbed, while separately derived osteoblasts deposit bone on what remains. Then, both the bone and cartilage are destroyed in a further restructuring of the antler to a denser bone. This essentially endochondral process differs in its particulars, but not in principle, from that of other non-membranous bones; Banks (1974) inter alios is of this belief.
    5. While the interior of the cartilage-like tissue contributes to ossification in whatever way it does, on the antler's lateral surfaces cells differentiate into osteoblasts and make a subperiosteal sleeve of bone, such as is seen on the developing mandible and penile bone and embryonic long bones. This appositional deposition of bone continues and by tradition has been termed intramembranous, as for example by Banks (1974), who characterized ossification in the antler as a combination of intramembranous and endochondral kinds (but with no metaplasia).
    The modes of ossification proposed for the antler can be summarized thus:
    1. Subperiosteal only
                         (a)--sides of antler *
                         (b)--tip of antler
                               germinal tissue--> preosseous tissue--> osseous tissue
    
    2. Via cartilage at the tip
                    (a)--replacement by bone    | both involve a narrowing of the
                                                | original column by (y) chondroclasia
                    (b)--transformed into bone  | and (x) shrinkage
    
    3. Endosteal   (a)--bone laid on primary spongiosa *
                   (b)--remodeling of primary spongiosa *
    
    *  Undisputed modes of ossification
    
    Actions (*), 1. (a) and 3. (a) and (b), have aroused little controversy, being sometimes described, sometimes not, while each author put forward his particular combination of processes I.(b), 2.(a) and (b), (x) and (y), with supporting data.
    There is enough evidence of erosive clastic cells acting upon bone and "cartilage" to put aside the possibility (x) of a shrinkage of the cartilage-like tissue and to pass on to a consideration of its other properties. But acceptance of a partial destruction means that any bone formed in its place ipso facto brings in step 2.(a) - a replacing or endochondral ossification. Thus, if conventional ostcoblasts cover the primary spongiosa with bone (which is accepted), and do this at the margins of spaces made wider by the erosion of the cartilage-like tissue (now also barely disputed), some of the ossification is of the replacing type.
    Banks (1974) drew from the extensiveness of the endochondral ossification the implication that other kinds, such as the direct metaplastic, are thereby excluded. However, Muller (1863), who first clearly described the main endochondral events, was puzzled by the existence of regions of transition where bone merged with calcified "cartilage."

    CARTILAGE, BONE OR CHONDROID BONE?

    We must now ignore the other processes involved and the interesting differences between endochondral ossification in the antler and elsewhere noted by Muller (1863), Gadow (1902), Modell and Noback (1931), Grubel (1937) and Banks (1971), in order to concentrate on the following questions (rephrased from the five put earlier):
    1. Is the transitional tissue a form of chondroid bone?
    2. What is its relation to the initial firm tissue?
    3. Are these tissues evidence for a metaplastic ossification, 2.(b), of the kinds tabulated, in addition to the osteoblastic kinds, 1.(a), 2.(a), 3.(a)(b)?
    4. Is there no metaplasia, but rather a special periosteal osteogenesis?
    5. Is the first firm tissue formed enough like cartilage to bring it into the ambit of secondary cartilages?
    I shall argue that: the apparently divergent answers can be reconciled by referring them to differing techniques and emphases on what was seen; the first tissue is very like cartilage, but changes to a kind of chondroid bone; this material, as with other chondroid bone, is suggestive of metaplasia; and the early tissue is sufficiently cartilaginous to justify its inclusion among the secondary chondrifications.

    As the tissue under the cellular cap differentiates, its cells enlarge, become more rounded or ovoid and deposit an intercellular matrix. Around the cells, capsules form which are metachromatic and alcianophilic. The extensive histochemical studies of Wislocki, Weatherford and Singer (1947), Mollelo, Epling, and Davis (1963), Banks (1974), and Frazier, Banks and Newbrey (1975, with this group employing pre-digestive techniques) demonstrate the matrix and especially the capsules to be rich in the mucosubstances histochemically characteristic of cartilage.
    This fact, added to the hypercellularity (Banks 1974), large volume and rounded shape of the cells (Molleto, Epling, and Davis, 1963), and their fine structure (Banks and Neal, 1970; Sayegh, Solomon, and Davis, 1974), disorderly arrangement (Banks, 1971), and alkaline phosphatase content (Wislocki et al. 1947; Ronning et al., 1990)), the later perilacunar mineralization (Banks, 1971) and the reduction in the interterritorial matrix (Banks 1974), lends strong support to considering the tissue as a kind of cartilage, endowed with many of the attributes of such secondary cartilages as the mandibular condyle's.

    If the antler's tissue is cartilage, does it nevertheless have other properties that might distinguish it from other kinds of cartilage and ally it to some degree with bone? This consideration was more or less dismissed by Newbrey and Banks (1975) when they wrote of the antler tissue and mineralizing somatic cartilage,
    "Banks and Neal (1970) however, have demonstrated from ultrastructural studies, that the differences are minimal."

    With their stress on proteoglycans, cellular morphology, and the correspondence of the possible parts played by matrix vesicles (Newbrey and Banks, 1975) and intra-mitochondrial materials (Sayegh et al., 1974) with events in primary cartilages, these authors drew attention away from the properties that had caused Landois (1865b) and his many successors to think of the tissue as bone rather than cartilage. However, Muller (1863), Robin and Herrmann (1882), Wislocki, Weatherford, and Singer (1947) were willing to concede the boniness of some of the cartilage, or the cartilaginicity of the bone.

    Three factors, two specific and one general, have impressed microscopists as conferring bone-like properties on the tissue, namely, the abundance of fibrils in the matrix, the small size and dark staining of some of the cells, and an overall resemblance of the tissue to bone or chondroid bone of the developing skull. Taking the last item first, Landois (1865b) wrote that the bones of the newborn mouse's skull vault provided pictures very similar to those of the early tissue of the antler, but he was too busy stressing its dissimilarity from hyaline cartilage to give it a name.
    Robin and Herrmann (1882) corrected this omission. They called the tissue tissu preosseux, comprising osteoblastes enclosed in a substance preosseuse. In the third section of their article, they discuss the substance preosseuse as it occurs generally in sites of intramembranous ossification like the facial bones, in the cranial vault and osteoblastic tumors, as a homogeneous, finely granular product of the osteoblasts. Robin and Herrmann's tissu preosseux was the osteoid of Virchow (1864), in other words, early woven bone. This tissue, although not fully mineralized, was not osteoid in its modern sense of the very mineral-poor material seen on new bone, more prominent in some circumstances, e.g., renal osteodystrophy (Bonucci, 1977), than others.

    Robin and Herrmann (1882), in fact, distinguished between the extensive tissu preosseux, or in sites of endochondral ossification le tissu dit osteoide, and a zone hyaline limitante de l'os, probably the modern osteoid border. That the former tissue was calcified is evident from the fact that acetic acid eliminated most of the granularity of its matrix. The acid treatment also changed the appearance of the matrix from fibrillar to homogeneous, in which condition, along with its sizable lacunae, the tissue could not be denied a certain resemblance to closely-celled cartilage, as occurs in fracture callus and tumors. However, the matrix stained with carmine and, without an acid treatment, was fibrillar and finely granular.

    American anatomists later adopted the term preosseous tissue, but adapted it in subtle but significant ways to mean only the early tissue of the antler rather than new bone in general, and within the antler, only the unmineralized regions of the tissue. Modell and Noback (1931) and Wislocki (1942) commented on the similarity between the formation and maturation of the antler's preosseous tissue and osteogenesis in the skull, for example, in the frontal bone of the six-month-old fetus (Wislocki, 1942). Wislocki was later (Wislocki et al., 1947) to modify his conception of the tissue in favor of something more cartilaginous, while still retaining the name "preosseous", thus giving it a meaning even further removed from Robin and Herrmann's.

    With a method based on silver carbonate, Modell and Noback (1931) revealed the matrix to be more strikingly fibrillar than had been evident from earlier carmine- and eosin-stained preparations. They went so far as to write that the "pericellular capsule is really an illusion produced by interlacing ribrils," but they did not employ any stains for proteoglycans. Wislocki (1942), who used azan, and Wislocki et al. (1947), with a silver technique and toluidine blue, were struck by the extent of both the collagenous fibrils and the non-fibrillar component of the capsules.
    More recent workers (Banks, 1974; Sayegh et al., 1974; Newbrey and Banks 1975) described the fibrils but paid them little heed in reaching a verdict on the tissue's nature. For those who have paid attention to the fibrils, the fibrils have immediately distanced the tissue from hyaline cartilage. On the other hand, the fibrils are not dense enough to take the tissue unequivocally into the category of fibrocartilage (Robin and Herrmann, 1882). In casting around for potential relatives of the antler tissue, Wislocki et al. (1947) rejected elastic and hyaline cartilage, but looked more favorably on fibrocartilage and the "chondroidal supporting tissue" that they observed in the lyssa of a slow lemur (Perodicticus potto):
    "Thus, we find the closest histological parallel to the preosseous tissue of the antlers in fibrocartilage and chondroid supporting tissue. However, the preosseous tissue of the antlers is bone formative, whereas the latter tissues are not normally described as being replaced by bone."
    One may add that not only is the fate of these tissues an issue separate from their histological similarity to antler tissue, but instances of their ossification are known and are discussed in Chapter 10. From their assessment of the tissue, Wislocki et al. proposed a new mode of ossification, "chondroidal" or "fibrovesicular," as occurring in the antler and sharing characteristics of endochondral and intramembranous types and involving metaplasia.

    Another departure of antler cartilage from typical endochondral cartilage is in its very low proportion of type II collagen (5%), but abundant expression of type I (Rucklidge et al., 1997).

    By now, it must be evident that the early tissue of the antler should not be too hastily equated with any other single tissue, since it combines properties of hyaline and fibrocartilage, chondroid, and young, large-celled bone. If it is to be accurately placed among the various classes of supporting tissue, that of chondroid bone cannot be omitted from contention. But now the zonation of the antler's growing tip needs to be described.

    ZONATION AND THE POSITION OF CHONDROID BONE

    The scheme of zonation below is based upon Banks's (1974) and Frazier, Banks, and Newbrey's (1975), but it will be appreciated that the number of divisions considered significant and the terminology chosen for them vary somewhat with each author's idea of the mode of ossification; compare, for example, Modell and Noback's (1931) Figure 1. Under the skin is:
    1) a periosteal-perichondral sheath of fibrous tissue and germinal cells;
    2) below this is a region of cellular differentiation and starting matrix synthesis;
    3) deeper is a columnated zone richer in matrix and with large cells; further removed from the tip the columns have cores that differ from their peripheral regions adjacent to the intercolumnar fibro-vascular tissue; and
    4) well away from the cap is a zone where the columns have more bone-like tissues upon and within them, the primary spongiosa.

    The tissues of zone 3 and the interior of the columns or trabeculae of zone 4 appear to be essentially the same, although there are more small and darkly staining cells in the deeper regions (Banks, 1974). The description of this tissue as having mostly large, rounded cells containing alkaline phosphatase and with proteoglycan-rich capsules, and a mineralized matrix permeated by many collagen fibrils, fits chondroid bone I.
    Numerous published illustrations affirm the resemblance of this tissue to other examples of chondroid bone I, for example, Figure 6 (Frazier, Banks, and Newbrey, 1975); Figures 12 and 13 (Banks, 1974); Figures 1 and 7 (Mollelo, Epling, and Davis, 1963); Figure 20 (Wislocki, 1942); Figure I (Belanger, Choquette, and Cousineau, 1967).

    Some of the likenesses drawn between the antler's tissue and other materials at various skeletal sites lead one to suspect that these may have been not so much bone as chondroid bone, thus lending indirect support to their antler look-alike's being chondroid bone. While Robin and Herrmann (1882) drew a parallel between antler histogenesis and that of the upper skull, they referred specifically to the margins of the cranial bones, and elsewhere to fracture callus and tumors, all common sites of chondroid bone I.
    Wislocki (1942) had G.A. Bennett look at his slides, and the pathologist showed him a very similar tissue in the extensive new trabeculae on the femur of a child with syphilitic periostitis. Zawisch-Ossenitz (1929b) had observed chondroid bone I on the human femur in its normal period of rapid osteogenesis before birth. It is therefore possible that the periostitis provoked bone formation at such a pace that a tissue more appropriate to fetal times reappeared.

    CHONDROID BONE AND CHONDROOSTEOID

    That four zones, and some subdivisions (Frazier, Banks, and Newbrey, 1975), can be discerned in the antler's tip signifies that the tissue(s) there is changing. How much of the tissue is chondroid bone? And does the chondroid bone turn into anything else? The tissue is initially cellular and then gathers matrix, which later calcifies. The unmineralized tissue has been given its own name, preosseous tissue, by Modell and Noback (1931). If, after calcification, it is chondroid bone, one could term its unmineralized precursor chondroid osteoid or chondro-osteoid, as has been done quite often for the material occurring in fracture callus. However, I have deliberately avoided drawing a distinction between chondroid osteoid and chondroid bone for reasons which also hold good for the antler.

    First, exactly how osteoid is to be defined is unsettled. Zawisch (1947) was correct but a little overwrought when she wrote,
    "The tragic fate of the word 'osteoid' becomes more and more obvious every year."
    Second, and related to the first, calcification occurs by increments, so that there is a problem of how to classify partly mineralized bone.
    Third, the majority of sightings of chondroid bone have been in decalcified tissue, without any direct evidence on the state of calcification.
    Fourth, even with methods to reveal the mineral, there is the possibility that the first fine crystals may have escaped, so that the technique reads low for the extent of mineralized tissue present.
    Fifth, chondroid bone I often accompanies rapidly growing membrane bone, which Boyde and Hobdell (1969) showed by scanning electron microscopy mineralizes very closely upon collagen synthesis, so that an osteoid state can hardly be said to exist. While chondroid bone might also calcify very quickly, some of the important unanswered questions concern its hardness in its various expressions and the relation of any mineral to fibers, proteoglycans, vesicles, etc. Not only for the antler, but for all instances of CB I, I have chosen to use chondroid bone for both the mineralized and unmineralized regions of the tissue.

    A possible objection to identifying the tissue here as CB I is that the formation of a cartilage-like tissue which then mineralizes is essentially the mechanism whereby chondroid bone II - mineralized persisting cartilage - develops. True enough. The category of chondroid bone itself is so sandwiched in between cartilage and bone, that it is no surprise that one ometimes seems to be splitting hairs to further separate CB I and II. The justifications for placing the antler's tissue in CB I are: what first develops is not hyaline, elastic or fibrocartilage, but a kind of cartilage more bone-like than these; soon after its calcification, the tissue is resorbed or, in part, perhaps undergoes a metaplasia, so that in neither case does the tissue persist, but is soon consumed in the fast growth for which the antler is noted. But, I admit the illogicality of using mineralization as the criterion for CB II, but neglecting it for CB I. CB I does, however, have organic characteristics of bone.

    CHONDROID BONE AND METAPLASIA

    The consensus that part of the CB I of the antler is being transformed into bone derives its strength from the deeper region of the third zone (Banks's 1974, chondrocytic), and the nature of the columns in the fourth zone or the primary spongiosa. Here is found the continuity of chondroid bone with bone; and here is seen a variety of cells, ranging in a spectrum from large, rounded, and encapsulated, to osteocyte-like ones, smaller, more elongated, angulated and darkly staining, on occasion with extensive processes. (There is a general problem with cartilage (and chondroid bone) in that fixation by immersion might have caused some of the apparent shrinkage and pyknosis of the cells, and some chondrocytes are naturally small and dark (Hwang, 1978).)
    Caution has to be exercised in interpreting small, dark cells as either degenerating chondrocytes or ones undergoing a transformation to osteocytes. Even those skeptical of any metaplasia, such as Banks (1974), have remarked on the heterogeneity of the cells in the tissue deep to the surface of the antler's tip.

    Since osteoblasts are present on the sides of the columns of chondroid bone, there is always the likelihood that all osteocytes seen have their origin in the osteogenic inter-columnar and inter-trabecular connective tissue. Furthermore, that the osteocytes are intermingled with the chondrocytes does not necessarily require the chondrocytic cells to have turned into bone cells. It could reflect a more disorderly pattern of erosion on the lower part of the columns, coupled with the original lack of symmetry in the dispersal of the cells throughout the matrix.
    This said, one has to conclude that with so many astute microscopists (Gegenbaur, Lieberkuhn, Kolliker, Strelzoff, Kassowitz, Landois, Robin and Herrmann, Wislocki, Weatherford and Singer, Goss, Gruber, Lojda, and recently Ronning et al., 1990 for reindeer antler)) convinced that some of the antler's chondroid bone/preosseous tissue/cartilage turns into bone, the antler cannot be excluded from the sites of possible osseous metaplasia. Again, what is needed is a study where individual cells are labeled so that their fate can be related to that of their enclosing matrix.

    CORNUGENESIS

    What occurs in the developing horn does not differ in its essentials - kinds of tissue present and the manner of their interactions - from those described for the antler. Robin and Herrmann's (1882) comparison of antlers with the bony cores of the horns of cows, rams, and giraffes, and with their skulls, gave few details of the early development of these structures. At the end of the nineteenth century several dissertations and monographs dealt with the structure of horns and whether they and antlers are homologous. The accessible articles are those of Fambach (1901, 1909), Gadow (1902), and Lankester (1902), and later papers by Atzkern (1923), and Bruhin (1953).

    The reports of cartilage (Gadow, 1902; Atzkern, 1923) are significant because: 1) the cartilage constitutes a secondary formation, and 2) may be, at least in part, chondroid bone, particularly where it joins the new bone, as is the case with other secondary cartilages early in their development. Atzkern (1923) suggested that new bone, separate from the skull, appears first, and only later, after birth and after fusion of the ossicone with the skull, does cartilage appear. However, Atzkern gave only a brief summary of his findings, which lead one to think that his samples were spread rather widely in time, and that his ordering of events may be in error.
    Thus, he described cartilage as being present at the horn's tip in the chamois antelope, as islands within trabeculae (goat and sheep), or partly free and enclosed in osteoblasts (cow, sheep, and goat). The variety could be interpreted as resulting from a haphazard sampling in a developmental sequence, because Gadow described a series of events closely matching that in the antler, with cartilage and "muco-cartilage" anticipating the bone, and being selectively destroyed by osteoclasts (seen also by Atzkern). The histogenesis of bovid-ovid horns deserves more investigation, to clear up this seeming contradiction and clarify the participation, if any, of chondroid bone.


    Chapter 14 PHALLIC BONES

    Occurrence
    Secondary cartilages
    Development of the penile skeleton in the rat
    The Anterior fibrocartilaginous process
    The Penile bone
    Chondroid bone
    Penile skeletogenesis in other species

    OCCURRENCE

    The extraskeletal bones are bones separate from the basic mammalian skeleton. The antler has already been discussed, because its development involves chondroid bone and the possibility of metaplasia. The degree to which these entities and secondary cartilage participate in the several other sites of extraskeletal bone or cartilage, as, for example, the heart (Ellenberger and Baum, 1932), is uncertain, since usually not enough is known about their histogenesis. The phallic firm tissues are an exception.

    Zoologists have long been aware that a majority of mammals possess a penile bone, and a goodly number of species a clitoral one. Those without the former include man, rabbit, marsupials, and the ungulates (Gilbert, 1892). Animals bearing a clitoral bone encompass orangoutang, mole, guinea pig, porcupine, bear and some others (Wiedersheim, 1909; Gerhardt,1905; Simokawa, 1938; Layne, 1954; Burt, 1960).

    There is a long tradition of French interest in the os priapi. In his comprehensive comparative study, Chaine (1926) cited the work of Daubenton (1767) on specimens collected for the Cabinet du Roi, de Blainville's (1839) osteography, and the histology done by Retterer (1887, 1914). In America, Burt (1960) has described in detail and illustrated the gross morphology of the baculum in North American mammals (emitting bats and cats), which had not been covered thoroughly by the earlier, European Zoologists.
    Burt paid some attention to clitoral bones and suspected "that the bone is present in most, if not all, of the species that have a baculum". The confirmed absence of this element in the female rat (Wiesner, 1934, 1935; Glucksmann and Cherry, 1972) suggests that one should modify Burt's proposition to this: if the male of the species has a baculum, the female may have one, but if the os priapi is lacking, then the female will be without an os clitoridis.

    SECONDARY CARTILAGES

    More recent studies such as those of Burt, although not histological, have used cleared specimens in addition to macerated ones, thus allowing discernment of any major cartilages present. This information, combined with the relatively few microscopic studies of penile structure and development, leads to the following conclusions.
    1. Cartilage is present in the mature penis as a separate distal structure - the anterior process - in the common laboratory rat (Wiesner, 1935); or as multiple distal prolongations of the bone in voles, muskrats, mice and cotton rats (Burt, 1960), and golden hamsters (Callery, 1951). These cartilages may undergo some ossification. Thus, the rat's anterior process becomes the "secondary ossicle" of Thyberg and Lyons (1948) or the distal part of the "penile bone" (Glucksmann and Cherry, 1972).
    2. The firm structure in the clitoris may be cartilaginous in some species of ground squirrel, or some individuals of a species (Layne, 1954). The clitoris of the cat also sometimes has cartilage (Ellenberger and Baum, 1932) or fibrovesicular tissue (Retterer and Neuville, 1913), and occasionally bone.
    3. The penis of some ungulates, although without distinct nodules of bone or cartilage, has large chondrocyte-like cells amidst the dense connective tissue in the glans. Schaffer (1930) classed this tissue among his chondroid tissues, but later indicated that he regarded penile chondroid tissue as a secondary formation very closely related to secondary cartilages.
    4. Whether or not cartilage is present in the mature penis, the microscopy of penile development in the few species examined - dog, rat, mouse, guinea pig - reveals that cartilage participates in the ossification of the penile bone. The exact nature of the process is disputed, except in the rat and mouse, but whatever the kind of ossification, the growth cartilage of the penile bone is secondary by its separation, temporal and spatial, from primary cartilages, and was so considered by Schaffer (1930).
    5. Another kind of secondary formation is the cartilaginous structure - the anterior process - that forms in the clitoris of the rat when given excess testosterone neonatally or as an infant (Wiesner, 1935; Glucksmann and Cherry, 1972), (my Figure 18).
    Fig 18

    The foregoing examples demonstrate the phallus to be an important site of secondary chondrifications. The close parallels between these and other secondary cartilages will become evident with the details of the processes of ossification and chondrification given below for male and androgenized female rats.

    DEVELOPMENT OF THE PENILE SKELETON IN THE RAT

    This account is based on the thorough study of Ruth (1934), the less detailed account of Wiesner (1934), and personal observations reported thus far only in abstract (Beresford, 1973, 1975b,c) or incidentally in papers concerned with other aspects of the penile skeleton (Beresford, 1970a,b; 1975a; Beresford and Clayton, 1977; Beresford and Burkart 1977). Thyberg and Lyons (1948) offered some useful photomicrographs of the developing bone.

    The Anterior Fibrocartilaginous Process
    This is a large elongated body of fibrocartilage developing rather slowly after birth from its own cellular blastema. It remains separate from the more proximal penile bone but is attached to it by dense collagenous tissue. Once well established, after one week of age, its collagen stains with the chlorantine fast red 5B of Lison's (1954) method, while its large cells react positively with the alcian blue. Later, it experiences partial calcification and replacement by very irregular endochondral bone, particularly in its dorsal region away from the urethra and the tip of the penile bone. The anterior process is a secondary chondrification.

    The independence of the anterior cartilage from the penile bone is shown by the former's being the only firm structure to develop: 1) when older female rats are given male hormone (Wiesner, 1935; Glucksmann and Cherry, 1972), and 2) when genital tubercles are transplanted into the brains of infant rats (Beresford and Clayton, 1977).

    The Penile Bone
    The bone forms to a timetable different from that of the anterior process. At birth a cord of cells extends forward from the distal end of the fused corpora cavernosa (together constituting the corpus fibrosum). One or two days later, the central-most cells in the cord become osteoblasts, laying down a matrix that within one or two days binds sufficient alizarin red S to be visible in cleared whole specimens. One or two days after the first appearance of bone matrix in histological preparations, chondrocytes and alcian blue-positive material appear on the proximal end of the bone (Figure 19).

    Fig 19

    The initial short solid rod of bone grows by accretion along its sides and at its tip, but at four days after birth osteoclasts eat into it. Some of these erosive cells work their way proximally into the cartilage where it is fused with the bone. From the time of this invasion of the cartilage, the bone grows in length mostly by the endochondral replacement of the proximal or basal cartilage. The growth in width of the bone's shaft is by the subperiosteal deposition of bone. Although osteoclasts erode some of the membrane bone first is joined by a transitional formed, the shaft soon becomes dense, but still well vascularized.
    Vessels enter the shaft from the marrow cavity in the bone's basal bulb, but because there are too many of them, their course in the bone is irregular, and some enter from the shaft's sides, it is incorrect to liken the whole shaft, as Ruth did, to one Haversian system. Indeed, the rat's skeleton is generally deficient in Haversian bone, as Ruth himself was later to note (Ruth, 1953; Enlow, 1962b; Singh, Tonna, and Gandel, 1974). There appears to be very little remodeling within the shaft of the mature bone, because along most of its central region small islands of residual cartilage matrix are evident when sections are stained with alcian blue, and resorption tunnels are absent.

    The manner of ossification in the rat's penile bone resembles that of the mandible's condylar region, with osteogenesis and bone preceding by one or two days a chondrification by cells in a particular region of the germinal tissue enclosing the bone. The cartilage developing proximally on the penile bone is secondary and matches that of the mandibular condyle very closely, but those of the antler and clavicle rather less so. A comparison of the four structures is presented below.

    First, several properties are common to them. A secondary cartilage of a basically hyaline variety undergoes processes of growth and endochondral ossification requiring a stratification of functions across the cartilage. Under a convexly-shaped fibrous cap, a germinal zone produces chondroblasts, which form a large-celled, matrix-poor cartilage with randomly distributed chondrocytes (see Figure 1, p. 2).
    This cartilage is eroded in an apparently haphazard manner by numerous multinucleated chondroclasts, aided perhaps by vessels and mononuclear cells. Erosion is rather more systematic in the antler. Little calcified cartilage is spared in the destruction, and the subchondral bone develops as very irregular trabeculae. The sleeve of bone that encloses the sides of the cartilage is very long in the early stages of chondro- and osteogenesis, but shortens as the growth cartilage adopts its later, more stable crescentic form, shorter perpendicularly to the zones within it than it is wide parallel with them.

    Although there are a common morphology and process of ossification, several differences exist among the four sites.

    1. The cartilage appears long after birth for antler formation, but early in fetal life on the human clavicle and mandible. A comparison of onsets is not very meaningful when the species differ. Thus, for horns the chondrification can begin before birth; and the observation that the penile cartilage appears in the rat at about two or three days after birth, the mandibular condyle's at three or two days before, must be assessed in the knowledge that the rat is born so immature (Moss, 1964b) that the timing of its birth cannot have the same significance as it has for man or guinea pig.
    2. The trabecular bone that replaces the cartilage is rapidly refashioned, but the degree to which the subsequent bone is subjected to remodeling differs for the locations. The penile bone's shaft and the major part of the antler are quiescent, while the ramus of the mandible and the clavicle are extensively reworked. However, the extent to which these later happenings are of concern to the preliminary secondary cartilage is questionable.
    3. In the antler and penile bone, the growth cartilage eventually disappears, while in the mandible's condyle and the clavicle some cartilage persists to participate in the articulations. At the mandibular coronoid process, the tissue which ultimately comes to occupy the site may be not so much bone as chondroid bone II (fibro) because of the firmness of the attachments there.
      A similar situation may exist in patches on the base of the penile bone. The proximal surface of the basal bulb of the penile bone, where it attaches tightly to the main erectile body, may not be completely devoid of cartilaginous properties (Figure 20). This is certainly so at the dorsal lip of the base, where fibrocartilage persists into maturity as what appears in a section as a spur, and chondrocytic cells continue for some way proximally among the dense fibers of the capsule of the corpus fibrosum. Elsewhere along the basal surface some cells in the superficial bone are large and ovoid and react positively with alcian blue staining.
      Fig 20
      When rats are castrated, the external basal surface is no longer seen to be smooth, but pocked by small lacunae, but when a synthetic androgenic hormone is then given, cartilage grows out from the eroded surface (Beresford, 1970b). This regenerative cartilage, like that on fracture stumps, constitutes another example of a secondary chondrogenesis. By contrast, the final, living firm tissue at the tips of the tines of antlers seems from all accounts to be typical bone.
    4. The mandibular condylar cartilage is said (Koski, 1968; Durkin, 1972; Meikle, 1973b) not to contribute much to the overall growth of the mandible. This is not true for the cartilages of the clavicle, penile bone, and antler, where they serve significant longitudinal growth. The clavicle might appear to be odd, in having a secondary cartilage at each end. However, the antler has one for each tine, and the mandible can have secondary cartilage in its condylar, coronoid, angular, and symphyseal regions.
    5. The mandible and clavicle together differ from the antler (Goss, 1963, 1970) and penile bone (Wiesner, 1935; Thyberg and Lyons, 1948; Glucksmann et al., 1976) in having no special sensitivity to gonadal hormones or anti-androgenic compounds.
    6. The growth cartilages of the rat's mandibular condyle and penile bone share a marked susceptibility to the ravages of hypervitaminosis A (Beresford and Cannon, 1970; Beresford, 1971), which may be linked to common mechanisms of growth and erosion. The effects of vitamin A on the developing antler and clavicle are, I believe, still unknown.
    The differences listed are not negligible, and serve as reminders that no one secondary cartilage can act, in all circumstances, as representative of even the closely related members of the class. To underscore this proviso, the secondary cartilages under consideration have other peculiarities. Thus, the first bone of the clavicle is somewhat cartilaginous, and the first cartilage has even less matrix than is usual among secondary cartilages.
    The course of the vessels through the antler's cartilage divides it into characteristic columns. The penile growth cartilage is fused with fibrous erectile tissue, having implications for the former structure's blood supply and mechanical loading for which few precedents or clues exist.

    Chondroid Bone
    The rat's penile skeleton has various kinds of chondroid bone. Chondroid bone II is present in two or more locations, chondroid bone I in another, and there are three places where the tissues are questionable as chondroid bone. The last are dealt with first.

    As mentioned, when endochondral growth at the base of the bone eventually ceases, the cartilage is replaced by bone. The superficial layer of this bone is not quite the same as more deeply located bone or the subperiosteal bone over most of the penile bone's shaft. How chondroid the basal bone is poses a specific form of the more general question regarding the nature of the mature tissues at skeletal sites where a secondary cartilage disappears, e.g., at the mandible's coronoid and angular processes, while one bears in mind that continued reshaping of the site may remove or bury the tissue that first takes over from the last of the cartilage. A clear description of how such transitions are effected is needed.

    The shaft of the mature bone contains small islands of cartilage matrix within its central core. In this it resembles the otic capsule of the temporal bone, where the absence of remodeling leaves the bone cartilaginous only to the extent of bearing long-standing remnants of cartilage matrix. In the penile bone, these are very small and hold no living cells, and so there is no justification for regarding the composite material as chondroid bone. In the inner ear and baculum, there is merely enduring bone with isolated rests of cartilaginous matrix. The persistence of the latter argues against an ability of bone matrix to encroach upon, and transform, such remnants to any significant degree.

    The third dubious chondroid bone comes in the early period of histogenesis in the second and third weeks after birth, when the sleeve of bone encloses a "wedge," "plug," or "carrot" of cartilage in the manner described for the early mandibular condyle. The boundary between bone and living cartilage constitutes a very thin-walled tube of a "tissue" (see Chapter 12, p. 216), where the cells are sometimes chondrocytic, sometimes osteocytic, and sometimes appear to be fronted by osseous matrix on one aspect, cartilaginous matrix on the other. The leading edge of the sleeve of ossification, closest to where the periosteum takes over from perichondrium, has as its lining a layer of CB I, where chondrocytes appear to be enclosed in bone matrix (my Figure 21).

    Fig 21
    Chondroid bone II is present in two forms in the rat penis. The anterior process of fibrocartilage experiences some mineralization to become CB II (fibro) before being excavated and partly replaced by bone. The penile bone proper is tightly fastened to the dense connective tissue in which it lies. It is to be anticipated that the insertions of the tissue will resemble other insertion structures in having chondrocytic cells close to the bone, and some mineralization of these cartilaginous zones.
    The site where these expectations are met is at a prominence on the dorsal side of the bone near to the tip, just before the tip is beveled off to fit under the overhang of the anterior process (Figure 22). On the hump, scanning electron microscopy (Figure 3 of Beresford and Burkart, 1977) revealed small open-topped domes similar to those seen on the mature mandibular condyle made anorganic with a solution of sodium hypochlorite (Takiguchi, 1978). In both locations, it is believed that the treatment removes the soft fibrocartilage, taking the visible surface down to the top of the mineralized zone or chondroid bone II, in which the domes are the mineralized lacunae enclosing the most superficial of the remaining chondrocytes.
    On the penile bone the hump occurs at the distal end of a linea aspera-like crest along the top of the bone to which connective tissue fibers attach. The dorsal hump may be the point that experiences major tugging from the erectile tissue behind the bone and the anterior process lying before it. The existence of Sharpey-fiber bone (Boyde, 1972) with open osteoblastic lacunae rather than chondrocytes on the surface, both elsewhere on the penile bone and at many other locations, demonstrates that chondrocytes and fibrocartilage are not the rule at all insertions and fastenings.

    Fig 23
    The most striking example of chondroid bone in the penis is that of type I, which appears after the first week after birth. Situated on the dorsal aspect of the distal tip of the young bony rod is a region of large chondrocyte-like, alcian blue-positive cells in a matrix reacting like new bone with chlorantine red 5B or eosin (Figure 23). The tissue, although at the surface, lies within the overall contour of the bone and merges smoothly into the bone on either side and deep to it.

    The chondroid bone on the tip of the baculum is relatively short-lived and small in amount. Two to three weeks postnatally it is no longer to be found. Studies with labeled cells or vitally labeled matrix have yet to be done, and so it is unknown whether all the chondroid bone is destroyed (Figure 24) and replaced by true bone, or whether chondroid bone experiences metaplasia into a bone of more typical appearance.

    Fig 24
    The position of the chondroid bone is very near the tip of a growing bone formed "in membrane" where it becomes attached to another firm structure, (although, as Ruth (1934) remarked, it is "endoblastemal" since there is no membrane). These circumstances mirror those under which chondroid bone grows at the sutural margins of the bones of the young cranial vault. The tight attachment of the eventually partly ossified anterior process to the penile bone has been likened to the beveled sutures of the skull (Beresford and Burkart, 1977). In this regard, it may be significant that the chondroid bone does not qppear immediately when the penile bone starts to be laid down, but a little later, as the anterior process begins to become firm by the deposition of matrix. Exactly what the brief presence of chondroid bone contributes to sutural development in the skull and on the baculum remains to be discovered.

    An abnormal formation of chondroid bone occurred in the clitoris of rats injected with high doses of testosterone propionate very soon after birth and many times thereafter (Beresford, 1973). If administered early enough in postnatal life or to the mother during gestation (Greene, Burill, and Ivy, 1939), the enlarged clitoris comes to hold both a rudimentary penile bone and an anterior process. The timing and the dosage determine how histologically abnormal the structures are (they are, of course, always smaller than the male's for the same age).
    Abnormalities that I have seen include: a premature mineralization and ossification in the anterior process; a fusion of the anterior process with the penile bone, thereby obliterating the normal suture-like junction (seen also in male infants given the excess testosterone (Figure 25)); the rare occurrence of the principal secondary growth cartilage on the proximal part of the ossicle; and the formation of a kind of chondroid bone in the interior of the ossicle (Figures 26 and 27).

    Fig 25,
    Fig 26,
    Fig 27
    From the values of a single dose reported as osteogenic by Glucksmann and Cherry (1972), the amount of hormone that I gave was far above that needed to recruit the blastemal population to osteogenesis. The abnormalities in the treated males indicated that the amount of hormone was unnatural. The excess hormone appears to drive other connective tissue in the direction of chondroid osteogenesis, whether it is the presumptive fibroblasts of the suture or the osteoblasts and chondroblasts of the clitoral bone primordium. That CB I is abundant in the virilized rat phallus and the forming antler may be less a coincidence, but more the expression of a cornmon action by testosterone on certain skeletal cells.

    The reports on some spontaneously hermaphroditic rats indicate that either a bone (Kikuchi et al., 1977), or a bone and anterior process (Greep, 1942), is present. The osteogenesis in these instances was probably provoked by male hormone, and if the hormone's level was modest it is likely that ossification proceeded fairly normally (for a male), and in part via a growth cartilage. Such a cartilage and any anterior process would be further examples of ectopic secondary cartilages. These abnormal clitoral bones and those in bitches (Grandage and Robertson, 1971), like the antlers of female deer (Wislocki, 1954) and supernumerary antlers in males (Wislocki, 1952), are ectopic, extraskeletal bones.

    PENILE SKELETOGENESIS IN OTHER SPECIES

    Ruth (1934) was prompted to investigate the rat because of the discordant accounts, based on various species, of the penile bone's arising endochondrally (Retterer, 1887, 1914; and the dissertations of Arndt (1890) (1911) cited by Schaffer, 1930), intramembranously (Jackson, 1902), or by mixed mode of histogenesis. Recent reports such as those of Eleftheriou and Stanley (1963) still leave obscure how the large canine baculum develops. In the mouse, the initial work was reported only briefly (Retterer, 1887), and his reference to cartilage at both ends of the bone in the mouse, guinea pig and dog could mean, for the distal end, the cartilaginous anterior process (present in mouse), or the chondroid bone of the tip (Clayton, 1977). The presence of any cartilaginous tissue may have contributed to the idea that bacula, in general, develop from a cartilaginous primordium. Also, the first bone formed is large-celled and lacks trabeculae, properties which give it a superficial similarity to cartilage. Glucksmann et al. (1976) and Clayton (1977) found skeletogenesis in the murine penis differed in no significant way from that of the rat, except that certain strains, e.g., ICR, naturally develop a tiny clitoral bone.

    Clayton's (1977) histochemical and ultrastructural examination of the developing penile bone in the mouse was the first intentional application of these methods to a site of chondroid bone I. As in the rat, the chondroid bone I develops at the tip of the baculum, first becoming evident at about four or five days of age and disappearing between the 14th and 16th days. The chondroid bone cells, while larger than osteoblasts, are somewhat smaller than the hypertrophic chondrocytes of the hyaline secondary cartilage at the bone's base. The chondroid bone cells are rounded, stain with alcian blue and also resemble the chondrocytes in their ultrastructure, having a large nucleus pale in chromatin but with prominent nucleoli, many short cell processes, a moderate amount of GER, many free ribosomes, and occasional cytoplasmic vacuoles (Figure 28).
    The matrix surrounding the chondroid bone cells varies in its density, having more ribrils and mineral where it merges with the bone deep to it (Figure 29). Sometimes the wall of the lacuna is well defined, in other places a clearcut lacunar margin cannot be seen all around the chondroid bone cell.

    Fig 28
    Fig 29
    Fig 30
    In contrast to the intervening bony shaft, the matrix of the chondroid bone stains well,but not quite as intensely as the basal cartilage with alcian blue at pH 1.0, aldehyde fuchsin-alcian blue (Figure 30), and high-irondiamine-alcian blue. However, the Paragon stain used on 1 um-thick sections in Epon-Araldite colors the CB I matrix the pinkish-red tinge typical of the very narrow seam of osteoid under the periosteal osteoblasts on the shaft of the bone and the osseous sleeve partially enclosing the basal growth cartilage. Also, the chlorantine fast red 5B of Lison's (1954) method stains the matrix similarly to the bone. Thus, the tissue at the tip of the mouse's penile bone is truly a chondroid bone, in having cartilage-like cells enclosed in a matrix combining the histochemistry of hyaline cartilage with the more plentiful collagen fibrils and some of the mineralization of bone. The fate of the chondroid bone is uncertain, because at 10 days of age, the medullary cavity of the still spongy shaft approaches it very closely, thereby allowing resorbing cells access to its deeper part.

    Other observations by Clayton were that the developing anterior process contains two kinds of cell, one smaller, darker and less rounded. This difference was seen in Paragon-treated semi-thin plastic sections but not in electron microscopy. In the anterior process, collagen fibrils were visible at one day of age in the quite extensive but lucent matrix, but the matrix did not start to react with alcian blue, alone or in combination, until a week after birth. Diastase digestion and blocking by phenylhydrazine before the use of the histochemical stains for polysaccharides led Clayton to believe that the residual staining in the basal growth cartilage might be due to sialoproteins.
    These materials are present in bone (Herring, 1977), and the extent of their participation in chondroid bone and secondary cartilages merits further work. The pericellular pattern of mineralization around the hypertrophic chondrocytes of the basal growth cartilage and their glycogen content match what Durkin et al. (1972) and Hall (1968a,c) have described in other secondary cartilages. Lastly, although osteogenesis and chondrogenesis may each be delayed by a day, the cartilage is secondary in the sense that it always follows bone. The murine penile bone therefore starts in an intramembranous way, to be closely followed and augmented by a cartilage serving endochondral ossification.


    Chapter 15 CLAVICLE

    Introduction
    Initial clavicular chondroid bone
    Hyalinzelliges chondroides Gewebe (Pseudoknorpel): early clavicular cartilage
    Mischgewebe: Chondroid bone I
    Conclusions

    INTRODUCTION

    In man, the large clavicle may be regarded as the exotic bone imbued with the novelty and controversy borne, in other species, by such bones as the antler and baculum. While clavicular development has been followed in many mammalian species, for example, by Bruch (1853), Broom (1899), Fuchs (1912) and Schaffer (1930), most work, and particularly that of concern to chondroid bone and secondary cartilage, is on man. The numerous papers are summarized in the lengthy introductions to three articles (Zawisch, 1953; Koch, 1960; and Andersen, 1963).

    What points need elucidation are: Does bone precede cartilage, thereby making the cartilages secondary? Is chondroid bone present? And, if so, is it evidence of metaplasia? What makes the first question difficult to answer in the mandible and penile bone is the closeness with which cartilage follows upon bone. But in the clavicle, rapid succession is less of a problem and, instead, the obstacle lies with the anomalous nature not only of the first firm tissue, but also of the one that next comes after it.
    In their efforts to ascertain the character of these early tissues, investigators have compared them with young bone and cartilage in the scapula and vertebrae (Schaffer, 1930), the mandible (Bruch, 1853), mandible and vertebrae (Koch, 1960), the mandibular condyle, penile bone and scapula (Zawisch, 1953), and scapula (Andersen, 1963), and of late have relied heavily on histochemical methods.

    The account of the first firm tissue began straightforwardly enough with Bruch's (1853) describing it as bone, both in man's clavicle and in the more rudimentary structures of dog and cat. He likened the histogenesis to that occurring in the mandible, where bone also preceded the cartilage, and referred to these ossifications as direct or not preformed, in preference to Kolliker's term, secondary, which was especially inappropriate for the clavicle because this was the first bone, direct or indirect, to develop. According to Koch (1960), the ancient Greeks' name for the clavicle expressed their knowledge of its priority. Bruch also remarked on another similarity between the mandible and clavicle, namely, that their articular cartilages become more fibrocartilaginous than hyaline.

    A decade later, Gegenbaur (1864) claimed that the human clavicle was an indirect or endochondral formation, but by 1883 he had reached a different conclusion, holding then that the bone arose not out of cartilage but out of an "indifferent tissue," in circumstances unique to the clavicle. Although the situation of initial osteogenesis - no prior membrane, rather a cord of mesenchymal cells - is in fact shared with the penile bone (Ruth, 1934; Zawisch, 1953; Beresford and Clayton, 1977), several reports subsequent to Gegenbaur's (1883) attest to the oddness of what the mesenchymal cells become and produce.

    The cells enlarge and synthesize collagen, prompting such names for the tissue as tissu comparable a du fibrocartilage (Florentin and Castelain, 1935), and a "peculiar form of precartilage" (Fawcett, 1913). The latter name is an unfortunate choice in light of the later development of cartilage in the clavicle, thus Fawcett (1913) used "precartilage," both in its conventional sense as the precursor to cartilage, and with the special meaning of the peculiar first clavicular tissue.
    Further hindrances to following and matching the accounts of the various authors lie in the disparity in the ages of the specimens examined, and the use by some anatomists of a single name for tissues that others identify separately. From the spatial distribution and the wide range of ages for which he reported the tissue, it appears that Koch's (1960) Wabenknorpel blankets the chondroider Knochen, Mischgewebe and Chondroid of Zawisch (1953).

    Despite the idiosyncratic nomenclature and its lack of histochemistry, Zawisch's account is perceptive and provides a useful basis for discussing the several clavicular tissues.

    INITIAL CLAVICULAR CHONDROID BONE

    Zawisch (1953) joined Bruch (1853), Fawcett (1913), Hanson (1920), Schaffer (1930), Fazzari (1934), Gardner (1971), and Andersen (1963) in characterizing the first tissue produced by the mesenchymal cells as bone, for which she adopted Schaffer's (1888) name Chondroidknochen, with these words,
    "of itself the development here of Chondroidknochen is nothing remarkable since every bone anlage is initially chondroid as Schaffer (1888) emphasized when he created the expression 'Chondroider Knochen.' But in the clavicular Anlage the cells are especially numerous, and whole groups and rows of osteoblasts are incorporated. One could speak of nests of osteocytes.. . . ."
    She noted that the tissue was not quite the same as early mandibular bone, and to a superficial inspection could have a "cartilagelike appearance".

    Koch (1960) made much more of the differences between the first tissues of the mandible and clavicle. He too noted the large, abundant cells growing in the clavicular blastema, but assessed the tissue as cartilage from the metachromasia with thionin of the sparse matrix and its positive PAS reaction, and an apparent parallel between the stages of development of its cells with those described for the human humeral cartilaginous primordium (Streeter, 1949).
    However, the sequence of cellular development was based more upon the cartilage that forms later at each end of the clavicle than on the fate of the cells in the initially bony middle section, which very soon falls to resorption. This confusion came about because Koch called all the early firm tissues, other than clear-cut bone and osteoid, Wabenknorpel, or honeycomb cartilage, from their scanty matrix.
    When attention is confined to the material of the rod formed in younger fetuses, 18 mm to 25 mm in crown-rump length, Koch's histochemical arguments for a verdict of cartilage were faulted by Andersen (1963) on several grounds. First, he attributed the metachromasia of the matrix to a probable contamination of the thionin with a red component, and dismissed unanalyzed thionin as too unreliable for use as a histochemical reagent. Second, the lack of any evidence for the presence of glycogen caused Koch to believe that the positive PAS reaction had to be from cartilage-typical mucopolysaccharides. Andersen, however, suggested that poor fixation and uncoated sections had allowed the glycogen to escape, since he himself found the primordial cells to be rich in glycogen. (Frazier, Banks, and Newbrey (1975) have put forward collagen as another source of PAS reactivity.) Third, the cells contain "alkaline phosphatase which is not present in chondrogenesis."

    His other reasons for doubting Koch's conclusion that the tissue was cartilaginous are less persuasive. For example, Andersen and Koch both estimated the mineralization of the tissue not from its staining with a mineral-specific stain, but on its affinity for toluidine blue and PAS (Andersen) and aniline blue and a metachromasia with thionin (Koch). These inferences are not sound histochemical practice.
    Incidentally, Koch seems to have used the metachromasia twice: as evidence of mucopolysaccharides and as a sign of mineralization. Andersen went on to offer the continued viability of the cells after calcification of the matrix, a calcification estimated as described above from the staining, as an indication that the cells could not be chondrocytes, but other evidence shows that matrix calcification does not necessarily kill chondrocytes.

    The most serious criticism that can be brought against Koch's interpretation was not made by Andersen. It is that he gave considerable weight to the appearance and histochemical reactions of the Wabenknorpel at later times, properties which are irrelevant to whether the initial tissue is cartilaginous or osseous. It seems to be true that the first firm tissue is bone, but an unusually cellular variety with even more resemblance to cartilage than other examples of newly formed membrane bone. One can use this similarity to justify the name chondroid bone, as Zawisch (1953) did, but the tissue is not quite the kind that is encountered as chondroid bone I in fracture callus, tumors, and cranial bones and will be specially designated chondroid bone (initial clavicular) to distinguish it from those instances of a definite intermediate or mixed tissue - CB I.

    HYALINZELLIGES CHONDROIDES GEWEBE (PSEUDOKNORPEL): EARLY CLAVICULAR CARTILAGE

    No analyst of the clavicle in fetuses with crown-rump lengths greater than 50 mm has questioned the presence of a hyaline kind of cartilage at each end of the bone. However, in addition to the controversy concerning the first tissue of the shaft, the character of the growing ends of the clavicle for the period between 20 and 50 mm CRL has differed enough from that in other bones to call for special names and explanations.
    The sheath of proliferating cells around the early clavicle differentiates at both ends into a material with large vesicular cells and a very sparse matrix, which from the start of its deposition was characterized by Andersen (1963) as
    "only an ordinary hyaline cartilage which merely acquires a more large-celled appearance than ordinary hyaline cartilage by its rapid passage through Streeter's phases (1949). The cells begin to undergo hypertrophy as soon as they have been formed from the perichondrium."

    Zawisch (1953) saw matters differently. This matrix-poor tissue reminded her of the similar tissue she had seen and described on the periosteal surfaces of developing femurs and then called Pseudoknorpel (Zawisch-Ossenitz, 1927, 1929a and b). Zawisch believed that the form of this tissue in the clavicle was its only expression in man. Its closest affinity, she thought, was with the hyalinzelliges chondroid Gewebe, described by Schaffer (1930) as not being a true connective tissue because of the absence of matrix.

    Zawisch's conception of the tissue as purely cellular is puzzling, since she went on to describe the cells as lying in lacunae of which the walls exhibited a capsular staining. Capsules lying back to back were fused because of the close spacing of the cells. Her claim of an absence of ground substance must refer to the apparent lack of interterritorial matrix. Since the lacunar walls are composed of matrix, the tissue is certainly not solely cellular. (Recent knowledge of the extent and significance of the glycocalyx makes the notion of a tissue composed only of cells untenable, even for epithelial and central nervous tissues.)

    Koch (1960) held that the metachromasia of the Wabenknorpel matrix observed by him and questioned by Andersen (1963), ruled it out as Chondroid, because Schaffer (1930) had employed an orthochromatic response as a criterion for distinguishing Chondroid from cartilage. What is unclear from Koch's account is whether this metachromatically reacting Wabenknorpel included both the initial bone and what Zawisch called Pseudoknorpel, or just the bone alone, which would make his objection of no account.
    Zawisch's pair of figures (5a and b), intended to bring out the differences between clavicular chondroid tissue and scapular hypertrophic cartilage, do more to convince one of the essential similarity of the two. Although the two tissues may differ in evincing signs of fibrils after staining with azan, Zawisch's claim that the Chondroid does not mineralize, like Andersen's (1963) claim to the contrary, did not rely on staining specifically for mineral. Calling the tissue in question early clavicular cartilage gives it a specific name without denying the claims that it be regarded both as a rapidly hypertrophying variety of hyaline cartilage, and as a relative of the chondroid tissue of lower vertebrates, although one probably more distant than Zawisch would have us believe. Her view that it meets the need for a tissue growing faster than either bone or cartilage needs to be confirmed by experiment.

    The cartilaginous nature of the tissue appearing at each end of a clavicle first formed as bone means that a secondary chondrogenesis is taking place. Schaffer (1930) portrayed (Figure 266) the clavicle as a site of secondary cartilage. His illustration, and those as well as the descriptions of other authors cited, reveal the cartilage to have the disorderly distribution of chondrocytes, the pattern of zonation (Andersen, 1961), the chondroclastic erosion, and the long bony collars typical of two other secondaries: the penile bone's growth cartilage and the mandibular condyle.
    Andersen (1963) could not accept the clavicular formations as secondary cartilages, on the ground that they and the preceding bone arose from the one skeletal blastema, but what concerned Schaffer in formulating the secondary category was not whether the bone and cartilage came from separate blastemas or shared a single one, but why any cells formed cartilage after the primordial cartilages were established and after other cells nearby had commenced osteogenesis.
    The layer of germinal cells under the perichondrium allows secondary cartilages to contribute to the appositional growth of their parent bones, a property called to attention by Symons (1965), who had the mandible and clavicle particularly in mind.

    MISCHGEWEBE: CHONDROID BONE I

    Although Andersen (1963) remarked on a gradual merging of the initial bone with the cartilage of the clavicular epiphyses, and Koch (1960) described the transition from his Wabenknorpel, i.e., bone, to epiphyseal hyaline cartilage as slow, only Zawisch (1953) described, discussed, and named the intermediary tissue.
    Because it has some cartilage-like cells in an osseous matrix, she named it a "Mischgewebe aus Knochen mit 'Pseudoknorpelzellen"' (Figures 6 and 7), and a "mit Pseudoknorpelzellen (Blasenzellen) durchsetzer Chondroidknochen" (Figure 14). The same kind of chondroid bone I is evident in Gardner's (1971) Figure 17 of a clavicle from an embryo of 28 mm CRL, although Gardner does not comment on its presence to the left of center in the bone.

    As had Kassowitz (1881), Fazzari (1934) took the chondroid bone to be evidence that cartilage was becoming bone by a direct metaplasia, in addition to a more typical endochondral osseous replacement of cartilage. Later observers have unanimously opposed this metaplastic interpretation of the chondroid bone in the clavicle, but only Zawisch attempted to explain how the material came about. In fact, she offered two accounts. For the chondroid bone portrayed in her Figure 14, she proposed an origin from blastemal cells participating in appositional growth, which differentiate only to the "lower" stage of chondroid bone.

    Her second route to chondroid bone was more roundabout. Osteocytes became lytic and aided osteoclasts and vessels in destroying some of the bone. Then, undetermined blastemal cells gained entry to some opened osteocytic lacunae and became chondrocytes, thereby bringing about a bone with chondrocytes as well as osteocytes. She contemplated the possibility that some osteocytes might become chondrocytes, but rejected it because no one had yet seen such a metaplasia of bone to cartilage. A scant few instances of the phenomenon have since been reported (Chapter 21).

    CONCLUSIONS

    The clavicle has two secondary cartilages, forming at each end of an established bone, to which they are joined by chondroid bone I. (Zawisch (1953) raised the possibility of a third secondary cartilage at the deltoid tubercle, which she did not observe but suspected of arising as an apophysis of secondary cartilage at a later time than the ages of her specimens.)
    The early cartilage becomes hypertrophic so rapidly that for a while it resembles the chondroid of fishes as much as it does most mammalian hyaline cartilage. But, with true chondroid bone I present, to call the secondary cartilage Chondroid can only bring confusion.

    The first bone of the clavicle is exceptional. Its high cellularity and histochemical reactions place it to the cartilaginous side of most early membrane bone, and could justify calling it initial clavicular chondroid bone, to distinguish it from the CB I later linking it to the secondary cartilages. However, in light of the wholesale use of chondroid bone (Chondroidknochen) for any "very cell-rich immature bone tissue" (Zawisch, 1953), it is better to keep CB I for the intermediate tissue, and bone for the initial firm tissue.


    Chapter 16 FRACTURE CALLUS

    Callus chondrogeneses
    The chondrogenic stimulus
    Callus chondroid bone I: by metaplasia or blastema
    Metaplastic interpretations
    Blastemal interpretations
    More evidence and an evaluation
    Chondroid bone on healing membrane bones
    Bony stumps

    CALLUS CHONDROGENESES

    Ziegler (1899) referred to the eighteenth-century authors who recognized cartilage as a participant in the healing of broken bones. Any cartilage arising in the callus of repair is clearly formed after the primary cartilaginous structures and is therefore secondary (Schaffer, 1930; Murray, 1957).

    The questions needing attention now are as follows: What kinds of cartilage are present in fracture callus? In particular, do they resemble those of other secondary cartilages? Is chondroid bone a constituent of callus and does it have any metaplastic implications?

    Four cartilaginous tissues may appear in a callus:
    1 . hyaline cartilage;
    2 . a cellular kind of hyaline cartilage- -the Zellknorpel of the nineteenth century;
    3 . fibrocartilage; and
    4 . chondroid bone I, known widely in the last century under Kassowitz's (1881) phrase, chondroide Modification des osteoiden Gewebes, but recently more often referred to simply as an intermediate tissue (Figure 31).
    That chondroid bone and fibrocartilage have been separately described for the same callus indicates that a distinction is possible.

    The cellular variety of hyaline cartilage has the histological characteristics typical of many secondary cartilages. For example, Murray's (1954) observation on fracture callus in guinea pigs,
    "The cells of the cartilage ... lay in thin-walled basophil capsules. Between the capsules was the rather sparse inter-capsular matrix which tended to be acidophil rather than basophil and which was delicately fibrillar."
    Pritchard (1963) observed,
    "The cartilage is hyaline in type but highly cellular and pleomorphic. Moreover, its cells and matrix give strongly positive reactions for alkaline phosphatase, like osteogenic cells and osteoblasts .... This form of cartilage is virtually identical with the so-called secondary cartilage which forms in the course of development of the membrane bones of the skull vault, face, and clavicle. It is also found in association with bone in tissue cultures containing osteogenic cells (Fell, 1933)."
    It should perhaps be added that any straight hyaline and fibrocartilage (Pritchard, 1964) are also secondary according to the non-morphological criterion of time. The cartilage newly growing in holes bored in bone, on amputation stumps and at pseudarthroses is also a secondary formation.

    THE CHONDROGENIC STIMULUS

    Fig 31
    Cartilage forms in fracture callus, because somewhere there are cells that can become chondroblastic. The principal somewhere is generally taken to be the inner zone of the periosteum (Hein, 1858; Hall and Jacobson, 1975). Tonna and Pentel (1972) demonstrated by tritiated thymidine labeling in the mouse's femur that the callus chondroblasts are progeny of the osteogenic cells of the periosteum, cells that are, in practice, osteochondrogenic. Why are some periosteal cells stimulated to make cartilage? The hypothesis of a mechanical stimulus, rubbing, with its components of pressure and shear and their fluctuation, has the evidence in its favor, reviewed earlier in Chapter 4.

    Another factor suggested is an inadequate vascularization (Wurmbach, 1927), but this might be related to mechanics by the physical disruption and obstruction of the blood supply (Pritchard and Ruzicka, 1950; Murray, 1954). Is low vascularity causal for chondrogenesis or coincidental with it? Ham (1930) and others cited by Hall (1970b) suggested that rapidly growing osteogenic cells can outgrow their blood supply, thus subjecting themselves to a relative ischemia, to which they respond by making the less oxygen-dependent tissue - cartilage.
    This story is not totally convincing. Despite their many years of phylogenetic practice, are callus angioblasts still lax in making vessels? Angiogenesis keeps pace with other growing tissues (Gullino, 1978), and in the skeleton enables osteogenic cells rapidly to form tumors composed of bone and other slowly deposited tumors of cartilage. Urist and Johnson (1943) drew attention to observations indicating that chondrogenesis starts not in poorly but in highly vascular regions, where the vessels must necessarily be pushed to the periphery when cartilage matrix is deposited, thus making the region later appear less well vascularized.
    Murray (1954) noted the development of subperiosteal cartilage in rats at sites where the cambial layer appears no different in its vascularity and cell density from osteogenic periosteum. Along similar lines, Altmann (1964) suggested that the rising pressure in an enclosed growing blastema could keep vessels out. Nevertheless, vessels, what they bring, and what they take away, undoubtedly influence osteogenic cells as these differentiate, perform and, in their turn, perhaps stimulate the vessels, as suggested by Trueta (1963) and others.

    CALLUS CHONDROID BONE: BY METAPLASIA OR BLASTEMA

    Where the callus is bulky, as in mobile fractures of long bones, considerable amounts of the special hypertrophic hyaline cartilage and fibrocartilage form, and are joined to new bone by noticeable zones of a tissue combining bony and cartilaginous properties. Animals producing much cartilage in mending fractures, such as the dog and rabbit (Bruns, 1886) and pigeon (Bonome, 1885), have longer boundaries along which this chondroid bone may occur than do species and sites generating only meager cartilage.
    Chondroid bone is commonplace in the illustrations of healing bones and sometimes the authors described it in their text. Examples of the figures are in Pritchard and Ruzicka (1950), Figure 5 of Plate XXIII (Pritchard, 1963), Figure 11 of Zadek and Robinson (1967), and Figure 7 of Anderson and Dingwall (1967) for animal fractures, and Figures 8-8 and 8-11 (Aegerter and Kirkpatrick, 1975) in man.

    In the last century, when the process of ossification was widely held to be by metaplasia, callus cartilage was assumed to transform into bone (Hein, 1858 inter alios), with chondroid bone as a natural concomitant. When the metaplastic hypothesis gave way to the neoplastic, whereby cartilage was destroyed to make way for newly formed bone, the cartilage of fractures was similarly interpreted as a provisional tissue to be resorbed and replaced. The persistent reports of the intermediate tissue - chondroid bone - have continued to prompt ideas that some of the cartilage may undergo a metaplasia, or, more recently, that, while the chondroid bone need not be the halfway stage from cartilage but might develop from the blastemal cells directly, it could itself then experience a conversion into bone.

    Many have favored one of these two metaplastic interpretations (Kassowitz, 1881; Ziegler, 1899; Haas, 1914; Schulze, 1929; Urist and Johnson, 1943; Pritchard and Ruzicka, 1950; Yamagishi and Yoshimura, 1955; Cabrini, 1961; and Bohatirchuk, 1969), but others who have acknowledged the existence of chondroid bone have dissented, for example, Asada (1927) and Murray (1957), believing a resorption of the chondroid bone to be the rule. The following examples represent both viewpoints, and give details of the observations and how the interpretations were arrived at.

    METAPLASTIC INTERPRETATIONS

    Ziegler (1899) described unsplinted humeral fractures in guinea pigs and newts, and an eight-week-old human fracture. He observed the formation of cartilage, which growing vessels selectively destroyed, leaving cartilage trabeculae. At the margins of these trabeculae the matrix started to stain red with van Gieson's method, while the cartilage cells became paler and disappeared, or shrank and became osteocytes surrounded by red bone matrix. Occasional cartilage cells remained within the bone trabeculae. He noted that, already before the vascular invasion, more fibrils appeared in the matrix of the cartilage, but he interpreted the redder staining with acid fuchsin as indicating mineralization, because, he thought, only mineralized tissue stained that red.
    This misunderstanding of what fuchsin stains makes it unclear whether he was looking at fibrocartilage - a frequent constituent of callus - or chondroid bone. His figures show large cells more like chondrocytes than osteocytes. If the redder staining of the cartilage seen later in healing was from a greater proportion of fibrocartilage rather than because cartilage matrix was becoming bony, this would disqualify one of his grounds for believing in a direct metaplasia.
    His other reasons - the merging of bone with cartilage and the continuum of cell types from chondrocyte to osteocyte - have another explanation in a common precursor cell's having given rise to bone, chondroid bone, hyaline and fibrocartilage in adjacent regions of the callus.

    Urist and Johnson (1943) remarked that the human callus held areas of hyaline and fibrocartilage. By staining for mineral with the method using silver nitrate, they showed (Figure 12) not only calcification of some hyaline cartilage prior to its penetration by osteogenic tissue, but the absence of mineral in the transitional "chondro-osteoid" tissue, a tissue, according to them, identical with that in the rachitic metaphysis (see Chapter 23). The pattern of mineralization was "apparently haphazard." They went on,
    "Resorption of fibrocartilage and cartilage continues, but a large part of the cartilage appears to lose its basophilic staining and gradually to disappear in the osseous tissue as though through transformation of the chondrocytes into osteocytes. This transformation, regarded by some authors as metaplasia, is a prominent feature of the ossification of the callus in man."
    Later in healing, "the chondro-osteoid disappears with improvement in calcification," so that by staining for mineral Urist and Johnson showed that their chondroid bone was more of a chondro-osteoid, but it could calcify and, in doing so, also appeared to turn into bone.

    Pritchard and Ruzicka (1950) compared fracture repair in the lizard, frog, and rat. In all three, the callus had a transitional area between bone and cartilage where
    "the cells were predominantly large and rounded as in cartilage, but they show the large juxta-nuclear vacuoles and intense cytoplasmic basophilia typical of osteoblasts. The intercellular matrix was of heterogeneous consistency, with a tendency toward the appearance of thin basophil capsules around the larger cells and an eosinophil matrix elsewhere. The original collagen fibers were incompletely masked by the matrix and many of them remained visible in van Gieson preparations."
    In the lizard and frog, the intermediate tissue became widespread as erosion ate slowly and at widely spaced points into the cartilage. Pritchard and Ruzicka (1950) confirmed Ziegler's (1899) finding that the loss of basophilia and fibrous transformation of cartilage matrix and the shrinkage of chondrocytes seem to precede erosion, but with a contradiction: Ziegler saw the phenomenon in fractures of guinea pig and man, but apparently not in newt; Pritchard and Ruzicka found it in the frog and lizard, but not in the rat.
    By staining for inorganic phosphates, Pritchard and Ruzicka showed the disposition of mineral in the intermediate zone in advance of irrupting vessels. They concluded, "an actual transformation of cartilage into bone was taking place." While they thought that the same process occurred in the normal epiphyseal lines of the lizard's femur and the frog's acetabulum, in those sites it is probable that the tissue, although similar histologically, had a different origin as chondroid bone II (see Chapter 10).

    Another paper on amphibian callus (Robertson, 1969) has a puzzling description of metaplasia. In the four-week-old callus of Rana pipiens, Robertson found hyaline cartilage and trabecular bone, of which he wrote, "the transitional region of osteoblastic activity with trabecular formation and the typical basophilic cartilage was abrupt," and "the abrupt change with little or no intermediate zone indicated a transformation of cartilage into bone, indicating a conversion of chondrocytes to osteocytes as suggested by Pritchard and Ruzicka (50)."
    His conclusion is remarkable because all other proponents of a metaplasia in callus have founded their belief on the presence of a significantly wide intermediate zone, whereas Robertson writes in one place of a transitional region, in the next he has none, and makes its absence the basis for metaplasia. The bone-cartilage boundary in his Figures 6 and 7 certainly is quite sharp.
    This, the osteoblasts, the consistent relation of the bone to the vascular network, and the acidophilia of the cartilage matrix around "foci of new capillaries," all bring to mind a process of resorption and canalization of the cartilage, followed by plain osteoblastic osteogenesis; but with the acidophil cartilage giving the misleading impression of pre-metaplastic change common around canals (Chapter 10, Patella and Cartilage Canals, p. 184).

    Yamagishi and Yoshimura (1955) described chondroid bone, cartilage, and its direct transformation into bone, in rabbits' tibial fractures held in various degrees of stability. Their paper is most notable for a new twist to metaplasi a. They maintained that mesenchymal cells in neutrally fixed fractures become osteoblasts - a method of osteogenesis they called "direct metaplastic bone formation." By contrast, the mesenchymal tissue under extensile fixation differentiates into fibrous connective tissue which then becomes "bony tissue by intramembranous bone formation," really a direct metaplastic bone formation, and certainly not typical membranous osteogenesis.

    Bourne (1944) intimated the presence of chondroid bone in the repair of holes bored in rodents' femurs. Thus, "some of the periosteal trabeculae had a cartilaginous appearance," and "there were some signs of the formation of cartilage from the osteoid trabeculae." In this instance, chondroid bone was taken by Bourne, quite legitimately, as indicating a metaplasia in the reverse direction to that usually read into its presence.

    Bohatirchuk's (1969) work on mammalian fractures showed by historadiography the presence of mineral around the chondrocytic and osteocytic cells of chondroid bone in undecalcified sections.

    BLASTEMAL INTERPRETATIONS

    In the tissues of healing fractures in the rat, Asada (1927) distinguished between: 1) a hyaline kind of cartilage, 2) osteoid and bone, and 3) a tissue transitional from osteoid to cartilage that he equated with Kassowitz's chondroide Modification des osteoiden Gewebes, but himself termed das chondroide Gewebe. It had more of a connective tissue character than the cartilage present, and its lacunae, while big, were not so large and round but narrower, and the matrix stained less well with hematoxylin. The chondroides Gewebe was in places trabecular and had calcified. These three principal tissues of the callus had, he thought, differentiated from the cellular blastema.

    Regarding their fate, Asada rejected Bruns's (1886) claim that both cartilage and chondroid bone could, in part, undergo a metaplasia to bone. The ossification in the cartilage appeared to Asada to be of a fairly straightforward variety, but that in the chondroid bone less typical. However, he saw chondroclasts and endothelial cells apparently resorbing the chondroid bone, while other regions of its trabeculae were clad in osteoblasts and an osteoid seam. Hence, he concluded that ossification proceeded by a substitution, but less rapidly than in the cartilage. Asada also noticed his "chondroid tissue" and osteoid in the medullary or internal callus, but unaccompanied by cartilage, circumstantial evidence that chondroid bone arises from the blastema independently of cartilage.

    Schulze (1929) detected fibro- and hyaline cartilage in broken human bones. He noted the merging of new cartilage with new bone to be so smooth as to suggest a common "genetic" (formative) source, a view shared with Ham (1930). Without being more definite as to why, Schulze then reversed himself and proposed that the cartilage experienced either endochondral replacement or a direct metaplasia.

    Murray (1954) observed a transitional tissue in the repairing broken fibulae of guinea pigs. His Figures 3, 6, and 7 illustrate cartilage merging gradually with bone. He gave the transitional tissue no particular name, but he did consider the possibility of a direct metaplasia. However, he wrote,
    "I have seen nothing in my preparations which convinced me that such a transformation occurred, the histological picture being always equally consistent with the theoretically more probable differentiation of the two tissues from different parts of a common blastema. . . ."

    Altmann (1964) encountered chondroid bone in two situations in his experiments on regeneration in the rat's hind-limb. He distinguished four tissues:
    Chondroidknochen (the large-celled bone of Schaffer (1888));
    Inselknochen (the mixed tissue with islands of basophilic matrix described as one of the telodiaphyseal formations by Zawisch-Ossenitz (1927, 1929a,b)); telodiaphysare Mischknochen (the other telodiaphyseal tissue described by Zawisch-Ossenitz); and
    callose Mischknochen (the mixed tissue appearing in fracture callus).
    He viewed these as very closely related members of a structural or developmental sequence, and interpreted the tissues and the smooth transition between them as evidence of their origin in a common germinal tissue.
    Only the Mischknochen coincides with the general category of chondroid bone I. Altmann saw it within the callus on the rat's fibula after placing a plastic tube over its broken ends (Figures 48 and 49), and under the artificially elevated periosteum of the rat's tibia (Figure 46b). While not a fracture, this second experiment provoked the periosteum to form a substantial layer of bony trabeculae, cartilage, and chondroid bone, such as occurs subperiosteally a short distance from a true break.

    Altmann contended that the hybrid nature of chondroid bone was expressed not only in morphology but also in a cartilage-like ability to grow expansively, to be followed shortly by mineralization to yield a bone-like rigidity. The evidence for an interstitial growth is lacking.

    MORE EVIDENCE AND AN EVALUATION

    In most of the papers just discussed, fixed samples of similar sites taken at intervals in the course of fracture repair were used to construct a feasible, but hypothetical, sequence of transformation of cartilage and chondroid bone to bone; but can one prove that cartilage cells known to be cartilaginous end up in bone?
    Urist, Wallace, and Adam's (1965) experiment pointed to an affirmative answer. They transplanted small pieces of callus to the anterior chamber of the eye of rats, having checked one half of each microscopically to be fibrocartilage. With tritiated thymidine they labeled nuclei either in the callus, or in the host cells that resorbed the callus and replaced it with an ossicle. Most transplanted cartilage was resorbed, but some chondrocytes persisted and may have transformed to osteocytes. They saw small labeled chondrocytes "embedded in an amorphous matrix more like bone than cartilage," i.e., chondroid bone, and labeled cells resembling osteocytes but with a cell capsule and metachromatic matrix, also indicating what they termed "chondroidal ossification." They conceded that, even with a nuclear label, a subjective element entered their conclusion that callus cartilage could in a small way experience direct metaplasia.

    Danis (1957) also had put grafts of callus into the anterior chamber of the eye. Because the bone resulting from homogeneic grafts did not last, he concluded that there was no induction, and thus the bone formed directly from the fibrocartilage. His brief note has no illustrations, and the difference in outcome between auto- and homografts is not conclusive evidence on the source of the osteogenic cells.

    Another aspect of the cartilage of fracture callus that may have led students of the tissue to believe it to be on its way to becoming bone is the presence of dark chondrocytes. Browne (1977) reports light typical cartilage cells and darker GER-rich chondrocytes in murine callus examined by light microscopy and TEM. The darker cartilage cells and any degenerative forms of both light and dark types may have been interpreted as osteocytes by previous observers using only light microscopy.
    This does not necessitate the conclusion that fracture callus lacks chondroid bone, since a tissue often seen has an osseous matrix enclosing chondrocytes. That some of the chondrocytes could be darker, and some degenerate, may, however, heighten the impression that the tissue has or is acquiring a bony nature. (The mixture of dark and light chondrocytes in rachitic cartilage (Riede, 1971) may likewise have contributed to the belief in a metaplasia by this tissue.)

    The evidence so far allows only these conclusions: 1) chondroid bone I participates in bone healing; 2) it might be made by blastemal cells; 3) it might reflect an osseous metaplasia of cartilage; and 4) that event 2 need not exclude the latter, 3.

    CHONDROID BONE ON HEALING MEMBRANE BONES

    Bones developing totally or in part by the intramembranous route may have cartilage in their healing fractures, and provide further examples of chondroid bone at its boundary. Koller (1896) regularly saw cartilage in the rabbit's mending zygomatic arch and mandible (confirmed by Craft et al., 1974), and occasionally in the supra-orbital margin.
    Fractures of the cranial vault heal with difficulty, if at all, but the injured bone margins do form meager amounts of callus. Following up Pritchard's (1946) observation of cartilage in the cranial callus of one rat, Girgis and Pritchard (1958) were able to provoke the infant rat's parietal bone to chondrogenesis by multiple cuts and scraping off the periosteum. Most of the cartilage was in the form of small nodules (their Figures 2 and 4) which merged into bone through zones of frank chondroid bone. Likewise, Figure 2 of Beresford (1969) shows a line of cartilage cells in the cranial callus of a vitamin A-deficient rat. Here, there are so few chondrocytes that all have adjacent osteocytes and bone matrix.
    As with secondary cartilages of normal cranial vault development, this reparative cartilage, by forming at the blunt edge, of necessity occupies a small space and thus tends more toward the category of chondroid bone than hyaline cartilage because of the proximity of bone matrix to all the cartilage cells. Cranial vault chondrogenesis does not require fracture, for Chang (1951) observed the formation of new bone and islands of cartilage under soft paraffin placed beneath the periosteum of the parietal bone of rats.

    Chondroid bone I is also evident in Figure 2 of Richman and Laskin's (1964) injured infraorbital bone, and Koller's (1896) description of one healing zygomatic arch (his Table 1).

    Other bones form in membrane and then grow further with the aid of secondary cartilages. Mending fractures of the penile bone's shaft (Beresford, 1970a) have a transitional tissue uniting the bone ends (Figure 32), and Sprinz' (1967) Figure 6 of the healing mandibular neck shows some chondroid bone (compare my Figure 31). In his later paper on healing in the same place, Sprinz (1970) drew attention to the tissue as a
    "calcified bone-like matrix also containing cartilage cells resembling that described as 'metaplastic tissue' by Haines and Mohuiddin (1968) beneath articular cartilage." Sprinz saw this tissue not only in the zone of repair but, after mandibular fracture, deposited on the articular surface of the squamosal bone.

    Fig 32
    After resection of the mandibular condyle and part of the attached neck in rats, Jolly (1961) saw a tissue intermediate between cartilage and bone and noted its similarity with the chondroid bone of fractures, and named it so. It formed the upper and superficial layers of the "callus" on the mandibular stump. It also occurred sometimes as the first tissue at a new center of bone growth that formed at a point inferior and anterior to where the condyle had been.
    Although "the cartilage or chondroid bone was replaced by immature bone," he did not specify how. The bone of the new center later fused with the callus of the stump to form a new condyle, but one often angulated to articulate with the squamosal bone inferiorly and away from the fossa. Sometimes chondroid bone formed on the lateral surface of the cranium opposite the new articular process.
    In light of Ham's (1930) hypothesis that the vascularity determines whether callus cells become chondroblasts or osteoblasts, Jolly interpreted the chondroid bone as having formed in a tissue of intermediate vascularity.

    BONY STUMPS

    When any elongated bone breaks, two stumps result. The regenerative behavior of a single stump, when the fellow stump is nearby, is similar but not quite the same as when one bone is resected or the bony pieces are badly dislocated, e.g., Hall and Jacobson (1975). Histogenetically, the regenerative response of the unmatched stump involves the same population of blastemal cells, able to form bone, cartilage, CB I, and fibrous tissue. Where new bone and cartilage adjoin, chondroid bone is to be expected, and was shown by Selye (1934) on a regenerating femur in the infant rat in his Figure 1, and by Jolly (1961) on the mandible.

    On the other hand, the inability of mammalian stumps to completely restore the status quo ante results in a mechanical instability that is reflected in an atypical pattern of chondrogenesis. Jolly's (1961) condylectomized mandible is notable not so much for the chondroid bone as for its occurrence at three separate sites. No doubt other instances would reward a search in the extensive literature on chondrogenesis at pseudarthroses, nearthroses, amputation stumps, and joint explantations (Urist, Mazet, and McLean, 1954; Krompecher, 1956, inter alios).

    In addition to hyaline cartilage forming by blastema on the stump (Person et al., 1979), if the mechanical conditions are favorable (Mooney and Ferguson, 1966), fibrocartilage may form in initially fibrous tissue on the bone-end. Such fibrocartilage thus arises metaplastically, and also falls into the secondary cartilage category, although functionally it may substitute for the primary articular cartilage. Severe erosion can denude a bone of articular cartilage and leave a kind of bony stump. Little (1973) portrayed cartilage regenerating from granulation tissue on the eroded bone of a severely arthritic human femoral head. Such new cartilage is, as she wrote, secondary.


    Chapter 17 TRANSPLANTATIONS OF SKELETAL TISSUES

    . Introduction
    . Chondrogenesis by periosteal grafts in vivo
    . In vivo grafts of bone and periosteum
    . Secondary cartilage from avian periosteum in vitro or on the CAM
    . Perichondral osteogenesis in transplant and explant
    . Stimuli for periosteal and perichondral switching
    . Osseous transformation in transplanted primary cartilage
    . Osseous transformation in transplanted secondary cartilage
    . "Metaplastic" changes in the matrix or cells of transplanted cartilage
    . Chondrocytic escape and metaplasia in the intact animal
    . Cartilage formed by grafted periosteum and regenerating tissue: signs of its metaplasia
    . Conclusions

    INTRODUCTION

    One might think that the facility with which cartilage or chondrogenic tissue can be observed in tissue culture or ear chambers would allow a resolution of whether any direct metaplasia of cartilage takes place. The occasions on which such an event has been seen are, however, very few, and the circumstances of transplantation introduce other difficulties. Thus, unless one takes such steps as feeling the hardness of the tissue with a sterile knife (Fell, 1928) or testing the specimen at the end of the culture period for von Kossa's reaction, one cannot distinguish bone from osteoid. This matters because cartilage matrix that has lost proteoglycans and now stains with eosin may be mistaken for osteoid.
    Rather than provide unequivocal answers to the metaplasia question, the many transplantations of skeletal tissues yield mostly additional circumstantial support for metaplasia, but also some valid examples of secondary cartilage and chondroid bone. Within the limited periods of culture, and with abnormalities in the nutrition and resorption of cartilage, chondroid bone II does not arise.

    Following Felts's (1961) example, I shall subdivide the analysis more by the tissue in which events occur than by technical procedure. A distinction to be stressed is between a transformation of cartilage to bone (metaplasia) and the diversion of cells expected to form bone to chondrogenesis and vice versa (novel differentiations).

    Many studies using in vivo transplantation or explantation to in vitro testify to the formation of cartilage by periosteum, taken alone or along with a piece of bone. Such cartilage fits well in the category of secondary cartilage: it is a new growth forming after the primordial cartilages and certainly separate from them, from cells more likely in their original site to become osteoblasts or fibroblasts than chondroblasts. The new cartilage is often accompanied by chondroid bone, interpreted by the observers either as indicative of metaplasia, or as evidence of the multipotentiality of periosteal cells.

    CHONDROGENESIS BY PERIOSTEAL GRAFTS IN VIVO

    The advent of serious microscopy circa 1850 gave new vigor to the long-standing debate on the role of the periosteum in the normally growing bone and in the callus after fracture. To ascertain the potential of the periosteum alone, many investigators grafted pieces to various sites, where the periosteum often survived, forming not only bone but sometimes cartilage and chondroid bone. Ollier was probably the first to tell of chondrogenesis by periosteal grafts, but Buchholz's (1863) finding cartilage arising from tibial, but not cranial, periosteum placed in the backs of dogs and rabbits, was the earliest example to be confirmed by microscopy.

    Bonome (1885) experimented with femoral and tibial periosteum and pieces of bone, including intramuscular and intra-ocular grafts, and an unsuccessful attempt at tissue culture. Periosteum, when transplanted intramuscularly in rats, formed cartilage, most of which experienced endochondral ossification, but in other regions it merged with bone (site D of his Figure 1) in a manner that led Bonome to believe that cartilage cells were turning into osteocytes.
    In another experiment with rats and rabbits, Bonome grafted pieces of bone from which he had removed the marrow. Some of the trabeculae of new bone growing on the pieces held not only large young osteocytes, but groups of chondrocytes, around which the matrix stained in the same way as osteoid. Bonome specifically equated this tissue with the chondroid modification of osteoid, i.e., chondroid osteoid or chondroid bone, described by Kassowitz (1881) as a participant in fracture callus.
    At the time, chondroid bone was no novelty, but rather was taken for granted as an inevitable accompaniment of metaplasia by faithful adherents of the hypothesis of metaplastic osteogenesis, such as Kassowitz (1881). Opponents such as Schaffer (1888), recognizing a potential of skeletal blastemal cells to form all kinds of supporting and connective tissues, while also familiar with chondroid bone, regarded it as but one of several expressions of the precursor cells' pluripotentiality.

    Against this background, the studies of transplanted periosteum continued. Grohe (1899) kept the periosteum of dead rabbits cool at 0-4 C for up to several days, before placing it in the muscles of littermates. The grafts formed bone, cartilage, and a chondroid bone, as described by Kassowitz, with a homogeneous carminophil matrix enclosing chondrocytes. The chondroid bone merged with hyaline cartilage, "cellular cartilage," and in another specimen, bone, causing Grohe to attribute to the cartilage a potential for metaplastic osteogenesis.
    His Figure 2 merits comment, because the chondroid bone is in the form of trabeculae with marrow in between. This appearance probably reflects the length of the period of transplantation, 28 days, allowing time for resorption to get well under way.

    Grohe's transplants of periosteum in cats and guinea pigs were neither osteogenic nor chondrogenic. Because of the rabbit's well-known ability to form skeletal tissues within injured muscle (Bridges and Pritchard, 1958) and the delays after death before Grohe transplanted the tissue, it is possible that the bone and cartilage seen in rabbits might have been a result of irritation, and solely of host origin. The results, however, match those of Buchholz (1863) and Bonome (1885) in dogs and rats, and Grohe discussed some other factors that may have been responsible for the species difference he saw.
    Incidentally, Grohe believed altered vascularity played a role in his example of periosteal secondary chondrogenesis. Morpugo's (1899) transplants of hens' tibial periosteum to the wattles or comb also formed cartilage and bone, of which the cartilage seemed to be experiencing an osseous transformation, but no details or illustration of the latter process were given.

    Tibia] periosteum of weanling rabbits when inserted autologously under the renal capsule sometimes formed nodules of cartilage (Cohen and Lacroix, 1955), but not when placed within the eye. Periosteum from adult rabbits produced less bone and no cartilage. The authors gained the same impression as had Buchholz (1863), that the cartilage formed when new bone was present in largest amount. They inferred that the bone had grown more rapidly when there was more of it, and proceeded to relate the chondrogenic response to the high rate of growth.

    IN VIVO GRAFTS OF BONE AND PERIOSTEUM

    The experiments just surveyed involved transplanting skeletal tissue into the same or another animal, which raises the general problem introduced when considering Grohe's paper: when cartilage or chondroid bone develops, is either one a product of the grafted cells, or has the tissue arisen from host connective tissue cells by a process of chondrogenic induction? The inductive question was first raised regarding the new bone rather than the rarer cartilage, because of the acute clinical interest in knowing what component of bone made it effective when it was grafted into defects and slowly healing fractures of bone.
    The experimental transplantations, ranging from those performed by Axhausen (1909) and others to those of Poussa and Ritsila (1979), have suggested that when major amounts of new bone and cartilage appear after the grafting of fresh autogeneic bone, these tissues mostly owe their existence to cells of the periosteum on the bone fragments. Marrow does hold osteogenic cells (see Chapter 8, Osteogenic Cells of Marrow: Experiments), and bone matrix can evoke ectopic cartilage and bone (Rohlich, 1941b), but the volume of skeletal tissues from these sources is minor in comparison with what periosteum can produce under favorable circumstances of transplantation. The discussion of induced cartilage and chondroid bone is deferred to Chapter 18, because, although they are a theoretical possibility in the experiments of this section, it is probable from the spatial relations of the firm tissues with the grafted periosteum that periosteal cells are their direct and main source.

    Although Axhausen's (1909) principal concern was with the role of the periosteum in the osteogenesis after bone grafting, he noted several times the development of cartilage and chondroid bone. The periosteum on femoral shafts placed in the muscles of rats formed a callus-like mass containing bone, cartilage, and chondroid bone of two varieties,
    "namentlich das Vorkommen von morphologisch ausgesprochenen Knorpelzellen in leuchtend rother (Van Gieson), also der Knochensubstanz tinctoriell gleichender Grundsubstanz, ist bemerkenswert und keinesweg selten; ebenso finden sich aber andere Stellen, an denen deutlich zackige Hohlen mit raumausfullenden grossen Kernen in blau gefarbter Grundsubstanz (Hamalaun-Eosion) liegen." In liberal translation, one kind had chondrocytes in bone matrix, the other had osteocyte-shaped cells in cartilage matrix. Both kinds of tissue merged with the fibrous and other connective tissues, but a metaplastic interpretation was not given to this continuity.
    When Axhausen grafted periosteum-free femoral shafts, the external callus failed to form, but the tissue toward the open ends of the shaft formed bone and cartilage and again:
    "Auch hier liegen die grossen, runden, blassigen Zellen theils in knorpeliger, theils in knocherner Grundsubstanz." His attribution of these new tissues to the marrow should be viewed with caution, in light of the extensive myeloid necrosis present. In a later description (p. 63) of the histological nature of the external "calluses," he wrote of a "Knochenmaschenwerk mit Beimischung von Knorpelgewebe und chondroiden Gewebe," using chondroiden Gewebe for chondroid bone in the same way as Grohe (1899) had.

    A finding of a quite different kind was seen just once, after an autogeneic transplant of a piece of periosteum-clad tibia in a rabbit. After 12 days, Axhausen noticed, aside from a large growth of new bone, that at one end of the implant definite chondrocytes, rather than fibroblasts, lay between coarse collagenous bundles in an outgrowth of the periosteum. This would be another instance of an apparent chondrogenic metaplasia of the fibrous region of a perichondrium, as has been seen in, for example, tibial development in the rat (Murray, 1954).

    Over the last 40 years, the brain has been a favorite site for grafting, because it offers some respite from immune reactions and has only the connective tissue cells associated with vessels. When whole mouse humeri, half mandibles, and other bones (Felts, 1961) are transplanted to the brains and other sites of living mice, the earliest periosteal response is to deposit large-celled cartilage (Figure 3).
    What was a one-sided regeneration attempted after fracture occurred when Ronning (1966) placed infant rats' mandibular condyles, some with a little ramus attached, in the brains of littermates: "occasional cartilage-like tissue was sometimes observed at the end away from the condyle. . . ."

    Simmons et al. (1973) achieved chondrogenesis from chips of cancellous bone from the rabbit's ilium. They implanted Millipore diffusion chambers loaded with the chips, with and without marrow from the host's femur, under muscle sheaths of other rabbits. Within the chambers cartilage and "osteochondroid" (Figure 3) developed. They attributed the tissues to the "few proliferative chondrocytes included with the original graft," so that this is not an example of secondary chondrogenesis. It has interest for the presence of chondroid bone, but whether this originated in osteoblasts or chondrocytes cannot be determined.

    Marrow, when transplanted, has formed chondroid bone. Bruns (1881) autologously placed femoral or tibial marrow under the skin in young dogs. After two weeks he found new bone and hyaline cartilage, which merged with each other. He interpreted the transitional regions as evidence of a direct metaplasia of the cartilage into bone, from which one may conclude that they were chondroid bone, although he did not call them that, referring only to a calcification of the ground substance and an acquisition by the cells of the form of "osteoid cells."

    His experiment throws another light on Fell's (1933) cultures of chick diaphysis without any periosteum. She supposed the osteogenic and chondrogenic cells were derived from endosteum. Bruns's results suggest that marrow cells should also be considered as a possible source of her new skeletal tissues.

    SECONDARY CARTILAGE FROM AVIAN PERIOSTEUM IN VITRO OR ON THE CAM

    Fell (1931a,b) cultured, in hanging drops, periosteum from embryonic bones of chicks. Not only did she see ossification centers, but also nodules of cartilage sometimes formed. A subsequent experiment (Fell, 1933) concentrated on the endosteal behavior, by taking pieces of the chick embryo's tibia stripped of periosteum and distant from the epiphyses. Cartilage developed in about 15 percent of her cultures.
    Glucksmann (1938), while confirming Fell's findings, attempted to manipulate the mechanical environment of the cells in culture. In a second such experiment (1939), he contrived various arrangements of chick bones in order to put periosteum under pressure, whereupon it formed cartilage. At the same time, some arrangements acted to displace viscous materials, and the cartilage formed at the resulting sites of displacement was also large-celled. Although that at points of pressure was small-celled, both constitute varieties of secondary cartilage.

    On the chick's chorioallantoic membrane Studitsky grew composite grafts of chick's long-bone cartilage artificially wrapped in chick or human periosteum, in order to examine the reciprocal actions of the tissues in skeletal histogenesis and morphogenesis. Control grafts of tibial or femoral periosteum alone, whether from chick (Studitsky, 1933, 1934a) or man (Studitsky, 1934b), often formed cartilage which went on to experience an abnormal endochondral ossification. To his surprise, one graft of chick's fronto-parietal periosteum produced a small ovoid body of "chondroid tissue" (Figures 3, 4; 1934a).
    When cartilage was included inside the periosteum, the latter almost always made not cartilage but bone, in abundance and with well oriented trabeculae. Studitsky suggested that tensions in its wrapping might provide a way for the growing grafted cartilage to influence periosteal behavior. Unlike Fell (1933), Studitsky (1934b) was reluctant to attribute the capacity for a dual response to any particular type of periosteal cell.

    Schaffer included among his secondary cartilages that of fracture callus, so that cartilage formed by the periosteum of fractured transplanted bone belongs in the secondary class on two counts. Prasad and Reynolds (1968) caused cartilage to form at the fracture site of chick tibia] shafts by adding insulin to the chemically defined medium, in which the bone fragments were cultivated.

    So far as the very varied experiments on periosteum - transplanted, cultivated, and disturbed in situ, Miller (1967) inter alios - can indicate, the formation of cartilage by periosteal cells depends partly on the donor's age and species and the location of the periosteum on the skeleton, and in part on the circumstances of the culture or the site of the implant, where factors favoring rapid cellular growth, pressure, and displacement, seem likely to lead to secondary chondrogenesis.

    PERICHONDRAL OSTEOGENESIS IN TRANSPLANT AND EXPLANT

    This phenomenon is the counterpart to periosteal chondrogenesis in demonstrating that periskeletal cells have at least a dual potential for becoming chondroblasts or osteoblasts. The transformation of perichondrium to periosteum that normally occurs along the surface of cartilages undergoing endochondral ossification is not dealt with, but other examples of such a change occur in the perichondrium of transplanted and explanted cartilage.

    Friedheim (1930) cultivated hyaline cartilage pieces from rat embryos in hanging drops. He placed lepromatous masses - as a natural pathological agent that might stimulate osteogenesis - on either side of some fragments of cartilage. A bone-like tissue (more strictly, osteoid) formed on the cartilage face against the leprous tissues. Within two consecutive sentences he termed the cells forming the bone variously as chondroblasts, mesenchymal cells, and fibroblasts. While the precursor of the osteoblasts is unclear, what emerges is that he was not describing a transformation of cartilage to bone. Instead, since his transplants included perichondrium, this experiment may be taken as an early example of perichondral osteogenesis.

    The mesenchymal anlage of the mouse's mandible differentiates in vitro to produce a bone on which the angular and condylar secondary cartilages form (Glasstone, 1968, 1971). At six days in culture these cartilages were "enveloped in membranous bone," the source of which was not discussed. A similar behavior is seen when established mandibular condyles of infant rats are transplanted intracerebrally. After five days, Ronning and Koski (1969) saw the articular surface sometimes to be "partly covered by bone," while Meikle (1973a) was more specific on the site:
    "frequently, the first evidence of chondrogenesis was a thin layer of bone matrix between the cartilage and the proliferative zone (Figure 11)." Stutzmann and Petrovic's (1975) Figure 5 shows bone in the same location five weeks after the condyle was grafted in another rat's testicle.
    Up till now the experiments, as told, suggest that the condyle provides an example of perichondral osteogenesis. There are some complications. Meikle (1973a,b) regarded the proliferative and articular zones of the condyle as derivatives of mandibular periosteum. Hence, osteogenesis in the proliferative zone, hitherto chondrogenic, could be construed as a not so remarkable reversion to an earlier periosteal role. Meikle's (1975b) intracerebral transplantation of rat metacarpals speaks against this construction, however, since the epiphyseal inner perichondrium also becomes osteoblastic, although it has no earlier periosteal history. And even if a switch is a reversion, its basis is no nearer an explanation.

    Next, Melcher (1971) looked carefully at the tissue formed on the condylar cartilage of cultured fetal and infant murine mandibles. He concluded that it was not exactly bone, rather only "osteoid-like." The details of his description identify the material as a chondroid kind of bone or osteoid:
    "osteoid-like material was seen to contain orientated birefringent fibers (Figure 5), and it was stained by the acid fuchsin in the van Gieson solution and by light green, but was less reactive to alcian blue than was the adjacent cartilage. The material was not stained by the von Kossa method, but osteoid and predentine known to have been deposited during the period of culture were not either. The lacunae of the osteoid-like material were large."
    In his discussion, Melcher commented "that the material could be fibrocartilage, but the decreased staining by alcian blue does not favor this." Melcher described the osteoid-like tissue as also "extending into the substance of the cartilage in some sites." His Figures 4 and 5 of this phenomenon could be regarded as suggestive of a transformation of condylar cartilage into chondroid bone. Ronning (1966) described, in only one sentence, some transformation of cartilage to bone in condyles placed within the brain.
    Melcher considered the brief reports of osseous transformation of cartilage in the condyles transplanted to sites in vivo by Ronning (1966) and Felts (1961), and noted the close resemblance of Felts' Figure 12 to his own observations. Melcher concluded that the tissue he saw in vitro was "osteoid-like," and it exemplified the dual formative potential of condylar perichondral cells, not a metaplastic ability of cartilage.

    STIMULI FOR PERIOSTEAL AND PERICHONDRAL SWITCHING

    As an example of perichondral osteogenesis, the grafted mandibular condyle may be flawed by the bone's being unusual, and perhaps partly the metaplastic product of cartilage, rather than originating de novo from perichondrium. However, for the sake of comparing the stimuli to skeletogenesis, one may take the position that the tissue is almost bone and it is derived mostly from erstwhile perichondrium.

    A puzzle arises concerning the factors provoking the unexpected differentiations: of osteoblasts from perichondrium, in the instances above, and of chondroblasts from periosteum, as discussed earlier. When periosteal cells turn to chondrogenesis, this is attributed in vivo (Wurmbach, 1928; Ham, 1930; Felts, 1961) and in vitro (Fell, 1933) to poor vascularization and the resulting hypoxia, and to an altered pH of the tissues (Brookes, 1966); only a few authors emphasize mechanical factors (Glucksmann (1939) and Hall (1968b) inter alios), when experimentally verified. By contrast, when perichondrium yields bone, or something very like it, Meikle (1973a, 1975) pointed to a lack of function, with resulting loss of stresses, as the prime reason why precursor cells have become osteoblasts.

    Although Meikle discussed vascularity, he did not follow it to what seems to be another reasonable explanation: that transplants to the brain are not merely well vascularized (Felts, 1961), but may receive excess oxygen which could promote an osteoblastic differentiation (Shaw and Bassett, 1967). On the other hand, Melcher (1971) found the condylar "osteoid" deposited regardless of the conditions of culture that he used, including differing oxygen tensions.

    Experiments using transplantation and explantation are not easily interpreted. Apart from the known retraction of clots, unsuspected contractile forces may act in cultures and the brain. Fell and Mellanby (1952) wrote of a pressure probably exerted by capsules of myogenic and connective tissues around cultured limb bones. Many cells are now known to hold contractile filaments, for example, derivatives of the neural crest (Trifaro, 1978), and these or other cells may make stimulants to contraction, so that it cannot be assumed that tissues in vitro are loaded only by gravity. It is unlikely, though, that cultures could generate the intermittent forces introduced by Hall (1968d) and Veldbuijzen, Bourret, and Rodan (1979).

    Meikle (1973b) quoted Brash's remark to the effect that in vivo condylectomies and similar experiments were mutilation procedures leading to consequences limited in their significance by the special circumstances bringing them into being. If what is left of the animal is "mutilated," how we are to regard and interpret the piece excised and transplanted calls for even more circumspection! The transplanted tissue is deprived of its "functional matrix" and other restraints, so that, while processes of erosion and differentiation occur, these may be excessive or abnormal.

    OSSEOUS TRANSFORMATION IN TRANSPLANTED PRIMARY CARTILAGE

    There are a few reports that transplanted cartilage occasionally turns into bone. Other experiments indicate changes in the matrix, or the cells, leading toward a more bone-like product, but the change does not constitute a full metaplasia of both components of the cartilage. First, here are the reports of a metaplasia proper.

    Fischer (1882) implanted the wing rudiment from eight-day chick embryos into a hen's wattle. About two months later, he studied histologically the resulting firm body in the wattle. Skin and muscle were gone, but a large mass of cartilage, partly calcified, remained. In the center of the cartilage were marrow and bone, with a prominent region of transition between calcified cartilage and bone. Hence, Fischer viewed the bone as the metaplastic product of the cartilage.
    He obtained similar results with implants of embryonic leg, cervical vertebral region, and sternum with attached thoracic wall. When he took embryonic cartilage after first removing the major soft tissues of the limb, the cartilages grew more in their natural proportions, so that he was able to distinguish a bony shaft from the epiphyses, and he again saw signs of bony metaplasia in the cartilage.

    His evidence for a metaplasia is not strong. In the first place, following a common fallacy of the time, he took the absence of a lamellar structure to the diaphyseal bone to be a sign that the bone was of metaplastic origin. Next, around some cartilage cells the capsular matrix had thickened and the cells were small and mildly stellate in the manner described for rachitic cartilage, thought at that time, from the work of Kolliker, Virchow and others, also to be a site of metaplasia. Third, Fischer's Figures 17 and 18 suggest more of a line of demarcation between bone and cartilage than an imperceptibly smooth transition.

    Since Seggel (1904) cited Fischer's (1882) paper, Fischer's belief in metaplasia may have influenced Seggel's (1904) assessment of his own transplantations of femoral condylar cartilage to the abdominal cavity of rabbits. The cartilage was invaded by vessels, and after 30, 50, and 80 days, had a central marrow cavity and was being replaced by bone. Of the ossification process, he noted briefly,
    "the cartilage is gradually replaced from its base by appositional bone, whereby chondrocytes also participate by changing directly to osteoblasts. This direct metaplasia is much less than the appositional growth." That the tissue was bone and not osteoid was indicated by its hardness and need for decalcification. However, the description of the metaplasia is so brief as to leave open the question whether the cells alone appeared to be becoming osteoblasts (as he implied), or whether changes in the matrix around the cells led him to think of the cells as osteoblasts.

    In connection with the viability of the cells in calcified cartilage (chondroid bone II), it is noteworthy that in Seggel's transplanted articular cartilage the nuclei of osteocytes of the underlying bone were gone by 16 hours, whereas the cells of the calcified cartilage layer were strikingly resistant, being still present 35-60 days later.

    Growth cartilages of the young rat's rib were chopped up to give a suspension of cells able to produce metachromatic matrix in vitro (Shimomura, Yoneda, and Suzuki, 1975). Freed, suspended chondrocytes were implanted within Millipore chambers intraperitoneally into other rats. Here the cells made cartilage, but no bone. When the chambers were perforated accidentally or by intent, bone formed inside, which the authors attributed, on the one hand, to in-streaming host cells, while on the other, they wrote of a "remarkable osteogenic potential" of the chondrocytes and its enhancement by "some unknown type of host cell". The failure of the chondrocytes in intact chambers to form bone and the events after rupture argue only for an inductive action by the chondrocytes on host connective tissue cells. The authors' own conclusion in unclear - "However, GC cells alone did not form new bone, but required the participation of certain host cells to initiate osteogenic activity."

    Subba Rao (1954) transplanted the xiphoid cartilage autologously to the omentum in six-month-old rats. Of one of 48 grafts he mentions "the conversion of an area of cartilage into osteoid tissue." His Figure 8 is at low power; even so, rather than a merging with cartilage, it shows a peripheral nodule of osteoid separated by a split from the closest cartilage. Hall (1970b) also was not convinced that this illustration depicted metaplasia.

    Tenenbaum, Thiebold, and Bolender (1976) described some hypertrophic cells in the interior of Meckel's cartilage as transforming into osteoblasts in two transplants from fetal rats to the chorioallantoic membrane. This occurred while the exterior of the cartilage underwent dissolution. The tissue in their Figure 6, from its larger cells, looks more like chondroid bone than the mesenchymally derived membrane bone to its left.

    Kahn and Simmons (1977a,b) have transplanted epiphyseal cartilage from quail embryos to the chorioallantoic membrane (CAM) of chicks: "the matrix surrounding some chondrocytes became more bone-like with respect to staining pattern, birefringence and collagen morphology (1977a).

    The point of transplanting perichondrium-free cartilage from quail was to be able to distinguish donor cells from those of the chick embryo's CAM in whatever new skeletal tissues formed. Bone developing around the cartilage had quail-type nuclei, indicating, Kahn and Simmons (1977b) claim, that chondrocytes can escape from their lacunae and "become phenotypically and functionally osteoblasts. Whether these cells initially dedifferentiated into a more embryonic cell type before redifferentiating cannot be determined from the present experiments".

    The authors also saw the formation of a chondroid bone for which the still entrapped chondrocytes seemed to be responsible: "some of the grafts also exhibited, in regions between peripherally located lacunae, a gradient in matrix composition ranging from cartilage to bone (Figure 3)." The changes toward anisotropia and acidophilia "suggested that the chondrocytes had begun to synthesize and secrete a new more bone-like product."
    Viewed with TEM, banded collagen appeared "within the lacunar spaces around many of the entrapped chondrocytes (Figure 5). Furthermore, in some of these lacunae, the collagen fibers appeared to be undergoing mineralization. It should be noted that we have not encountered anything comparable to this phenomenon in our EM survey of normal avian endochondral bone formation."
    There are two normal findings that might be related. Gay et al. (1976) saw immunological reactions typical of both type I collagen and type II around the largest hypertrophic cells of the human infant's normal growth plate. In the chick's epiphysis, type I collagen appears in the apparently uninvaded lacunae of hypertrophic chondrocytes (von der Mark and von der Mark, 1977b), suggesting to the authors that "the initial layer of osteoid in lacunae of calcified cartilage is synthesized by hypertrophic chondrocytes before degeneration."

    The formation, late in the cartilaginous sequence of normal epiphyseal growth, of any collagen of the kind typical of bone raises anew the role of metaplasia in long-bone development, believed to have been resolved by Muller (1858). The chondroid bone described by Kahn and Simmons (1977a,b) certainly appears to be an example of a metaplasia in the osseous direction by avian epiphyseal chondrocytes, but whether the chondroid bone proceeds finally to bone is unclear, because on their evidence chondrocytes may escape and become osteoblasts. Hence there may be two metaplastic routes to osseous materials: direct, in situ, to chondroid bone; and indirect, by migration, to osteoblasts and then bone. But the new bone need not be the final form of the chondroid bone.

    OSSEOUS TRANSFORMATION IN TRANSPLANTED SECONDARY CARTILAGE

    The five-day-old mandibular condyle of the rat, when transplanted intracerebrally by Ronning (1966), displayed "a direct transformation of cartilage into bone." Ronning's Figure 5 legend states that "the cartilage cells seem to submerge into the bony tissue without any distinct endochondral ossification apparatus." This is mentioned and shown as occurring at the periphery of the condyle. Now, this is a region that Schaffer (1888) drew attention to as a site where, in vivo, cartilage merged with bone in a way likely to give an impression of metaplasia. Br> It may be that what Ronning drew attention to in his transplanted condyles is that same phenomenon, or an accentuated or deranged form thereof, as occurs generally in development when a leading edge of periosteal sleeve bone extends onto condylar or epiphyseal cartilage. Ronning was not alone in his observation. Meikle's (1973a) Figure 12 exhibits (unlabeled, on its right side) the same kind of cartilage-bone transition as that on the left of Rbnning's Figure 5.

    Another kind of cartilaginous transplant - fracture callus - is of more than usual interest, because what is grafted is not only secondary cartilage but, in part, may already contain some chondroid bone. This latter possibility is not mentioned by the investigators; each characterized the transplanted tissue as fibrocartilage. Danis (1957) transplanted to the anterior chamber of the eye callus tissue that he estimated from its color, elasticity, and appearance under the dissecting microscope to be fibrocartilage. Autologous and heterologous callus tissue resulted in an ossicle, but only "autologous" ossicies persisted for several months. From this difference, he concluded that the bone was not induced, since if it were host bone, it should not have been rejected even when evoked by homologous tissue. Thus, the bone was a direct product of the cells of the fibrocartilaginous graft, which "ossified itself," i.e., underwent metaplasia.

    His brief report can be disputed on two counts. He made no microscopic study of the tissue at the time of grafting, which might have revealed the presence of osteoid or chondroid bone. Second, the difference between the homologous and autologous ossicles - trabecular rather than lamellar bone; fibrous, not hemopoietic, marrow - is open to another interpretation. It can be taken as indicating that homologous tissue was at a disadvantage in inducing bone and hence left fewer and less mature ossicies, but nevertheless ones of host origin. Thus, the varying structure of the ossicle is not incontrovertible evidence of a different origin, graft versus host, that Danis supposed it to be. He noted that his results contradicted Urist and McLean's (1952) finding no difference between the bone induced in the anterior chamber by autologous versus homologous tissue.

    In a later experiment, Urist, Wallace, and Adams (1965) sought to distinguish transplanted cells from induced osteoblastic cells by tritiated-thymidine labeling of the callus before its grafting into the anterior chamber. This labeling was done because the formation of new bone began on the surface of the transplanted fibrocartilage, so that whether the osteoblasts involved came from donor or host could not otherwise be certainly determined (Urist and McLean, 1952). Half of each callus specimen was taken for histology, and proved to consist of fibrocartilage and hyaline cartilage, granulation tissue, and "spindle-shaped fibrous connective-tissue or mesenchymal cells."

    Urist and McLean (1952) favored a host or induced origin for the bone, since: 1) they observed the events typical of endochondral osteogenesis accompanying absorption of the transplant; and 2) resorption of cartilage and osteogenesis proceeded inwards into frozen or boiled callus, which could contain no potential donor cells. When the grafted live callus tissue was already 3H-thymidine labeled (Urist, Wallace, and Adams, 1965), the eventual ossicle had very few labeled nuclei, and these were in small chondrocytes in a bone-like matrix, suggesting a "chondroidal ossification" - the transformation of young cartilage cells into osteocytes.
    The authors introduced several examples claimed to be metaplasia into their discussion of chondroidal ossification, but concluded that no major metaplasia of callus cartilage to bone occurs, although this may happen on a minor scale. There remains the possibility that these few chondroidal bone cells were already differentiated to that state (though without mineral in their matrix) at the time of their transplantation - 11 days post-fracture.

    Another secondary cartilage has been transplanted, viz., that on a membranous bone of the embryonic chick's skull (Hall, 1972b). Hall wished to know the fate of the cartilage, when, with its membrane bone, it was immobilized, by paralysis in ovo, by being grafted onto the CAM, or by explantation to organ culture. The quadratojugal bones grafted or explanted at 12 days of age had by then a well established secondary cartilage. After five days, areas of this cartilage were enclosed in bone and hence not subject to erosion.
    Instead, they "were apparently being transformed into osseous tissue," at the time "poorly calcified in comparison with bone," but with a matrix "losing acid mucopolysaccharide and acquiring collagen and the staining properties of osteoid." After 10 days the matrix stained more strongly bone-like with chlorantine fast red, although the cells still had the size and appearance of chondrocytes. Between 14 and 21 days, "the cells within this tissue had come to resemble osteocytes," contained alkaline phosphatase, and were evidently alive, while the collagenous matrix around them exhibited calcification.

    Although he had not labeled the cells, Hall believed that the avian secondary cartilage had, without any dedifferentiation of the chondrocytes, undergone a direct transformation to a "bone-like tissue." This term in his summary conflicts with the "bone" in the title of the paper, and expresses the text's more cautious appraisal of the final tissue. His Figure 16 depicts cells like young osteocytes, but their osteocytic nature was gauged only from their size and positive reaction for alkaline phosphatase. For example, no mention was made of their having processes in canaliculi. Hall's discussion of other studies where chondroid bone arose indicates that he considered the avian cartilage to have experienced a metaplasia to a more bone-like tissue than the chondroid bones described by Gussen (1968a) and Moss (1961).
    However, it is not clear that the transformation described by Hall went to completion in the sense of leaving a bone indistinguishable from that formed conventionally by avian osteoblasts; and the tissue seen earlier, after five days of immobilization, clearly is a variety of chondro-osteoid, from its chondrocytes and weak mineralization. Kahn and Simmons's (1977a,b) finding for grafted avian epiphyseal cartilage is similar.

    Only some of the foregoing experiments provide any basis for believing in a metaplasia of transplanted cartilage to bone. The following studies of cartilage in vitro and transplanted in vivo offer even fewer grounds, and illustrate, in the main, changes that particularly lend themselves to misinterpretation. For example, an early and obligatory step in any metaplasia of cartilage to chondroid bone or bone is a reduction of the high content of proteoglycan, which at the least would make room for materials such as collagen and mineral; but perhaps also, as Hall (1972a) suggested, might "leave the cells susceptible to modulation to other skeletal cell types." While some loss of sulfated proteoglycans must occur for cartilage to become like bone, the loss itself does not constitute a metaplasia.

    "METAPLASTIC" CHANGES IN THE MATRIX OR CELLS OF TRANSPLANTED CARTILAGE

    Roulet (1935) cultured long bones from five- to 10-day-old chick embryos. He noted that some of the cartilage appeared more fibrillar and stained with aniline blue. He saw this occur directly under the periosteal collar of bone and around the "degenerating hypertrophic chondrocytes" of the interior where endochondral ossification was proceeding. He believed that, in both sites, by a dissolution of chondromucoid material, an unmasking of the collagen of the cartilage took place, and then this collagen was used to construct some of the apposed bone, partly as fibers and possibly also going through a soluble phase. He saw the hypertrophic chondrocytes as mostly dying, so that the osteoblasts arose instead from mesenchymal cells.

    His proposal can be viewed as an aberrant kind of metaplasia whereby cartilage matrix is used to form bone but the cells do not contribute. While not without some possible truth, particularly in tissue culture where materials cannot escape so readily and may be reused, it is unlikely that cartilage matrix materials would make up any more than some of the new bone right at their junction. (Knese (1956) has discussed this aspect of bone-cartilage boundaries.) More recent cultures of cartilage make one doubtful of Roulet's conclusion. For example, the continuity of bone fibrils with cartilage collagen fibrils is not necessarily evidence that the one is turning into the other. Melcher (1972) saw a continuity between the degenerating Meckel's cartilage and the adjacent soft connective tissue. Melcher also offered observations on the chondrocytes' hydrolytic enzyme content and uptake of labeled nucleotides and amino acid, indicating that they were alive and destroying the matrix, the opposite of Roulet's situation where the collagen of the matrix supposedly outlasted the chondrocytes.

    That the chondrocytes may not all die raises a major problem - discovering what they do next - which is to be kept in mind while examining other reports concerning cartilage matrix in culture. Fell and Mellanby (1952) exposed cultured avian limb rudiments to excess vitamin A. Of the skeletal tissues, the cartilage was most affected by the hypervitaminosis, displaying a loss of metachromasia with toluidine blue and a marked increase in its affinity for van Gieson's fuchsin. The intercellular partitions thinned, and groups of cells became enclosed in one large capsule. The experimenters were surprised to see many of these cells in mitosis.
    In mouse rudiments the cartilage was somewhat similarly affected by the vitamin A. The cartilage cells dissolved their surrounding matrix and eventually became free, and "seemed to wander into the medium where they were lost." There was no suggestion that the now fibrous cartilage matrix was related to bone, as Shaw and Bassett (1967) claimed, when they cultured 11-day-old embryonic chick bones under various concentrations of oxygen.

    Shaw and Bassett's experiment is cited as demonstrating that oxygen tension influences not only the rate of osteoblastic activity, but also the differentiation of osteoblasts from chondrocytes. What is the basis for this latter contention? Shaw and Bassett cut through the epiphyseal cartilage of the explanted tibias. In culture they found,
    "for a distance of eight to ten cells in from the cut surface of the explant, the underlying cartilage lost its metachromasia, became diffusely cosinophilic, and, frequently, appeared shrunken. Occasionally, a single large cartilage lacuna in this region was occupied by several cells. Other lacunae did not appear to be enlarged but contained three to four cells. The chondrocytes found in such altered lacunae frequently were stellate and resembled osteoblasts. In and about the lacunar spaces of such altered chondrocytes, coarse fibers occasionally were observed. In other lacunae, occupied only by cell remnants, numerous nonoriented, positively birefringent fibers were found. These fibers stained red with the van Gieson technique, and subsequently were identified as broad, 640 A-banded collagen fibers."
    In their discussion Shaw and Bassett remarked, "it is possible that certain chondrocytes responded to a higher oxygen concentration and changed nutritional status, altering their metabolic capacities to function as osteocytes," based upon the large collagen fibers' being a product of the cells of the original cartilage. The picture the authors (1967) described is not substantially different from that typical of other degenerating or cut cartilages, as described, for example, by Fell and Mellanby (1952) and Weiss and Amprino (1940). Furthermore, since thick collagen fibrils occur around old chondrocytes (Dearden, Bonucci, and Cuicchio, 1974) and those exposed to cortisol (Dearden, Mosier, and Espinosa, 1978) and its analogues (Lewinson and Silbermann 1978), Shaw and Bassett's acceptance of these as indicative of osteogenesis by what had been chondrocytes can no longer stand. What they describe, and their arguments based upon it, together do not constitute a strong case for osseous metaplasia within cartilage.

    The situation becomes more complicated, if chondrocytes escape alive from a degenerate matrix, as, for example, appears to happen with excess vitamin A, or in grafted quail cartilage (Kahn and Simmons, 1977b). If such cells become osteoblasts, they can form a new matrix. This could be construed as an orthodox metaplasia, still in keeping with Virchow's original definition, "the cells persist, but the tissue changes." When Barratt (1973) cultured pig articular cartilage with excess retinol for 10 days or more, the matrix deteriorated to the point where it released the chondrocytes, which took on an "elongated fibroblastic form."
    These "fibroblast-like cells in these (erosion) cavities and sometimes also in the proliferative region, acquired an osteoblastic appearance, rounding up and becoming strongly basophilic; they deposited a non-metachromatic fibrous network which stained red with van Gieson's stain." Barratt was, however, skeptical of a true osteoblastic metaplasia: "it is doubtful whether the apparent proliferation of osteogenic cells seen in the invasion cavities of the pig explants should be regarded as a true osteogenesis; it is more probable that the histological picture represents a selective collagenous regeneration by the liberated chondrocytes."

    Another site where such skeletal cellular transformations appear to take place is the pubic symphysis, where in many species pregnancy and aging bring about special changes in the bone, cartilages, and soft connective tissues of the joint. Crelin (1969) reviewed the work on the pelvis of the mouse, and his paper in 1954 cited other papers dealing with the guinea pig's pubis and its reactions to hormones. Earlier investigators had written of metamorphosis and metaplasia in the pubic bone. For example, Ruth (1935) considered the unmasking of the fibrils in the hyaline cartilage of the rat's symphysis in old age to be a fibrous metaplasia. Ruth (1936a,b) took his idea further for the more labile tissues of the guinea pig's pubic joint. There he suggested (1936a) that in the normal development of the female symphysis the chondrocytes "take on the nature of fibroblasts," and that after pregnancy and delivery (1936b), "the abundant active young fibroblasts are metamorphosed into osteogenic cells along the line of bone formation. . . ."
    This constituted an early phrasing of the concept, articulated by Crelin (1969), of the connective tissue cells of the pubic joint as modulating entities, and furnished a route whereby, via fibroblasts, chondrocytes could become osteoblasts. This pathway was speculation on Ruth's part, but Crelin and Koch (1967) believed that they had put such transformations of the chondrocytes on a sure footing by the use of tissue culture and tritiated-thymidine labeling.

    Their experiment is commonly held to prove that chondrocytes can become bone cells, but other investigations since then have provided the means to challenge what at the time seemed an invulnerable conclusion. They provided 3H-thymidine to murine pubic rudiments cultured at their 13-day-old mesenchymal stage. Following the rudiments as they continued to row in vitro revealed persistent labeling in chondrocytes of the pubic rami, but a loss of label from mesenchyme, which the authors presumed to have resulted from a dilution by repeated mitosis.
    The cartilage cells hypertrophied, their matrix dissolved, and the cells were freed, as described earlier by Crelin and Koch (1965). In the vicinity of the disintegrating cartilage were large multinucleated cells, with some nuclei bearing radioactive label. These cells were taken to be chondroclasts. When endochondral bone was formed, "intensely radioactive osteoblasts, derived from transformed chondrocytes, were found." Other osteocytes, "which showed only a small amount (of radioactivity) were the progeny of chondrocytes that had undergone a number of divisions."

    What are the implications of these interpretations? And are there alternative conclusions?

    1. If osteocytes are as heavily labeled as chondrocytes, this means, as the authors implied, that the chondrocytes transformed without dividing. Holtzer et al. (1972) believed that drastic changes in a cell's activity do not take place without one intervening cycle of division (or more; Yamada, 1977).
    2. The lightly labeled osteocytes could have re-used label lost by rapidly degenerating chondrocytes. Tonna and Pentel (1972) held that such a reutilization of label contributes significantly to the intensity of radioactivity in callus cells in vivo. Within the confines of tissue culture, a recycling of label is possible.
    3. Crelin and Koch (1965, 1967) submitted, as another witness to the central "endochondral" osteoblasts' arising from chondrocytes, the proposition that there is no other source, since the continuous solid nature of the periosteal bone excludes other cells. However, their (1965) Figure 25 (lower left) seems to have a gap occupied by cells in the periosteal bone. In their second report (1967) there was a large, early, labeled mesenchymal population of which they lost track because of dilution of the label. If the periosteal bone were not the seal they thought it to be, periosteal cells could have entered, divided, picking up shed chondrocyte label, and become the marked osteoblasts observed by the authors.
    4. Another study by Tonna (1972a) is relevant to the labeled nuclei in the "chondroclasts." Using electron microscopy to observe the fate of osteocytes liberated by osteoclasis, he noted a lysis of their cytoplasm, but an incorporation of their pyknotic nuclei within the osteoclast. What happened thereafter to the nuclei he was unable to determine. (Soskolne (1978) suggests that the osteoclast digests the whole osteocyte.) Extrapolating from bone, this suggests that the labeling over some of the chondroclast nuclei, although indicating inclusion of a chondrocytic nucleus, need not mean that the whole chondrocyte necessarily has become a part, and a functioning part, of the multinucleated cells seen by Crelin and Koch.
    5. Were Crelin and Koch (1967) right in calling the multinucleated cells chondroclasts? Hypertrophic chondrocytes in culture appear to degrade their own matrix under conditions of excess vitamin A (Fell and Mellanby, 1952) and in artificial media (Melcher, 1971), when they increase their reactivity for acid phosphatase (Melcher, 1972). On what are the "chondroclasts" to act, if there is no longer any cartilage matrix (Crelin and Koch, 1967)? The cells could simply be fused chondrocytes.
      Crelin and Koch's (1967) claim that chondrocytes became both chondroclasts and osteoblasts within the interior of the bone raises a question of why the factor supposed to turn them into chondroclasts, for example, does not make all of them differentiate that way. This difficulty might be disposed of by the evidence, reviewed by Hall (1975b, 1978) and Owen (1978), favoring the osteoclast's origin in monocytes. If chondroclasts were to stem from the same source, the large cells seen by Crelin and Koch either were not chondroclasts, or if they were, had not come from chondrocytes but from cells of a blood line included in the original graft.
    6. While there is evidence from various experiments that chondrocytes of cartilage disintegrating in vitro can divide (Fell and Mellanby, 1952; Crelin and Koch, 1967), take up tritiated thymidine (Melcher, 1971), and also proline (Melcher, 1972), it is questionable that chondrocytes behave thus in the intact animal, and the reason is this: In culture, the destruction of hyaline cartilage departs from the normal by having no participation by blood vessels, sometimes no calcification of the matrix (Melcher, 1971), and perhaps lessened contributions by blood-borne cells, although Melcher et al. (1978) have evidence that osteoclasts can form de novo from mononuclear precursors in vitro.
      When, in vivo, agents outside the cartilage - 'clasts, macrophages, endothelium - erode it, they do so in a selective way that may locally eradicate the chondrocytes. If this is so, when the cultured chondrocytes themselves play the principal role in degrading the matrix, they may escape alive, but to an illegitimate existence and a behavior that is unrepresentative of what happens normally.
    Although other interpretations thus can be made of Crelin and Koch's (1965, 1967) work, their original conclusion stands as one of several possibilities needing further investigation. The pubic symphysis of certain mammals is clearly a challenging site for hypotheses on the relations between connective tissue cells. In extrapolating findings in the pubis to elsewhere in the skeleton, it is well to remember that the joint is physiologically distinct in its "unique hormonal response" (Crelin 1963), and should be regarded as cautiously as other so-called special cases of sex hormone-dependent skeletal elements, such as penile bones and antlers.

    CHONDROCYTIC ESCAPE AND METAPLASIA IN THE INTACT ANIMAL

    Virchow (1853), Muller (1858) and other early microscopists thought that chondrocytes survived the destruction of primary cartilage and joined, or became, the population of cells that would form the endochondral bone and hemopoietic marrow. The number of the holders of this idea in its chondrocyte-to-osteoblast form greatly increased in the nineteen-sixties. Hall (1978) reviews their evidence. The work in vitro is open to the criticisms detailed above for Crelin and Koch's (1967) experiment, but before attempting a general assessment, here are more reports derived from intact animals.

    Lufti (1971) saw in the chick's tibia:
    1) hypertrophic chondrocytes of a healthy appearance;
    2) in the resorption spaces a cell population which "shows all transitions through the cartilage cells being liberated . . . " - a conclusion based principally on a continuum of nuclear morphology from hypertrophic cartilage to the marrow;
    3) a sharing of one lacuna by two chondrocytes, taken to be a sign of "recent division;" and
    4) "the walls of the marrow processes ... are lined by bone," partly as lamellae covered by osteoblasts and "partly in the form of 'cartilage bone.' This latter resembles bone in its general staining properties but its cells have evidently been derived from the hypertrophic cartilage (fig. 8)."
    These observations and interpretations are typical of the argument for a cellular metaplasia, and are discussed in turn below.

    In the formation of the cartilage canals of the chick's tibial epiphysis, along part of the canal's wall, "the adjacent cartilage matrix appears to disintegrate, liberating its cells which apparently dedifferentiate into mesenchymal cells" (Lufti 1970). At other regions of the wall, mesenchymal cells apparently are forming cartilage. Lufti suggested that such processes of dissolution and apposition at the canal's wall allowed for a relocation of the canal during growth. Later, the canals may be plugged by fibrocartilage, so that the following is a conceivable sequence:
    hyaline cartilage cell--> mesenchymal-like cell--> fibrocartilage cell, which could constitute an indirect metaplasia.

    Such a fate for cartilage cells may not be confined to birds. Barrie (1978) postulates the same sequence as Lufti's for hyaline cartilage lying free as a "loose body" in joints, in a kind of natural in vivo system of culture. (Blenkinsopp (1978) suggests another route to cartilaginous "loose bodies" - by a metaplasia of synovium.)
    Second, Friant (1958) described some of the cells of Meckel's cartilage in the mole as transforming into cellules conjonctives (connective tissue cells) which then contributed to the ossification of the mandible. She believed the same sequence occurred in the tarsier, but not in the golden hamster, where ossification was typically endochondral. While it is possible that the cells in vivo can survive the dissolution of Meckel's cartilage, Friant's contention was founded on only one fetus.

    To conclude from the observations of Lufti (1971) and others that there is a transformation of cartilage cells to osteoblasts or other cells is weak in the following ways.

    1. Although some hypertrophic chondrocytes show signs of life and activity and may even divide right up to the time resorption reaches them, they could die during or shortly after their liberation.
    2. The claim that some chondrocytes stay healthy while their matrix is destroyed is made more often for avian cartilage, Howlett (1979) inter alios, than for mammalian, although Hall (1978) cites Cooper (1965) as noting the reverse in vitro. Should the two classes differ in their chondrocytes' behavior, the observations in mammals, although similar, must have another explanation. But opinion on chondrocytic survival in birds is still divided. Thus, Silvestrini, Ricordi, and Bonucci (1979) note a consistent degeneration as the chick's uncalcified tibial cartilage is eroded.
    3. Mamnialian chondrocytes in vitro can escape from their lacunae, e.g., under the influence of retinol (Barratt, 1973) or when the matrix is degraded by collagenase (Green, 1971), then divide and produce a metachromatic matrix. But even if they do escape in vivo, there may be restraints on their resuming activity.
    4. The evidence that the supposedly liberated cartilage cells become osteoblasts relies on "transitional forms," and the appearance of radioactively labeled osteocytes in a region where earlier the chondrocytes were tagged. However, a label can be transferred independently of the cell in which it was first incorporated. Second, arguing from cells loosely categorized as transitional is the insubstantial cellular equivalent of taking the intermediate chondroid bone as proof of a metaplasia of cartilage to bone.
    5. The bone laid down in the cartilage's empty lacunae can, if enclosing a single large osteocyte, look like the product of a transformed chondrocyte (see Chapter 22, Globulus Osseous), but to call it cartilage bone (Lufti, 1971) is to prejudge its origin.
    6. Since osteoblasts have one source among the cells growing in with the invading periosteal buds, the justification for another origin needs to be especially sound. Two sources must bring confusion in interpretation, as when the undifferentiated mesenchymal cell is offered as an additional source of macrophages, osteoclasts, fibroblasts, etc. The dedifferentiating, potentially metaplastic hypertrophic chondrocyte therefore merits continued skepticism: life in one place is not survival in another, and survival is not transformation.

    CARTILAGE FORMED BY GRAFTED PERIOSTEUM AND REGENERATING TISSUE: SIGNS OF ITS METAPLASIA

    Studies using transplanted cartilage have not led to a firm conclusion that the cartilage, or its cells alone, can turn into bone. How different a matter is it for cartilage that develops after the grafting or explantation of periosteum? Some of the chondroid bone appears to be a stage in the direct metaplasia of such cartilage towards bone, which observers occasionally have been able to watch in progress.

    Echoing the earlier work of Bonome (1885) and Grohe (1899), Cohen and Lacroix (1955) wrote briefly of the hypertrophic cartilage formed by periosteum placed under the rabbit's renal capsule: "other areas of the cartilage nodules showed metaplasia of cartilage to immature fiber bone, without trabeculation, but nowhere did this process extend beyond a very thin lamina."

    (Fischer and Parker (1929) kept avian, cranial, perichondral chondroblasts in long-term serial culture. Despite their "fibroblastic" form, the cells, if changed to plasma, without embryo-extract, made nodules of a tissue, at first fibrillar, then becoming hyaline (Figure 21) and hard, until finally "one reached a tissue that, to a marked degree, resembles bone." The strong hematoxylin staining of the matrix and the knorpelahnlichen oder knochenahnlichen Zellen left the authors undecided whether it was woven bone. Their summary's knorpel-bis-knochenahnliche Grundsubstanz further identifies the tissue as chondroid bone, so that the chondroblasts had achieved a partial metaplasia to bone.)

    Fell's (193lb) First reference to anything like chondroid bone in her cultures of chick periosteum and endosteum was merely in passing and can be gleaned only from details of two exceptional instances. One culture of 10-day periosteum produced a nodule of a tissue very similar to hyaline cartilage, except that its matrix stained green rather than the expected pink, when stained with safranin and picro-indigo-carmine. In another periosteal transplant, instead of cartilage nodules isolated from the new bone, a bent plate of bone had a small mass of cartilage inside the bend, which "gradually merged" with the bone.

    Although Roulet (1935) observed some abnormal cartilage in vitro that has already been assessed as not chondroid bone, he also reported what could be confirmation of Fell's (193lb, 1933) sightings of chondroid bone. Within the periosteum of a cultured tarsal bone of the chick, cartilage developed (Figure 19), but with further passages the chondrocytes stayed alive and kept their form, but the ground substance appeared gradually to diminish, while the matrix became fibrous and took on an osteoid appearance.
    His Figure 20 shows osteoid and cartilage with a transitional zone between them. The osteoid matrix holding chondrocyte-like cells could be chondroid bone. This arrangement he saw in stained sections; there is no mention of his observing any actual transformation while the cells were alive. Roulet introduced this finding by referring to the old metaplastic theory of osteogenesis, and clearly regarded the behavior of the periosteal cartilage as an example of metaplastic ossification,

    A better described and more closely followed example was obtained by Fell (1933) using cultures of tibial diaphysis with endosteum, but neither periosteum nor cartilage, present at explantation. After an annular outgrowth of cells had appeared, the original fragment of bone was excised, leaving the outgrowth. Thus the new tissues seen later arrived de novo from a cellular blastema. Observations on the living tissue revealed that "chondrogenesis and osteogenesis usually took place merging imperceptibly through a series of intermediate cell types." The most common site of chondrogenesis was the central hole from which the bone had been taken. This area of cartilage diminished, and osteoid and finally calcified bone took its place:
    "Further study showed that this replacement of cartilage by osteoid tissue was effected by a direct transformation of the cartilage, the cells of which gradually decreased in size resuming the appearance of osteoblasts, whilst the matrix assumed the opaque, yellowish look characteristic of osteoid tissue".

    In these specimens, when fixed, sectioned, and stained, Fell clearly described chondroid bone; e.g., of the tissue seen between cartilage and inwardly encroaching osteoid she wrote:
    "The cells of this transitional tissue varied in appearance. A few, though not encapsulated, resembled chondroblasts, being large, round and vacuolated, others possessed a thionin staining capsule but otherwise resemble osteoblasts, whilst the majority, some of which were encapsulated with a film of cartilaginous matrix, were intermediate in appearance between osteoblasts and chondroblasts. The intercellular material was fairly plentiful and, with the exception of the cartilaginous capsules, was almost colourless in preparations stained with thionin and eosin."

    Her brief discussion comments were perceptive. She noted that the hypertrophic nature of the chondroblasts formed by the endosteum left relatively thin capsules, and that "this comparatively low differentiation of the cartilage may explain its capacity for direct transformation into bone." The "remarkably strong resemblance" of this cartilage to that in fracture callus, and the presence of the same intermediate tissue in both situations, did not escape her notice. The "perfect graduation" between cartilage and bone she attributed to both tissues' having their origin in the same "indifferent type of skeletogenous cell," and "the factors responsible for the differentiation of cartilage in a fractured bone may be identical with those which induce chondrogenesis in the endosteal cultures."

    Fell, in effect, described two kinds of chondroid bone: the first, the tissue joining concurrently formed cartilage and bone; the other, the intermediate stage of the slow transformation of cartilage to bone. Glucksmann (1938) confirmed in his cultures Fell's observation of a transformation in the cartilage of endosteal origin. The control limb-bone rudiments cultured by Fell and Mellanby (1952) for their experiments on hypervitaminosis A had islets of cartilage within the bone, when the periosteal bone was thick. The cartilage merged with the bone, and "in such specimens the histological picture somewhat resembled that of callus in which cartilage and bone are irregularly mingled," but no transformation was mentioned.

    The chance to watch a transformation of cartilage to bone (Fell 1933) also befell Clark and Clark (1942), but for fewer specimens. They cut holes in the cartilage of rabbits' ears in order to insert windows for observing regenerative phenomena. Within the newly formed marginal cartilage, bone was seen on four occasions at the time the chambers were removed, but in one of those instances the bone developed near the center within the area subjected earlier to regular microscopic examination.
    The bone watched in vivo developed "spontaneously" in two small areas "in the midst of the patch of new cartilage." The texture of the matrix changed and later osteocyte lacunae and canaliculi could be made out; and blood vessels invaded the tissue. New cartilage in a second chamber also experienced a similar limited metaplasia to bone, but the bone appeared later than it had in their first specimen, and after several weeks was absorbed. They reported observations through the window but undertook no subsequent histological study of the tissues. Clark and Clark's example constitutes a possible instance of sequential metaplasia, metaplasia in a tissue - cartilage - that itself arose by metaplasia. This would have come about, if their supposition that the new cartilage formed by a metaplasia of connective tissue cells were true.

    CONCLUSIONS

    Skeletal tissues transplanted and explanted from many sites in birds and mammals sometimes form unexpected tissues: cartilage and chondroid bone from periosteum, endosteum and/or marrow; bone or osteoid from perichondrium; bone and chondroid bone from cartilage. Any new cartilage belongs in the secondary class, and its origin is open to the usual two explanations: a novel or deviant differentiation of a connective tissue stem cell, however named - scleroblast, osteo-chondroprogenitor, etc. - or the metaplasia of committed cells in the graft, such as fibroblasts or osteoblasts. It may also be that chondrocytes escape from cartilage held in vitro and participate anew as skeletal precursor cells. In most instances, the multipotentiality of either a resident surface stem cell or a dedifferentiated osteoblast, chondroblast, or fibroblast can account for the untoward cartilage and bone, and the chondroid bone I frequently described, without invoking a direct metaplasia of cartilage.

    Tissues intermediate between bone and cartilage, or appearing to be, can be a result of other processes. In cartilage present at grafting, a loss of proteoglycans and abnormal collagen synthesis by the chondrocytes can lend the matrix an osteoid look. However, cartilage derived from cells proliferating after grafting has been observed transforming toward bone, thereby introducing the possibility of sequential metaplasias - connective tissue--> cartilage--> bone.


    Chapter 18 BONE AND CARTILAGINOUS TUMORS

    . Introduction
    . Secondary cartilages
    . Instances of neoplastic secondary cartilage
    . Sources of cartilaginous tumors
    . Virchow's hypotheses
    . Tumors on membrane bones: role of previous secondary cartilage
    . Experimentally evoked secondary cartilage
    . Chondroid bone in tumors
    . Nomenclature
    . Chondroid bone II
    . Chondroid bone I
    . In malignant tumors (skeletal)
    . In malignant tumors (extraskeletal)
    . In benign tumors
    . With experimentally induced ectopic skeletal tissues
    . Metaplasia in tumors?
    . Creeping substitution

    INTRODUCTION

    From the immense literature on tumors, only particular articles are used to cover the following questions:
    1. Are any or all cartilaginous tumors secondary cartilages?
    2. Does the existence of cartilage in a tumor present the same kind of problem of explaining its differentiation as for other secondary cartilages?
    3. Are the primary or secondary cartilages of development in any way related to the subsequent formation of tumors?
    4. Does chondroid bone occur in bony or cartilaginous tumors? If so, is it type I or type II, or can both be found?
    5. Does metaplasia occur? And is chondroid bone evidence for such a happening?
    6. Does chondroid bone seen during development play a role in the later formation of tumors?
    7. Is it possible experimentally to evoke new growths of bone or cartilage to aid in answering some of these queries?

    SECONDARY CARTILAGES

    Any abnormal growth of cartilage is a formation after the development of the primary cartilaginous skeleton, and hence is in one way a secondary cartilage. The original concept of secondary cartilages also took into account that the circumstances of their differentiation and growth differed from those of primary cartilage. Before the notion of secondary cartilages was properly established, Virchow (1863) drew a distinction among cartilaginous tumors based on the differentiation of the new chondrogenic cells. His ecchondromata were growths arising from an existing cartilage, for example of the airway, rib, vertebral, or symphyseal cartilage; and the etiological difficulty lay in explaining the abnormal growth.
    By contrast, enchondromata were cartilaginous neoplasms in bone or soft connective tissues, and they entailed two difficulties. The first problem was to account for the unnatural growth. The second problem, a heteroplastic one, was to explain why cells that had not exhibited a cartilaginous nature were now producing cartilage. This apparent hurdle of differentiation is one of the aspects distinguishing secondary cartilage from primary. (Enchondromas now in modern usage are benign growths of cartilage within bones (Lichtenstein, 1972).)

    In the temporal sense, all tumors with cartilage are examples of secondary cartilage. If one includes the factor that secondary cartilages differentiate spatially as well as temporally away from the primary ones, then those cartilaginous new growths - Virchow's enchondromata - also meeting this criterion can be considered to be more secondary, in sharing with normal secondary cartilages a common metaplastic histogenesis, than tumors arising from primary cartilaginous bodies.

    INSTANCES OF NEOPLASTIC SECONDARY CARTILAGE

    What kinds of cartilaginous tumor fall into this second, more restricted, but more harmonious category of secondary tumorous cartilage? The tumors can be divided by location into those in, on, or close by bones, and those in soft tissues away from the skeleton.

    Cartilaginous or cartilage-bearing tumors on or in bones, but excluding outgrowths of epiphyseal, articular and synchondrial cartilages, are the osteochondroma, chondroma, chondromyxoid fibroma, chondroblastoma, chondrosarcoma, osteochondrosarcoma (i.e., osteosarcomata with cartilage). The chondroblastoma usually develops at an epiphysis, which might be thought grounds for its exclusion, but occasionally a chondroblastoma occurs in the metaphysis (Aronsohn, Hart, and Martel, 1976), or in an unspecified intraosseous location (Steiner, 1979).

    Other firm growths arise from the periosteum and sometimes stay demarcated from the bone. Although Virchow (1864) referred to them distinctly as "discontinuous and therefore mobile, periosteal exostoses," the custom of distinguishing them was dropped until about thirty years ago, when they were given adjectives expressing their parosteal or juxtacortical position. Thus, cartilage can occur in the parosteal osteoma (Copeland, 1965) and the parosteal osteosarcoma (Harkness, 1964; van der Heul and von Ronnen, 1967; Ahuja et al., 1977) and comprises most of the periosteal chondroma (Cooke and Pearce, 1976; Fornasier and McGonigal, 1977) and juxtacortical chondrosarcoma (Goldman and Perzik, 1967; Jokl, Albright, and Goodman, 1971). Moving a little away from the skeleton, chondromas and chondrosarcomas sometimes form in the synovium (Lichtenstein, 1965; King et al., 1967).

    In soft organs of the body cartilage is an infrequent participant in benign and malignant tumors. Virchow (1863) remarked that it was relatively common in tumors of the breast and testis, more rare in the lachrymal gland and kidney. Other instances in these and other organs are reviewed by Geschickter and Copeland (1949), Nicholson (1950), Collins and Curran (1959), and Willis (1962).

    Other formations consisting of heterotopic or ectopic skeletal tissue alone may have cartilage present, but as entities are not now ordinarily viewed as tumors. Some have acquired specific and over-lengthening names; thus, Tracheopathia osteoplastica has became Tracheobronchopathia chondroosteoplastica (Kolling, 1976), and what Virchow was content to call an osteoma of soft tissue is now "extra-osseous, localized non-neoplastic bone and cartilage formation (so-called Myositis ossificans)" (Ackerman, 1958).
    While these cumbersome names make points regarding the extent, differential diagnosis, and prognosis for limited growth that are crucial to the handling of the cases, the fact remains that in, for example, myositis ossificans there is a new growth of bone, and the longer name reflects no advance over osteoma in our knowledge of why bone or cartilage has formed. Following the practice of Geschickter and Copeland (1949) and some present-day dermatological and oral pathologists, I include here for this non-clinical purpose such new growths of bone as "tumors," in order to take note of the presence every so often of cartilage in myositis ossificans traumatica (Leriche and Policard, 1928; Seemen, 1929; Geschickter and Copeland, 1949; Willis, 1962) and myositis ossificans progressive (Collins and Curran, 1959; Bona et al., 1967). Aside from its pathological occurrence in man and animals, ectopic bone and cartilage can be induced to form experimentally. This cartilage also meets the criteria for secondary cartilages.

    SOURCES OF CARTILAGINOUS TUMORS

    Virchow's Hypotheses
    Virchow (1863) deliberately named the enchondromata cartilaginous tumors, formed where "truly no cartilage should be present," and then set about explaining the puzzle of how the cartilage cells had arisen. His observations and thoughts are a potent influence today and deserve brief summary in their original form and sequence, because they give fair and considered treatment of the likely sources and factors:
    1. Most chondromas occur in bones, but not at the permanent, articular cartilage. [It should be noted that his class of enchondrona included some chondrosarcomas.]
    2. Chondromas develop in soft organs, where no cartilage existed.
    3. Chondromas of bones develop in young individuals: half the cases in the first twenty years of life.
    4. In some multiple enchondromas there is an hereditary tendency.
    5. The above observations suggest the prime error is in the development of the bone - an irregularity causing a predisposition to later tumor formation. Such an irregularity could well be a remnant of the original cartilage anlage remaining unossified [remembering that he believed in the direct metaplastic ossification].
    6. While investigating the skeletal tumors, it often emerged that there was a history of rickets. Later it was shown that florid rickets only preceded a few cases of enchondroma. In rickets, areas of cartilage can remain within the bone, raising the possibility that this irregular rachitic ossification or a similar disturbance could provide the predisposed tissue.
    7. An included cartilage piece could serve as the nucleus for a tumor somewhat in the way that a tooth germ in the jaw can give rise years later to a tooth.
    8. Such cartilage rests as the source were only a hypothesis. [He had never seen a tumor arise from one.] Moreover, some tumors appear very late in life, without any previous sign of abnormality. Also, even if something of the sort suggested gives rise to the tumor, the tumor's later course may be a completely heterologous one, i.e., with a change in cell type.
    9. The age at which enchondromas are most frequent in the various bones is related to the sequence of ossification in those bones, in that where ossification is late and irregular, enchondromata are more common, pointing again to a disturbance of growth as a causal factor.
    10. There is no satisfactory accounting for enchondromata of the soft tissues.
    11. A role for trauma is suggested by the more peripheral bones being more prone to cartilaginous tumors than the central ones, and sometimes from the case history.
    In his second volume on tumors, Virchow (1864) discussed the same problem of origin in terms of the question, whence came the cartilage cells of osteochondromata (numbered among his osteomata)?
    1. Noting again that many of the tumors are common in childhood and grow along with the skeleton, it is likely that they arise as deviations in development.
    2. Since most bones are entirely preceded by cartilage, one would expect that a portion of the primordial cartilage could make an independent growth anywhere within the bone. However, the osteochondromata overwhelmingly are to be found by the epiphyses.
    3. The epiphyses are the site of a longstanding cartilage - the growth plate - perhaps early in life some stimulus acts on the periphery of the plate to cause abnormal lateral growth. If such were the case, the tumor would be more an enchondroma, arising in cartilage, and turning into an osteoma by ossification, but such a sequence is only speculation.
    4. Another point to be considered is that periosteum can form cartilage not only in facture callus, but at sites of persistent pressure such as result from dislocations. Thus it is always possible for cartilage to grow from periosteum without any earlier abnormality in the development of cartilage.
    Virchow's ideas of the possible etiological roles played by inheritance, developmental rests, mechanical stimuli, trauma, and rickets were adopted and passed on, usually without heeding Virchow's carefully worded caveats and counter-arguments. Cohnheim (1889) extended the hypothesis of embryonic cartilaginous remnants into a general theory of neoplasia.

    Though Cohnheim's doctrine fell into wide disrepute among pathologists as was acknowledged by Geschickter and Copeland (1949), these authors continued to rely heavily upon Cohnheim's hypothesis to account for cartilaginous tumors. Geschickter (1965) was still citing Cohnheim as one of his two references, and attributing osteochondromas to "an unutilized and persistent form of embryonic tissue."

    In their book, Geschickter and Copeland (1949) first rejected Cohnheim's theory of embryogenic abnormality, but later rooted central chondromas in "persisting islands of cartilage in an arrested state"; osteochondromata at tendon insertions in "the persistence of extraskeletal blastema"; chondrosarcomata in "the survival of primitive perichondrium and periarticular strands of precartilaginous tissue"; cartilaginous variants of osteogenic sarcoma in "persisting portions of perichondrium from which the periosteum is derived"; and myositis ossificans in "fibrous strands in the muscle, or tags of precartilaginous embryonic connective tissue (blastema) displaced from the primitive periosteum,."

    Why did the authors think that they were not using Cohnheim's hypothesis in its basic form? Of neoplasms at tendon insertions, they wrote,
    "This relatively undifferentiated fibrous tissue persisting at sites where chondromyxosarcomas arise does not necessarily represent fetal-cell rests in the sense of Cohnheim, although this form of sarcoma may arise centrally from cartilaginous rests within the marrow cavity. The evidence educed here favors the view that the persistence of this tissue about periarticular points provides a normal growth center which functions in maintaining tendon length in keeping with increased skeletal growth."

    How far, in fact, had they departed from Cohnheim? They were making an exception of only one of their sites of persisting blastemal tissue, claiming that at tendons such tissue functions rather than is dormant. In effect, they were describing the modification to Cohnheim's basic mechanism represented by persisting undifferentiated mesenchymal cells. Such a substitution was more explicit in Cahn's commentary to Blum's (1954) case of maxillary osteochondroma:
    "If, on the other hand, as many pathologists believe today there is little basis in fact for the Cohnheim theory, then we can have recourse to the primitive mesenchymal cell as the forerunner of the chondroblast."
    The hypothesis of rests as the source of cartilaginous tumors may be better examined in membrane bones, for the small amount of cartilage involved in these bones' development is relatively confined to known locations. One might expect this pattern to be reflected in the distribution of chondrogenic tumors. Tumors on Membrane Bones: Role of Previous Secondary Cartilage?
    As they develop, many of the intra-membranous bones acquire secondary cartilages, sometimes quite small and often overlooked in the case of the maxilla and skull vault, others such as the mandible's obvious and wellknown. Most of these secondary cartilages experience endochondral ossification, and of these some are not completely replaced by bone, e.g., the mandibular condyle; others are, e.g., the human coronoid process. These secondary cartilages widen the range of potential contributors of cells to future cartilaginous tumors of bones such as the mandible and maxilla. For chondrosarcomas of the temporomandibular joint, Richter, Freeman, and Quick (1974) envisaged these several sources:
    "remnants of Meckel's cartilage, secondary cartilage area of chondroid bone, remnants of cartilage from rickets, fibrocartilage of the articular head or disk, metaplasias of connective tissue, or periosteum."
    One notes that to Virchow's hypotheses of cartilage rests, rachitic disturbance, periosteal metaplasia, and existing cartilage, and Cohnheim's idea of a displacement of nearby cartilage (Meckel's), Richter et al. (1974) have added "secondary cartilage areas of chondroid bone."

    This last item is an example of the commonly practiced fusion of two concepts which overlap but do not coincide. Miles (1950) had earlier given separate attention to the chondroid bone in considering the etiology of maxillary chondrosarcoma. Miles discussed two theories of how cartilaginous tumors arise in facial bones - by a metaplasia, or from vestigial rests of cartilage - but came to no conclusion. He illustrated a secondary cartilage regularly found at the lateral aspect of the maxilla by the malar process. He believed this formation to be a more likely source of residual cartilage than any entrapment of the nasal septum by the palatal halves, as suggested by Roper-Hall and Adcock (1939).
    Miles also drew attention to areas of another tissue - "chondroid bone" - which "is distributed in the jaws in no consistent pattern, except for showing some preference for alveoli and the mandibular angle." His Figure 4 depicts chondroid bone I by the maxillary deciduous canine tooth germ. He regarded the maxillary secondary cartilage and the regions of chondroid bone as possible sites of rests, "since with such heterologous growths one cannot leave Cohnheim's hypothesis out of consideration."

    The principal attraction of the hypothesis of rests has been the semblance of its accounting for the higher frequency of chondrogenic tumors towards the epiphyses of long bones and, it was supposed, at the condyle, coronoid process, and symphysis of the mandible, and in maxillary alveolar bone. Miles noted that the two chondrosarcomas he followed seemed to start in alveolar bone of the anterior maxilla, a predilection Chaudhry et al. (1961) believed they had confirmed. However, Mikata, Iri, and Inuyama (1977) and Terezhalmy and Bottomley (1977) report chondrosarcomas apparently starting in the hard palate; and Sato, Nukaga, and Horikoshi's (1977) series of chondrosarcomas, while displaying a predominance of lesions in the anterior and palatal regions of the maxilla, had a very wide distribution.

    While many mandibular chondrogenic tumors form at what seem to be sites of earlier secondary cartilage, e.g., coronoid process and mandibular condyle (Allan and Scott, 1974; Nortje, Farman, Grotepass, and van Zyl, 1976; Koller, 1896; Pap and Friedman, 1959; Cooper and Finch, 1974), others are seen in the body, ramus, and angle (Lanier, Rosenfeld, and Wilkinson, 1971; Chaudhry, Robinovitch, Mitchell, and Vickers, 1961; Looser and Kuehn, 1976; Grotepass, Farman, and Nortje, 1976; Martis, 1978; Schulz, Maerker, and Delling, 1978, High, Frew, and Glass, 1978; Brady, Sapp, and Christensen, 1978).
    In Sato, Nukaga, and Horikohi's (1977) review of Japanese cases, chondrosarcomas of the molar and ramal region well outnumbered those at the condyle, and there was none at the symphysis and coronoid process. Thus, no consistent pattern of distribution favoring the hypothesis of rests has so far emerged among these various rare facial tumors. Shira and Bhaskar (1963) earlier arrived at the same conclusion.

    "Seem to be sites of earlier secondary cartilage" above is deliberately phrased, because at the symphysis and coronoid process the cartilage is replaced by bone. This bone is subsequently reshaped (Enlow, 1968, 1975) with a relocation of anatomical landmarks in such a way that the bone of which they were earlier composed is destroyed, and, without vital labeling, no one can be sure what relation the new cells on and in the bone have to the secondary cartilages and perichondrium of earlier development.

    What favoring of sites exists could have another basis than rests, which Geschickter and Copeland (1949) started to develop -
    "The majority of osteogenic neoplasms occur at sites which correspond to developmental patterns, rather than to embryogenic abnormalities, such as was postulated by the Cohnheim theory." They subsequently reverted to Cohnheim's doctrine without establishing the point that regardless of the relation of the later cells to earlier ones, at a particular position on a bone, circumstances, say mechanical, such as acted in development to evoke the normal secondary cartilage, might repeat themselves sufficiently to once again specify cartilage as the tissue of growth. That tumorous cartilage appears more often at certain surfaces of the bone than at others may reflect, not a pattern of distribution or displacement of rests, but that the loading of the bone, or whatever, more often reaches a threshold to engender chondrogenesis on those surfaces.
    As proponents of this kind of pathogenesis, Cooper and Finch (1974) suggested of a coronoid osteochondroma that, "the production of cartilage rather than bone may have arisen as a result of the stresses of continued mandibular movements." Allan and Scott (1974) regarded their coronoid osteochondroma in a related light, as, "an exuberant osteochondrogenic response by the periosteum to trauma stimuli - a reactive hyperplasia," related in the sense that trauma has mechanical effects, albeit unknown, on the cells. As Virchow, Kassowitz (1881) and others perceived, if periosteal and other connective tissue cells can experience a cartilaginous differentiation or metaplasia, this eliminates the need for rests.

    The facial bones are not the only membrane bones to have cartilaginous tumors form on them or close by. A few benign and malignant cartilage-bearing tumors form intracranially under the occipital, parietal and frontal bones (Chorobski, Jarzymski, and Ferens, 1939; Berkmen and Blatt, 1968; Scheithauer and Rubinstein, 1978; Giantriglia, Pompili, and Occhipinti, 1978; Alvira and McLaurin, 1978). They differ from those of the facial bones in usually being separate from the skull, when excised or found post mortem, but have also been attributed variously to displaced cartilaginous rests, undifferentiated mesenchymal cells, or a metaplasia of fibroblasts. Chorobski et al. gave the fullest discussion of the etiology.

    In summary, a response of periosteal cells to some stimulus, perhaps similar to those evoking secondary cartilages in development, can account as much as the hypothesis of rests and their displacement for these chondrogenic tumors of membrane bones. If the reacting cells were stem cells, their chondroblastic venture would be a novel-stem-cell differentiation; if they are osteoblasts or fibroblasts, a metaplasia. Thus, Katenkamp, Stiller, and Waldmann (1978) favor a UMC as the stem cell for the various cells seen in osteosarcomas by TEM. Whereas Schulz, Maerker, and Delling (1978) tentatively designate the anaplastic osteosarcoma cell as a malignant osteoprogenitor with a significant chondroblastic potential, and a possible origin by the dedifferentiation of an osteoblast (Schulz et al., 1977).

    EXPERIMENTALLY EVOKED SECONDARY CARTILAGE

    With the pathological formations of cartilage so far discussed, one can only speculate after the fact, and with the tumor often grown large, about the source of the cartilage. To what extent can neoplastic secondary cartilage be produced by experiment, where at least the site of first chondrogenesis can be discerned? The intra-tibial inoculation of infant rats with Moloney sarcoma virus leads in about two weeks to a metastasizing osteosarcoma, at the growing margins of which a "chondroid differentiation" occurs (Olson and Capen, 1977). Their Figures I-D and 3 indicate that some of this tissue is frank cartilage. Of course, rats of this age have abundantly cartilaginous tibias undergoing active endochondral growth, but from the position of first appearance of zones dense to X-ray associated with focal lysis, the authors suggest that the neoplasms arise from the metaphyseal and diaphyseal endosteum.

    An example of the evocation of a benign cartilaginous growth from more mature bone followed the scraping of the periosteal surface of femurs in mice by Miller (1967). The same procedure in rats and guinea pigs resulted in only bone formation, although cartilage appears after fractures in these species. Also, the reaction of the rat's femur and tibiofibula to prolonged venous stasis included "a deposition of new layers of an admixture of cartilage cells and woven bone around the circumference of the outer aspect of the cortex" (Abdalla and Harrison, 1966).

    Cartilage can be induced by a number of agents to form away from the skeleton, sometimes alone, sometimes along with bone (Bridges, 1959). This ectopic cartilage is secondary by appearing after and apart from primordial cartilage, but also by its morphology, e.g., after the implantation of alcohol-fixed skeletal muscle, Bridges noted, "The cartilage was of the hypertrophic type with large cells and relatively scant matrix." Ostrowski and Wlodarski (1971) in reviewing bone induction bring up more examples of the experimental evocation of heterotopic cartilage, including the actions in mice of human amniotic FL cells (Anderson and Coulter, 1967) and WISH cells.
    Bone matrix has osteogenic and chondrogenic inductive powers. Rohlich (1941a,b) implanted femoral and tibial shafts stripped of marrow and periosteum and killed with alcohol. After some months, small amounts of new bone and cartilage were found on the old endosteal surfaces of the tubes. This inner location suggests that, although the animals were rabbits, the induction was not owing to injury to the muscle outside. Furthermore, new skeletal tissue only occurred when the dead bone had experienced erosion, leading Rohlich to suggest that erosion liberated an osteogenic factor from the bone matrix, a hypothesis endorsed and developed by Urist (1965, 1971).

    Urist developed a somewhat different system of induction by demineralizing bone or dentin's matrix before implantation. These materials evoke cartilage formation when placed intramuscularly in animals, and demineralized bone evokes cartilage from muscular connective tissue in vitro (Terashima and Urist, 1977; Anderson and Griner, 1977). Urist et al. (1977) in a recent adaptation of his system transplanted devitalized osteosarcoma from the Dunn mouse into intramuscular pouches, whereupon cartilage and bone were induced.

    CHONDROID BONE IN TUMORS

    Nomenclature
    In bone pathology names are used as nowhere else. Thus, chondroid is applied to tissue that is hyaline cartilage, e.g., Lowry and McKee (1972). On the other hand, Lichtenstein (1972) used it thus; "As the qualifying adjective 'chondroid' implies, these tumors were not all composed of full-fledged cartilage, as seen in endochondroma or the usual chondrosarcoma. In fact, some were composed essentially of poorly differentiated spindle connective tissue showing only focal areas of cartilage or chondroid matrix microscopically." Concerning the histopathological differentiation of these two matrices: "As for the fields of cartilage differentiation, these may be chondroid (exhibiting slight tinctorial metachromasia with azure A or methylene blue stains) or more hyaline and partially calcified."
    Weinmann and Sicher (1955) also distinguished chondroid from cartilage in both osteochondroma and osteogenic sarcoma. They used chondroid according to Schaffer's meaning of a large-celled matrix-poor tissue other than hypertrophic hyaline cartilage and, indeed, they specifically likened it to the tissue of sesamoids (a site of Schaffer's (1930) chondroid).

    A second problem of terminology is that names like Osteoidchondrom (Virchow, 1863) and osteochondroma suggest not only tumors composed of bone and cartilage, but those consisting of a tissue intermediate between the two. When one reads Virchow's (1863) writing of Osteoidknorpel as the major constituent in osteochondromas, one's first thought is that this is a neoplasm of chondroid bone. However, this is not what he meant (see Chapter 2, Knochenknorpel), nor what subsequent observers have seen.

    Chondroid bone I appears never to be the sole or major tissue of a tumor, but rather participates in proportion and manner similar to its contribution to fracture callus: minor in amount and transitional in position between cartilage and bone or (often in tumors) osteoid. Virchow remarked on such transitional regions in a variety of skeletal and soft tissue tumors, but never gave the tissue a particular name, probably because he thought the state to be a temporary one as cartilage became bone by metaplasia.

    Virchow (1863), after discussing cartilaginous tumors growing from permanent cartilage (Ecchondrome), sought to establish a rational nomenclature for cartilaginous neoplasms arising in bone and other non-cartilaginous tissues (his heteroplastic chondromas). On the inner surface of cranial vault bones and under the diaphyseal periosteum of growing long bones, Virchow had observed a tissue very similar to bone and one which he regarded as having an osteoid nature. With the metaplastic conception of osteogenesis prevailing then, Virchow viewed this osteoid as participating in intramembranous osteogenesis in a way similar to the role of cartilage in long bones, namely, in being a precursor that would transform into bone by mineralization.

    One result of this overdrawn parallel between intramembranous and enchondral ossifications was that Virchow was led to regard tumors composed of his relatively soft osteoid tissue or Hautknorpel (Knochenknorpel) as a form of chondroma - the osteoiden Chondrome (Desmochondrome) - to be grouped alongside true enchondromas in a general class of heteroplastic chondromas, although they might have no cartilage in them. The Osteoidchondrom appeared for many years in the pathological literature, at first signifying, as intended, a tumor composed of osteoid, but later the term also came to encompass osteochondromas and osteosarcomas as they are now understood. By 1878, Ziegler was using Osteoidchondrom for what reads as an osteochondrosarcoma, while Borst (1902) continued using it in Virchow's sense for what would now be viewed as an osteoid osteoma or osteoblastoma.

    The categorization of tumors was and is confused by the widespread occurrence of mixed forms. From the outset, Virchow appreciated that there was no rigid distinction between his osteoid chondromas and true chondromas, since there are many mixed tumors composed of both tissues, apart, or merging into one another. The intermediate zones of the latter would be chondroid bone I as understood here. For many years the tissue had no name of its own, and the names that seem to refer to chondroid bone mostly identified osteoid or bone. For instance, Funkenstein (1903) was still lamenting the persistence of pathologists in employing Osteoidknorpel and Knochenknorpel as synonyms for osteoid tissue, and the absurdity of their calling bone verkalkter Osteoidknorpel.

    Although pathologists usually failed to name chondroid bone, its occurrence in tumors is evident from their illustrations and detailed descriptions. Many instances could be cited, but a few will do to show its presence in benign and malignant tumors, in and away from the skeleton; in ectopically induced firm tissues; in evoked malignant tumors; at and removed from sites of developmental secondary cartilage. Attention will also be paid to whether the pathologist named chondroid bone as such or as something else, and whether the tissue was interpreted from its nature and intermediary location as evidence for a metaplasia.

    Chondroid Bone II
    One variety of chondroid bone results when the resorption of endochondral ossification spares areas of calcified cartilage wide enough to hold cells, leaving chondroid bone type II (hyaline), for example, in the slow ossification of lower vertebrates, and as osteogenic substitution dwindles under mammalian articular cartilages. In many tumors the pace of growth is too fast and the cells are too abnormal to allow proper mineralization of the bone, and osteoid is frequently observed (Sela and Boyde, 1977; Delling, Schulz, and Seifert, 1978). Also many osteociastic and giant cells may be present to hasten destruction of the tumor's early tissues. Nevertheless, chondroid bone II might be expected in the more languid growths.

    Three cases illustrate its presence. Many osteochondromata take the form of an articular cartilage-like cap, under which endochondrally formed trabecular bone becomes slowly more dense. At the bone-cartilage boundary of a maxillary osteochondroma pictured (No. 6) by Blum (1954), on the bone side of the tidemark is a tissue with chondrocytes, such as is regularly seen in normal older epiphyses. The legend to Figure 8 of Allan and Scott's (1974) report of a mandibular osteochondroma, indicates successive bands of calcified cartilage in the caps, with bony replacement mainly at the expense of the innermost band, thereby leaving the more superficial ones intact and still mineralized. Deeper in the tumor, "the center of the lesion consisted of lamellar and immature varieties of bone (Figure 8) and contained entrapped, persistent islands of calcified cartilage." Their Figure 8 depicts these islands as large with numerous lacunae and cells, and identified in the legend by the authors as chondroid bone.

    A third example is in Figure 259 of Weinmann and Sicher (1955) showing part of a maxillary osteochondroma, where the calcified cartilage labeled is fibrocartilage, and hence an example of chondroid bone II (fibro); see also Figure 3 of a zygomatic osteochondroma (Pool et al., 1979).

    Chondroid Bone I
    In Malignant Tumors (Skeletal) The malignant tumors of the skeleton may grow fast, perhaps increasing the likelihood of cartilage and chondroid bone I. In tumors of the femur and humerus that were probably malignant, Ziegler (1878) described transitional regions between bone and cartilage and took them to signify a metaplasia of cartilage to bone. In the humeral lesion, cartilaginous tissue was present within the marrow cavities and enlarged Haversian canals and appeared to blend with the bone. He interpreted this merging of tissues to be a sign of a metaplasia of bone into cartilage. Other things being equal, this is as logical an interpretation of chondroid bone as metaplasia in the cartilage-to-bone direction. More recently and in juxtacortical positions, van der Heul and von Ronnen (1967) reported "chondroid areas" (meaning cartilage-like) in half the 16 cases of osteosarcoma they examined. Their Figure 17, stated to be of endochondral ossification, does not make a good case for this event, but reveals cartilage merging into chondroid bone, having small cells among larger ones and a darker matrix than the cartilage, as is seen in Dahlin's (1978) Figure 19-17 of chondroblastic osteosarcomas (my Figure 33).

    Closer to, but not necessarily at, sites of earlier chondroid bone and secondary cartilage are tumors of the jaws. Dahlin (1978) finds nearly half the osteosarcomas of the jawbones to be chondroblastic. As shown in his Figure 19-23, right, the cartilaginous cells sometimes produce "a homogeneous acidophil osteoid tissue" around them (my Figure 34). Remberger and Gay (1977) could not find type II collagen in the chondroblastic areas of osteosarcomas. Chaudhry et al. (1961) reported a tumor of the maxillary ridge with myxoid, chondroid, and an odd bony tissue, in which "were areas in which metaplastic transformation of chondroid into atypical osseous tissue was apparent." Their Figure 7.B, illustrating the merging of a tissue with anaplastic chondroid cells into irregular atypical bone, brings home a point hitherto avoided,

    Fig 33
    Cartilaginous and bony tumors follow, rather than contradict, the patterns observed for secondary cartilages and chondroid bone in non-neoplastic situations. If the tumor's cells were that abnormal, they would not have been able to make the matrices that brought them to attention as examples of secondary cartilage and chondroid bone. However, there is a dimension of abnormality to the cells of tumors, which in malignant tumors reaches extreme forms. In particular, the chondroid bone of a malignant growth can be expected to have a considerable variety of expressions as its cells and their products are also involved to lesser or greater extent in the neoplastic derangements, whatever these may be.

    Tissue somewhat of the kind illustrated by Chaudhry et al. (1961) in a maxillary tumor is described in Figure 2 of Richards and Coleman (1957) from a case of osteogenic sarcoma extending from the mandibular second premolar to the second molar. Hyaline cartilage resembling fetal cartilage merges, via chondroid bone, into an osteoid with osteoblasts. For a tumor with illustrated chondroid bone at an actual site of a second cartilage, the only example rewarding a brief search was that of Nortje et al. (1976) of chondrosarcoma of the mandibular condyle. Their Figure 9 has hyaline cartilage with areas of ossification, none too clear, but suggestive of chondroid bone.

    Fig 34
    In Malignant Tumors (Extraskeletal) Virchow (1863) was struck by the variety of bony and cartilaginous tissues in his "chondromas" of the soft tissues, which included entities such as the malignant osteochondrosarcoma of the thyroid gland. There, aside from osteoid and sarcomatous regions, Funkenstein (1903) described cartilage-like cells in a weakly eosinophilic matrix, sometimes fibrillar in texture. Other parts of the matrix were trabecular, with some trabeculae close to cartilage in nature, others held many round or irregularly elongated cells in a more eosinophil ground substance. This trabecular tissue was often continuous with purely cartilaginous bands, but they were separated by an irregular blue line.
    On the face of it, he took this line to indicate a limit to calcification, because he wrote, "One can with justification designate such formations as Knochenknorpel [he meant chondroid bone I] on account of the bone-like trabecular form and calcification but also their undeniable kinship with cartilage." His lengthy discussion of Cohnheim's and metaplastic hypotheses regarding the etiology led to a circumspect conclusion that the bone, cartilage, and chondroid bone were a metaplastic product of the sarcomatous cells, themselves originating by a reversion of connective tissue cells.

    Livingstone and Sandison (1962) observed in an osteogenic sarcoma of the thyroid,
    "the 'chondroid' tissue appears to be firmly cartilaginous, with disordered chondroblasts and areas of calcification in places... The nature of the 'chondroid' matrix is interesting: although there is a resemblance to 'osteoid' the staining qualities and reticulum pattern are entirely consistent with its essentially cartilaginous nature. Some of the bone present in the tumor has been formed by metaplasia of non-malignant stroma, but in other areas there is direct transition from undifferentiated tumor cells through chondroid to woven bone formation." So, Livingstone and Sandison, while describing chondroid bone, left it nameless, but did use it as evidence for a metaplasia of cartilage to bone.

    Smith and Taylor (1969) illustrated chondroid bone (Figure 4) in the human breast without naming it, but Hager and Lederer (1977) refer to and illustrate chondroosteoide Strukturen. Cotchin's (1958) widely cited paper on mammary neoplasms of the bitch noted admixtures of cartilage to bone and osteoid in malignant tumors. While invoking stromal metaplasia as a possible way for the firm tissues to have originated, Cotchin did not concern himself with any further metaplasia of the cartilage to bone.

    In Benign Tumors The evidence that chondroid bone I participates in benign tumors is less strong than for the malignant neoplasms. Although Virchow (1863) described transitions between cartilage and the osteoid of his "osteoid" tumors, this category included ones that had sarcomatous areas and had metastasized. When the benign neoplasms are kept separate, for example, by Geschickter and Copeland (1949), their report of transitional regions in osteochondromata may refer to calcified cartilage abutting or within bone, i.e., to chondroid bone II. The same holds for Lichtenstein's (1972) description of enchondromas within bone: "heavily calcified areas, particularly where they border on the interlobular vascular spaces, tend to undergo osseous metaplasia." Although there the metaplasia proposed involved calcified cartilage, of another benign condition - synovial chondromatosis - he wrote that "the cartilage foci, as noted, may become calcified or converted to bone."

    Next, Geschickter and Copeland's (1949) Figure 261 shows chondroid bone I at the boundary between bone and cartilage in an osteoid osteoma. Their legend has the cartilage being converted into bone by creeping substitution, i.e., metaplasia. Cotchin (1958) reported "osteoid and chondroid tissue merged in trabeculae of mixed structure" in canine benign tumors. In some human chondroblastomas, Dahlin (1978) saw cartilage merging into bone (Figure 4-19) in a way suggestive of metaplasia. Last, an opercular osteochondroma of the jewelfish comprising mostly hyaline cartilage had "some transformation into osteoid tissue" (Nigrelli and Gordon, 1946).
    That CB I is less often reported in benign skeletal tumors may have two explanations: it is not there, perhaps because chondroid bone is only formed when osteogenic or chondrogenic cells are multiplying and synthesizing rapidly; or it is present early in small amounts, but is resorbed by the time the tumor has grown to the point of demanding excision and microscopy.

    Chondroid bone I also is present in such induced or evoked benign growths as heterotopic bone and fracture callus. Chondroid bone joins in the benign ossicles of myositis ossificans, although this condition is no longer viewed as a tumor (Lichtenstein, 1972). The illustration (Figure 271) claimed by Geschickter and Copeland (1949) to show "confluent areas of osteoid trabeculae and cartilage in myositis ossificans" seems to have trabeculae only of bone and chondroid bone. Hirsch and Morgan's (1939) first four figures depict extensive merging of cartilage with bone in the several cases of traumatic myositis ossificans in which they saw considerable cartilage.

    With Experimentally Induced Ectopic Skeletal Tissues A condition resembling myositis ossificans or muscle "osteoma" can be induced in animals. The animals are usually little. The minute nodules of induced bone and cartilage match the small dimensions of the muscular site of grafting and the focal origin of the inducer, e.g., a transitional epithelial cyst. Occasional nodules are mixed, with areas of cartilage and bone separated by a zone of chondroid bone of a necessarily narrow width. Such chondroid bone is around part of the perimeter of the amniotic cell-induced cartilage of Figure 6 of Ostrowski and Wlodarski (1971), and where cartilage and bone merge in a transitional epithelium-evoked ossicle depicted in Figure 6 of Beresford and Hancox (1967).

    Other examples of chondroid bone arose in Urist's decalcified-bone-matrix system for the induction of bone and cartilage. Buring and Urist (1967) loaded Millipore chambers with minced skeletal muscle (including endomysium) and decalcified lyophilized bone matrix, and implanted them intramuscularly in rabbit. Inside the chambers, hyaline cartilage formed and a tissue whose matrix "had the faintly eosinophilic or metachromatic (azurophilic) quality that is characteristic of 'chondro-osteoid'." They likened this tissue to the chondro-osteoid observed by Shaw and Bassett (1964) in tissue cultures.

    When the decalcified, lyophilized, isogenic bone is implanted free in muscle (Buring, 1975), after 19-23 days the first sign of osteogenesis by the host rat is a "chondrosteoid tissue;" see Figure 5 of Koskinen, Ryoppy and Lindholm (1972). The amount of the intermediate skeletal material around free implants is increased by giving rabbits disodium-ethanehydroxy-1,1-diphosphonate (EHDP) (Plasmans, Kuypers, and Sloof, 1978). In the treated group they sometimes saw "a band of osteoid-like tissue containing cartilage cell islands ... After 3 weeks there was a marked proliferation of this irregular tissue which contained but few fibers and showed a strong affinity to Alcian blue (Figure 6). The cells could not be exactly classified. They showed some resemblance to both osteoblasts and osteocytes (Fig. 7)." Their name for CB I was "chondroitic tissue," as in the legend to their Figure 5.

    When the inducing tissue is viable, e.g., a fresh homograft of murine metaphysis (Upton, 1972), its imprisonment in a Millipore chamber sometimes results in the material which it itself deposits being an "osteochondroid." To explain the contrast between the "immature bone" and osteochondroid formed in the chamber with the darker, normal bone induced outside, Upton suggested that the oxygen supply to the chamber's interior is decreased.

    METAPLASIA IN TUMORS?

    As has been noted, many pathologists have assumed that chondroid bone I, in the presence of and physically joining cartilage and bone, is a result of a transformation of cartilage to bone. Although the microscopic picture could with equal justice be construed as showing a metaplasia in the reverse sense, very few pathologists (Ziegler (1878) inter alios), have espoused this interpretation. Since the tissue is seen only, on the single occasion of biopsy, excision, or autopsy, there can be no observing of steps in a sequence, were there to be a metaplastic sequence.

    An equally valid interpretation of neoplastic chondroid bone I is of a tissue formed de novo from similar prolific cells to those that give rise to the bone and/or cartilage alongside the chondroid bone.

    To study the histogenesis of malignant bone and chondroid bone, and whether tumorous cartilage and chondroid bone can experience metaplasia, one needs a reliable experimental evocation of neoplasms in animals. In the system of Olson and Capen (1977) using a sarcoma virus, their Figure 3 leads one to believe that chondroid bone takes part in the osteosarcomas induced. Timmer et a]. (1968) were able to bring about osteosarcomas in mice injected with radioactive 45Ca. Of these osteosarcomas they wrote, "A few small areas of swollen tumour cells were seen, resembling in a way cartilage. However, the scanty intercellular substance lacked the characteristics of true cartilage." That such cartilage-like areas reacted metachromatically with toluidine blue adds to the impression that the authors were describing either chondroid bone or a region of cartilage (secondary) in the osteosarcomas. They offered no illustration of the tissue.

    CREEPING SUBSTITUTION

    It may be worth noting that Geschickter and Copeland's (1949) use of 'creeping substitution" as a synonym of metaplasia is not innocuous. It leaves unclear (even if known) whether the creeping is to refer to place, from one side to the other, or to the transformation's happening very slowly but simultaneously throughout the cartilage. Another objection is that the term, as first employed, is now discredited (Weinmann and Sicher, 1955).
    Barth (1893) introduced the concept that he shortly afterwards named schleichender Ersatz (1895). He trepanned dogs' skulls and replaced the bone, sometimes after maceration in alkali. With or without this treatment the isolated bone died, but new bone formed on its dural face and sometimes fused it to the intact and living cranial margins. Although he noted a clear line of demarcation between the new and dead bone, he somehow gained the impression that new bone was growing into and at the expense of the old, without any osteoclastic resorption.
    He compared this fusion to the laying down of endochondral bone on calcified cartilage and suggested that components of the dead bone serve as a raw material for the construction of the new. He did make a connection with metaplasia, writing, "Thus, here we do not have a resorption in the usual sense followed by a substitution by new bone, rather it is a matter of, if you like, a kind of metaplasia, a substitution of new bone tissue for old." We still know little of what happens at the interfaces between new and old bone, new and dead bone, bone and cartilage, but any wholesale replacement by the mechanism and on the scale proposed by Barth does not appear to occur to justify this, the first, application of creeping replacement.

    Later, Urist and McLean (1952) reinterpreted creeping substitution around bone grafts as a "process of new-bone formation by induction," while Mosiman in the discussion to that paper, held to the older notion of the term. Metaplasia as a term has troubles enough, without burdening it with having Barth's unconfirmed creeping substitution as a synonym.


    Chapter 19 PISCINE CHONDROID BONE

    Introduction
    Selachian calcified cartilage (CB II)
    More truly intermediate chondroid bone
    Bony fishes and chondroidal ossification

    INTRODUCTION

    On four counts fishes have a special role for classifying skeletal tissues.
    First, not only do fishes possess several tissues ambiguously intermediate between bone and cartilage, but also forms lying between bone and dentin, and enamel and other hard tissues (Orvig, 1951).
    Second, Rose (1897) sharply distinguished echte Hartgewebe, bone and various kinds of dentin, from verkalkte Bindesubstanz, calcified fibrillar connective tissue and cartilage, because of the former's production by embryonic cells and the latter's by calcification in an already constructed tissue, e.g., the vertebral cartilage of sharks and rays. The same two routes to hard tissues underlie my separation of CB I and II.
    Third, tissues otherwise very close to those of tetrapod vertebrates can lack what is generally held to be an essential characteristic of the tissues, e.g., the absence of cells in most teleost bone (Moss, 1961, 1963).
    Fourth, the role of fishes as the forerunners of the terrestrial vertebrates, and the many fossil fishes amenable to histological examination, have led to numerous attempts to elucidate which was the first skeletal tissue in phylogeny.

    The answer, of course, requires, among other factors, that one can distinguish the skeletal tissues from one another. The difficulty of this task for the early workers such as Owen (1840) and Ko1liker (1859) lay with enamel, bone, dentin, and their related expressions. At that time, the intermediates between bone and cartilage received less attention, in part because the chondroid bony tissues of fishes are not as distinctively different from their related forms in other vertebrates as is, say, osteo-dentin (Kerebel et al., 1978) from mammalian dentin.
    Also, Kolliker and his contemporaries brought to their study of fishes convictions on the nature of cartilage and bone and how they interact in endochondral ossification, gained from much experience with mammalian osteogenesis. Thus, although earlier isolated observations pointed the way, e.g., Schmid-Monnard's (1883), an emphasis on the special chondroid bones of fishes did not come until well into this century (Kyle, 1927; Wurmbach, 1932; Orvig, 1951; and Moss, 1961).

    Kyle's (1927) introduction, while focused on fishes, is pertinent to all chondroid bone:
    "To describe the nature of bone seems to be an easy exercise; nevertheless there is hardly a more difficult question in zoology. Textbooks usually begin the description of bone with an enumeration of the typical characteristics, the matrix with Sharpey's fibers and the anorganic matter, lacunae with osteoblasts, Haversian canals, etc. Then the exceptions are mentioned, whereupon one finds out that any of the components can be absent, until finally only the matrix remains, and that perhaps could be missing. When one reads the description of the softer material, cartilage, one discovers that this material can become even richer in fibrous material, until finally a distinction between cartilage and bone is hard to find".

    Several circumstances in fishes involve varieties of chondroid bone. Selachian endoskeletal cartilage calcifies and not only persists but is added to. The tissue experiencing mineralization takes on various forms, e.g. areolar, and can be hyaline or fibrocartilage. A very similar tissue occurred in the placoderms.
    By contrast, the teleostian endoskeleton may be deficient in mineralized cartilage, even in those bones ossifying endochondrally (Moss, 1961), but particular cranial bones in part form by a distinctive transformation of a cartilage-like chondroid directly to bone. This metaplasia requires a kind of chondroid bone as a stage in the transition.

    SELACHIAN CALCIFIED CARTILAGE (CB II)

    The cartilaginous skeleton of sharks, skates, and rays experiences a consistent localized calcification. That occurring at the tissue's surface - the Rindenverkalkung - generally takes the form of a mosaic of completely or partly separated flattened units, tesserae or prisms (Figure 35), which early on claimed the attention of J. MulIer (1834) and other authors cited by Orvig (1951). Hasse (1882) summarized the early reports of his century, Kemp and Westrin (1979) those of the twentieth.

    Fig 35
    Another notable mineralization develops around the notochord in the vertebral centra, in patterns almost distinctive enough to aid in the taxonomic classification of Selachii (Ridewood, 1921). According to him, a zone of parenchymal or close-celled cartilage, formed in the notochordal sheath by invading cells, calcifies in the form of a double cone. Hyaline cartilage surrounding the cone then partly mineralizes as an investing layer, as radiating lamellae, or as tubes arranged concentrically around the cone. Other regions at the periphery of the centra may mineralize in some species.

    The spinal tissues made hard by mineralization are dense ligamentous or ensheathing tissue, hyaline and a more cellular form of cartilage, fibrocartilage, and hyaline and fibrocartilage traversed by dense Sharpey-like bundles of collagenous fibers. These hard tissues exist elsewhere in the Selachian skeleton, where their mechanical implications took the interest of Bargmann (1939) and others cited by him. He noted that the jaw of Myliobatis aquila comprises a cartilage with a peripheral system of inverted pyramids of calcified material; it is a hyaline cartilage into which an extensive dense perichondral tissue inserts anchoring fibers. The bases of the pyramids comprise the typical mosaic of Rindenverkalkung, but their apices join with an "endochondral" lattice of calcification running internally through the cartilage.
    Each strut of the lattice has mineralized, longitudinally oriented fibrils, but some struts end blindly in soft cartilage. Bargmann followed up the earlier suggestion that the hard, fibrously bound, superficial plates of cartilage lying in a softer matrix were a device for absorbing pressure, by proposing that the pieces' connection with the hard internal lattice provided a further means of redistributing and dissipating pressure. Also, the imbedding of the perichondral fibers in the pyramids gave the overlying dental and oral tissues a firm fastening to the jaw. He remarked further that the endochondral calcification appeared to follow the regular pattern of vessels in the embryonic cartilage.

    The endoskeletal hard tissues accordingly can be distributed in a pattern of an interrupted cortex buttressed by an incomplete internal framework. The mineralized tissues bear living cells (Moss, 1977b; Kemp and Westrin, 1979), collagen fibrils and fibers, anchor external collagenous bundles, and become a permanent reinforcement of the fish. Therefore the tissues take on the role and nature of bone so much so that some, such as Stark (1844), Williamson (1851), Goette (1878), and Kyle (1927), have regarded them as such.
    To bring another factor into the argument, Kyle (1927) had chemical assays performed on the large-celled, hard, vertebral tissue of Selache maxima L. and Lamna cornubrica. From the overwhelming predominance of phosphate over carbonate, and the presence of cells at the site of calcification, he concluded that the tissue met his two necessary criteria and therefore was bone. It was further specified, from its large lacunae, as an Areolarknochen, depicted, for instance, in his Figure 26 from Acanthias vulgaris. Other microscopists, such as Ridewood (1921) and others mentioned by him and Orvig (1951), have balked at such a classification, insisting that the tissues are clearly recognizable as calcified fibrous connective tissue or kinds of cartilage. A compromise is to regard these hard cartilages in general as cartilages, but by virtue of their mineralization and persistence, also as examples of chondroid bone II (hyaline or fibro to the extent of their fibrous collagen content).

    MORE TRULY INTERMEDIATE CHONDROID BONE

    The bulk of the calcified cartilage of fishes can be assigned to the widely acknowledged categories of cellular, hyaline, and fibrocartilage, with or without Sharpey-like bundles. However, Orvig stressed that certain selachian cartilages are more than usually bony, and that typical calcified cartilage was sometimes joined to bone by a region of transitional tissue, in both fossil and recent fishes.

    Many selachians have extensions to the pelvic fins - the claspers - believed to be used for copulation. Huber (1901a) reviewed the studies on this organ. He found the several supporting cartilages to be hyaline and elastic, and mostly to develop late as a secondary formation in connective tissues. At the end of certain of the subsidiary cartilages was a peculiar hard tissue whose matrix shared the appearances of cartilage and dentin. Peripherally the tissue was crossed by wide canals opening at the surface, and the deeper lying matrix had irregular lacunae holding connective tissue cells. Huber (1901a,b) called the tissue Chondrodentin.

    Chimaera has a frontal "clasping organ," the report on which by Stephan (1900) is summarized by Orvig (1951) thus:
    "between the perichondrium and the calcified subperichondral stratum of the cartilage there is in this organ, a fairly thin zone of what Stephan calls 'fibro-cartilage ossifie' (op. cit, pl. 6, fchc, fig. 3). This zone does not consist of either typical bone or of calcified cartilage, but of a hard tissue of an intermediary type, which in a superficial direction becomes rather bone-like and in a basal direction passes over gradually into calcified cartilage."

    The unusual tissue lining the cartilage of the claspers in Chimaera and Selachii is considered by Orvig (1951) as not a
    "true bone tissue but some kind of calcified fibrous cartilage (Reis 1895a), more or less intermediary between calcified cartilage ... and ordinary perichondral bone tissue. This calcified fibrous cartilage may contain blood vessels and is frequently pierced by calcified or uncalcified fibres of Sharpey. Its cells are somewhat irregular in shape, somewhat elongated, but they seem to be without distinct canaliculi.... According to Reis (1895a pp. 386-387, pl. 12, fig. 2) the same sort of hard tissue as lines the 'frontal clasper' in Chimaera is also met with in the corresponding 'clasper' of Squaloraja (see Fig. 19 of the present work)."
    Orvig's Figure 19 of the clasper of this fossil elasmobranch depicts a strikingly osseous tissue, because not only are there vascular spaces but "the special chondrocyte-spaces" have canaliculi.

    Paleozoic fishes experienced some of their cartilaginous calcification in a globular form (Orvig, 1951), although there could be mineralized structures intermediate between globules and prisms. From published figures he suspected globular mineralization also occurred in teleosts and amphibia, e.g., the frog (Tretjakoff, 1929). Globular calcified cartilage is a very old hard tissue, and Orvig's observation that it was present in the Ordovician has been confirmed for the armor of a heterostracan by Denison (1967) and Halstead (1973).

    Placoderms had a hard endoskeleton in addition to their massive carapace. Many of the endoskeletal elements consist of cartilage with bony replacements and additions. In these substitution bones the globularly calcified cartilage is usually set off distinctly from the peripheral subperichondral bone. However, in certain representatives, e.g., Plourdosteus canadensis (Woodward), some regions of the bone-cartilage boundary have a transitional hard tissue, "which is not bone and not calcified cartilage either" (Orvig, 1951). In describing his Figure 16, he wrote
    "In the basal part of the perichondral bone layer close to the subperichondral calcified layer, the cell-spaces are sometimes of a somewhat irregular or rounded shape, in that they possess a few short canaliculi or else are without distinct canaliculi. In contradistinction to all the other cell-spaces in the bone tissue now under consideration, which doubtless contained true bone cells, the irregular or rounded cell-spaces just described must have housed cells which were intermediate, more or less, between bone cells and cartilage cells. The latter cell-spaces do not occur only where they may be expected, viz. at those places where the perichondral bone layer merges fairly gradually into the calcified cartilage underneath, but are also frequently met with in the basal part of the perichondral layer where this layer is well bounded off towards the subperichondral layer."
    From his description, the tissue has more the nature of a chondroid bone I - a bony matrix with at least some cells chondrocytic - than CB II.

    Although the calcified cartilage (CB II) of the deeper layers in the same specimens stained with alizarine, picro-fuchsin, and malachite green in the same manner as the bone, with crossed Nicol prisms "the calcified cartilage in question is easily distinguishable from bone." The transitional layers were too narrow to be investigated in detail by the last technique, but Orvig speculated that
    "it is nevertheless quite imaginable that these layers originally consisted of fibrous cartilage, or some other tissue intermediary more or less between connective tissue and cartilage, which lay at the basal boundary of the perichondrium and calcified simultaneously with the perichondrium and the subperichondral stratum of the cartilage." (Some acanthodians also displayed a gradual transition from perichondral bone to deeper, globular, calcified cartilage.)

    The third of the tissues placed by Orvig (1951) in the category of "hard tissues transitional between bone and calcified cartilage" are ones described by Wurmbach (1932) in extant Selachii, not in the claspers, but in regions of the vertebrae. In his study of vertebral development in Acanthius vulgaris, Scyllium canicula, Pristiurus melanostomus, and Galeus canis, Wurmbach was struck by how many were the expressions of cartilage, including one no different from bone. Although he discussed cartilage under two headings, hyaline and acidophil, he concluded that all the forms he saw lay on a continuum. The principal variables were the contents of chondromucoid material and collagen, which were inversely related to each other. Other less significant variables were the cellularity and calcification.
    Starting from the bony side, the range ran: bone-like inner appositional lamellae on the dorsal arch of Galeus and the same in Scyllium, "which are no longer distinguishable from bone;" lateral appositional cartilage of the middle piece of the dorsal arch of Galeus; the anterior and posterior appositional cartilages of the same; the middle zone (of the notochordal sheath) and the intermediate of Galeus, - etc., on to end with the hyaline arch cartilage.

    The more bony members of the series lay toward the outside of the cartilage, where it grows by the incorporation of perichondral fibrous tissue. The appositional tissue facing the spinal cord, "from its low cellularity and lamellar deposition, together with its strong calcification combined with an absence of interstitial growth, is very reminiscent of coarse-fibered bone. Indeed, in Scyllium a fine-fibered cell-free lamellar tissue forms here that is no longer to be distinguished from bone."

    Wurmbach took the ability to grow by intussuception (interstitially) to be the criterion by which cartilage is to be separated from bone, since, he maintained, there is no difference in their origin - in mesenchyme - nor are they demarcated by morphology, not even in tetrapods. He noted that in the mammalian skeleton Zawisch-Ossenitz (1929a,b) had found chondroider Knochen, and suggested that, according to the mode-of-growth criterion, the tissue she saw probably should be reckoned as cartilage, while the inner appositional cartilage of his selachians would then be bone. Wurmbach ended that section by emphasizing that he had not seen any transformation of cartilage into bone, rather that his findings supported the concept of bone, cartilage, and connective tissue as being expressions of a single mesenchymal entity and differing only quantitatively from one another.

    In assessing their valuable TEM and SEM study of three species of shark, Kemp and Westrin (1979) consider the problem of distinguishing bone from cartilage, particularly at the calcifying superficial surfaces of selachian endoskeletal cartilages. The cap of a tessera typically has a fibrous matrix with elongated cells, like but noticeably larger than the perichondral fibroblasts. Mineralization proceeds bone-like along the coarse collagen fibrils:
    "Now calcification may proceed directly in the matrix of perichondrial fibroblasts without the delay resulting from appositional transformation of fibroblasts to chondroblasts. Therefore a calcified matrix develops around well differentiated collagen fibrils and fibers (Sharpey's fibers). Direct scleroblastic activity of inner perichondrial connective tissue cells could qualify them to be classified as osteoblasts rather than chondroblasts. Their activity produces a cap zone surmounting the earlier base of calcified cartilage. Histologically the tesseral cap could be considered a type of bone containing fusiform osteoblasts. Thus the tesserae may be interpreted as blocks of calcified cartilage which in their later stages are surmounted by a thin veneer of bone."

    Fig 36
    The only reason for regarding this very bone-like layer in their Figure 15 (my Figure 36) as chondroid bone rather than bone would be its origin, by a transformation of perichondrium, its mineralization resembling that overtaking other fibrous tissue, e.g., tendons, when chondroid bone II (fibro) develops. Further study may reveal that, as in placoderms, some selachian superficial bone may merge via something more intermediate with the deeper calcified cartilage.

    BONY FISHES AND CHONDROIDAL OSSIFICATION

    Kolliker gave Muller (1858) part of the tail and a fin from a specimen of Polypterus bichir, in which Muller found only the kind of endochondral ossification typical of other classes, and closest to that of frog. Muller apparently did not examine the cranial bones instrumental in leading Leydig (1854) to believe in a peculiar direct conversion of cartilage to bone, when Leydig had earlier examined the same specimen.

    Somewhat later, Schmid-Monnard (1883) looked at osteogenesis in around 30 species of teleost. He showed that the striated acellular tissue taken for dentin by Kolliker was more a bone crossed by bundles of fibers. He confirmed Gegenbaur's finding that osteogenesis started well outside the cartilage in the perichondrium (see Moss (1961)). Schmid-Monnard showed that a bone, the squamosum, in salmon was a membrane bone, whereas in pike it was a composite of dermal bone and bone derived from cartilage. Thus, despite Kolliker's claim, the histogenesis of a bone cannot be a criterion for resolving questions of homology between bones.

    For now, Schmid-Monnard's most interesting finding was at the articulation of the squamosum in the pike. The tissue there differed from hyaline cartilage. Although the cells were like chondrocytes, there was much less matrix and it appeared a little fibrous. At the free surface, this "articular cartilage" (as he settled on calling it) had a proliferative layer, while on the deeper aspect its intercellular substance underwent a sclerosis to become bone. The cells exhibited all transitions from cartilage cells to osteocytes, with a smaller size and notched lacunae, but no canaliculi were present. The bone formed by this direct ossification of the cartilage was essentially the same as that developing in other ways, and therefore was not merely calcified cartilage. That the osteocytes lacked processes in canaliculi was not significant, he maintained, because this was also true of some of the osteocytes of other piscine bone undoubtedly derived from osteoblasts.

    From Schmid-Monnard's description of the tissue and hesitation in calling it cartilage, it is clear that he was seeing chondroid and its direct transformation into bone - chondroidal osteogenesis, as named by Moss (1961, 1962), who applied several histochemical methods to the lower jaw of various teleosts, where the phenomenon occurs, and to the operculum.
    Moss described chondroid and how it is unlike cartilage in its staining, for example, in not staining with mucicarmine or thionin. He wrote that the osteocytes produced by this metaplasia of chondroid cells are identical in shape with osteocytes of periosteal origin, without indicating whether canaliculi are present. He did mention elsewhere that canaliculi were to be seen in fresh whole mounts of bone, but were unstained by Schmorl's method. He emphasized the distinction between chondroid and secondary cartilage (Moss, 1958), while observing that they can both be transformed to bone. Two other relevant points emerge from his papers on chondroidal osteogenesis.

    Muller (1858), Schmid-Monnard (1883) and other early writers on teleost osteogenesis regularly mentioned a calcification of cartilage in such bone as forms endochondrally. Moss (1961), on the other hand, was struck by the paucity of calcification before the resorption of the hyaline cartilage, also present in the jaw; and he included undecalcified and von Kossa-stained preparations for his study. If the cartilage does not mineralize, chondroid bone of the second type - persisting calcified cartilage - would not be expected in teleosts.

    Chondroidal osteogenesis raises an obvious semantic problem. Bone formed from chondroid has a just claim on "chondroid bone," but chondroid bone, among other names, has been widely used for a tissue intermediate between bone and cartilage. Does this latter tissue participate here? As chondroid becomes bone, it passes through a state of being chondroid bone in this second, intermediate sense. While it does experience the mineralization typical of type II, the half-way tissue is a true intermediate and experiences further metaplasia to become bone, properties which favor categorizing it as type I.

    Do teleosts manifest this ability to form CB I in any other way? In the repair of fractures of the lower jaw, Moss (1962) often saw what "appeared to be direct transformation or modulation of some of these cartilaginous callus cells into functional, if not histotypical osteoblasts. Furthermore, in the cellular fish, these same cells continued on to become osteocytes." Incidentally, in light of the apparent scarcity of calcified cartilage during normal endochondral growth, one notes that among the diverse cartilaginous tissues in the callus, Moss saw calcified cartilage.

    Moss's (1961) bibliography included Lowenthal's (1924) report of chondroidal ossification. Omitted were Stephan (1900) and Haines (1938b), who noted significant calcification of hyaline cartilage prior to endochondral ossification, and specifically referred to the intermediate chondroid bone under teleost mandibular articulations. They identified two routes to the chondroid bone: by a metaplasia of cartilage, and as a periosteal neoformation.
    Unlike Moss, Haines (1937b) found calcified cartilage frequently in the mandible of various teleosts, particularly underlying its fibrocartilaginous articular region, e.g., Figure 6. Calcified cartilage also participated in endochondral ossification in fishes' branchial bones (Haines, 1934, 1938a); Haines described the precursory tissue as cartilage and fibrocartilage. He did not mention chondroid, but he confirmed Stephan's (1900) observation of chondroid bone in teleosts.

    Below the fibrocartilaginous articular surface of the mandible of Trigla capensis, the Cape gurnard,
    "is a peculiar tissue which Stephan has shown to be a calcified fibrocartilage (Fig. 16). The cells are rounded and irregularly scattered or elongated and arranged in irregular radiating rows. These lie in a matrix which stains red with eosin and haematoxylin, not blue as does typical calcified cartilage. At the margin of the formation the tissue passes without interruption into the neighbouring bone. Some of the cells are surrounded by a thin capsule of blue-staining cartilage, others have no such capsule, but all are quite clearly cartilage cells. Thus this tissue consists of cartilage cells embedded in a bony matrix.
    "As Stephan has pointed out (1900, p. 373), the origin of this tissue can be followed quite clearly at the line of transition from the fibro-cartilage which gives origin to it. The cells of the fbrocartilage become enlarged and lose their flattened shape, so as to form the rounded or elongated cells of the calcified tissue. The fibres undergo the changes seen in the ossification of any fibrous tissue, that is they become calcified, and the calcification tends to mask their fibrous nature. The formation of this tissue from fibro-cartilage resembles the formation of ordinary bone from periosteum, but in the one case the included cells are cartilage cells and in the other they are osteoblasts."

    In addition to this metaplastically derived chondroid bone, Haines and Stephan detected in its vicinity another intermediate tissue formed de novo from the germinal periosteum:
    "At the periphery of the formation another kind of mixture of bone and cartilaginous tissue is found. The border between bone and calcified fibrocartilage is not clear-cut as is that between bone and true calcified cartilage, but is irregular, so that groups of cartilage cells lie in the bone. Stephan (1900, p. 369) has accurately described and figured these forms, and has traced their origin back to the region where the periosteum joins the fibro-cartilage. This junction of the mother tissues is not sharp, so that from the transition zone bone and calcified fibro-cartilage may be formed alternately.

    In summary, the cartilaginous fishes have much CB II (hyaline and fibro), including calcified hyaline cartilage with prominent entrapped Sharpey's fibers. Chondroid bone I as an intermediate between calcified cartilage and bone participates at the boundaries of the vertebrae and clasping cartilages of particular selachians. Another CB I served as a transitional tissue between the perichondral bone and globular calcified cartilage of some placoderms. No suggestion of a metaplastic origin for the above tissues has been made recently, which contrasts with the longstanding assessment of the skull of some teleosts.
    There, parts of the jaw and some other bones form by a direct metaplasia of cartilage or chondroid to bone, which may then become acellular, with a kind of CB I participating as an intermediary in the osseous transformation - Moss's chondroidal osteogenesis. Elsewhere, the bony endoskeleton develops in membrane or endochondrally, but by a process apparently not always requiring a mineralization of the cartilage, so that CB II is not extensive despite the often slow pace of endochondral replacement.


    Chapter 2O CAVIAN DENTAL CHONDROID BONE

    The teeth of herbivores differ from those of carnivores, and omnivores such as man. In many herbivores, cementum partially covers the enamel; according to Jones and Boyde (1974), Havers (1691) was familiar with such coronal cementum in the horse. In most species cementum is very like bone, regardless of whether it is coronal or radicular. The guinea pig and the capybara (Weidenreich, 1930) are exceptional in having, in addition, a cartilaginous kind of coronal cementum, which, if cementum is almost bone, thereby falls within the province of chondroid bone. Kolliker (1889) noted this peculiarity of the guinea pig's teeth, but Brunn (1891) first gave the material detailed attention under the name Knorpelcement.

    Cavian molar teeth in cross-section have the form of a thickened N, the clefts of which are lined by the chondroid cementum. Although the tissue is not as hard as dentin or enamel (Gottlieb and Greiner, 1923) and is more brittle (Santone, 1935), it is calcified. The tissue is very like cartilage. The illustrations of all those cited here show a tissue populated by numerous, large, mostly ovoid cells (Figure 37). Hunt (1959) identified it simply as "cartilage," whereas Brunn (1891), who regarded it as a form of verkalkter Hyalinknorpel, drew attention to the paucity of matrix, the resulting proximity of the cells and the disorderly distribution and sometimes angular form of the cells; all these distancing the tissue a little from the hyaline cartilage usually experiencing calcification. From the cellularity and lack of obvious collagen fibers, Santone (1935) gave the tissue the status of "embryonic cartilage." To the extent that the chondroid cementum is cartilaginous, it belongs in the class of secondary chondrifications,

    Fig 37
    The bone-like attributes of the tissue are more evident chemically than morphologically. Nevertheless, when it forms within mesenchymal condensations of the dental sac, the first deposits are somewhat like trabeculae (Figure 25 of Santone, 1935; Figures 19 and 20 of Listgarten and Shapiro, 1974), which enlarge, leaving only some narrow vascular channels. Listgarten and Shapiro subjected the tissue, which they dubbed "cartilage-like cementum," to thorough biochemical, light and TE microscopical study. Their work confirms the peculiar and intermediate nature of the Knorpelcement.

    The tissue has a high mineral content (62%), relative to which the collagen - represented by hydroxyproline and hydroxylysine residues and visible fibrils - is low, and the moisture high, in comparison with bone, cementum, and dentin. It is close to bone in its degree of mineralization, which is achieved rapidly, apparently by the focal action of matrix vesicles. Despite the close resemblance of its cells in light and TE microscopy to chondroblasts, its hexosamine content is low and matches that for bone. On the other hand, unlike bone it has only a little collagen (14% by weight), much of which is non-fibrillar. Also in its metachromasia and water content, it is akin to cartilage.

    Chondroid cementum is thus an example of blastemal chondroid bone, differentiating directly to that state from mesenchyme. As CB I it is atypical, because the cells appear soon to degenerate, it stains poorly for collagen, and it does not undergo resorption or metaplasia. In this persistence, until its loss from dental attrition, it lends itself to further study. Listgarten and Shapiro (1974) relate the degeneration of most of the cells to the high level of mineralization, but the apparent survival of a few is of interest.

    Why Caviidae should have chondroid cementum is obscure. Unlike other cementum, it has few Sharpey's fibers to act as a strong anchorage (Listgarten and Shapiro, 1974). Gottlieb and Greiner (1923) told a now familiar story - - that the rapidity of dental eruption called for the fastest-growing firm tissue, believed to be cartilage, to fill the developmental grooves. Santone (1935) questioned this idea, because the rabbit with similarly constructed (Brunn, 1891), continuously erupting molars, has only the usual bone-like cementum. Santone suggested that, in the guinea pig, the chondroid cementum not only occupied the clefts to make the tooth compact, but did what cellular, collagen-poor cartilage usually does, namely, absorbs direct pressure, here arising during chewing.


    Chapter 21 OSTEOCYTIC OSTEOLYSIS: Mistaken Chondroid Bone?

    Introduction
    Osteocytic osteolysis: Belanger and colleagues' work
    Evidence against osteocytic osteolysis
    Avian medullary bone
    "Chondroid bone" from excess PTH and vitamin A deficiency
    Chondroid bone in osteogenesis imperfecta congenital
    Osteopetrosis
    Bone-to-cartilage metaplasias?
    Conclusion

    INTRODUCTION

    The idea that osteocytes can destroy the bone in their vicinity dates back to Volkmann (1863) and before, and was resurrected by Zawisch-Ossenitz (1927), Lipp (1954a), and Ruth (1961) without attracting much notice. Then, some experiments with parathyroid extracts by Belanger and his colleagues (1963) gave the concept of osteocytic osteolysis new vigor (Belanger, 1971), until Boyde (1972) made a detailed argument that the evidence for the phenomenon was inadequate.

    There are three reasons for venturing to enter the controversy. First, if osteocytes, i.e., late osteoblasts, switch their activities to destruction, they are performing a cellular metaplasia, and if they then reverse their role to synthesis, they would have achieved a reversible metaplasia or modulation.
    Second, for bone, as a tissue, to experience metaplasia to cartilage, not only must the cells change, but the necessary transformation of the matrix requires its controlled breakdown. If osteocytes have either minor or no lytic powers, the prospects for a metaplasia of bone are slim.
    Third, Belanger and others' (1963) account of osteolysis included chondrocytic cells within the bone resembling those described by Zawisch-Ossenitz (1927, 1929a,b) in developing femurs, thereby endowing the "osteolytic" bone with chondroid features. Even if the cells remain osteocytic, if they enlarge their lacunae enough, the bone will appear large-celled and, on that property alone, be more easily mistaken for a cartilaginous tissue. An analysis of the early experiments of Belanger et al. (1963) will serve to introduce the pitfalls in interpretation to which large-celled and proteoglycan-rich bone and the activities of its osteocytes are subject.

    OSTEOCYTIC OSTEOLYSIS: BELANGER AND COLLEAGUES' WORK

    Belanger et al. (1963) stained sections of bone from young rats, chicks, and dogs for acid and neutral polysaccharides and examined the lacunae and matrix by microradiography and alpharadlography. They classified the osteocytes of tibial and parietal bone in two groups: small and large. The latter "show a hypertrophy which is mostly cytoplasmic," and exhibit metachromasia and basophilia. This characterization of large osteocytes as "hypertrophic" was the crux of their hypothesis of osteocytic osteolysis. When several species of animals were given parathormone, rats were pregnant, dogs infused with EDTA, or chicks received Norethandrolone, more of the large osteocytes were present, and acid proteoglycans appeared to spread out into the matrix around the large lacunae housing the cells. Belanger et al. interpreted these findings as evidence for a hyperactivity on the part of the osteocytes. In other words, a large osteocyte is truly an hypertrophied version of an earlier one that was smaller. They did not consider an alternative explanation - that a large bone cell is one that was large as an osteoblast and failed to become smaller.

    The manner they described by which the "hypertrophic" osteocyte erodes the matrix did not call for the cell to do other than continue synthesis:
    "The mature, hypertrophic osteocyte presides over salt removal through changes induced in the organic matrix (osteolysis) ... These changes lead to a loss of density partly explained by the secretion of acid mucopolysaccharides." Thus, the question of the osteocyte's changing its role to the extent of becoming a kind of uninuclear osteoclast and altering its synthetic program at the time did not arise.

    Their findings are vulnerable on several points. The nearest that they came to a normal chick for comparison appears to be rachitic birds administered one dose of vitamin D3. They left unexplained the uneven distribution of large osteocytes seen by them in the "normal" tibias of other species. Zawisch-Ossenitz (1929a,b) had already shown that these large cells could be attributed to earlier special histogenetic events in particular regions of the periosteum. In their treated animals and in two human cases of osteogenesis imperfecta, they assumed large osteocytes to be enlarged forms of ones that would in normal circumstances have been smaller, but had no proof that these were not osteoblasts which had never shrunk. Some of their manipulations would have provided both the stimulus and the time for the laying down of new bone, in which large lacunae and proteoglycan-rich osteocytes are normal. A newness of formation could also account for the low mineral density of the bone in which the large cells lay.

    The two routes, destructive and formative, to large lacunae were not unrecognized by workers in the field. Baud and Auil (1971), in putting forward their "osteocyte differential count" as a way to estimate the degree of osteolysis in a bone, noted, "it is important to distinguish between the enlarged osteocytes, resulting from the osteolytic process, and the large irregular osteocytes which are found in woven bone, fracture callus, and other sites of rapid osteogenesis (Jowsey, 1968), and in fluorotic alveolar bone (Baud and Alami, 1970)."
    They offered no practical means of making the distinction, except that they left the implication that large lacunae of formation are confined to the situations they listed. This sounds like more helpful information than it actually is. In the first place, there are other instances of such bone, for example, the medullary bone in laying birds or animals treated with certain steroid agents, some chondroid bone on the surfaces of both intramembranous and endochondral bones in development, e.g., on the femur (Zawisch-Ossenitz, 1929a,b), and in the human disease of osteogenesis imperfecta fetalis where much of the bone is primary (Jowsey, 1963).
    Second, this last abnormal example points to one general difficulty. In most small laboratory animals, the rat in particular, the skeleton has little Haversian (secondary osteonal) bone, so that its bone, though in places appropriately dense, is closer to woven bone than to lamellar, making it more difficult in the young animals commonly used for experiments to make a formative-resorptive distinction among osteocytes in large lacunae.

    Another defect in the evidence is that most of the support for an osteocytic osteolysis comes not from normal animals, but ones in which resorption has been provoked by such means as reducing the calcium in the diet or giving an excess of parathyroid hormone (Jande and Belanger, 1973) or vitamin D (Baylink et al., 1973), or otherwise eliciting a secondary hyperparathyroidism, and from human victims of kidney disease (Bonucci et al., 1976). Such insults result in a marked increase in osteogenesis involving the deposition of woven, large-celled bone. Early in the revival of the hypothesis of osteocytic osteolysis, it was pointed out that some of the large lacunae seen frequently in the bone of humans suffering from hyperparathyroidism or rickets were in new bone rather than altered old tissue (Riggs et al., 1965).

    There are claims of an osteolytic role for osteocytes based on more physiological circumstances, namely, the pregnant rats of Belanger et al. (1963), hibernating bats of Whalen, Krook, and Nunez (1972) and hibernating ground squirrels of Haller and Zimny (1976, 1977). Nevertheless, whether in the normal or the treated animal, to be convincing each proposed instance of osteocytic osteolysis must establish: 1) that the region of bone involved was present when the stimulus to osteolysis came into play; 2) that bone around the osteocytes has been destroyed; and 3) that the osteocytes were the active agents of destruction. These conditions have yet to be met all together in one study, and the third has not truly been satisfied in any experiment. Indeed, a body of evidence compels one to be skeptical of the destructive osteocyte.

    EVIDENCE AGAINST OSTEOCYTIC OSTEOLYSIS

    1. Scanning electron microscopy of osteocytic lacunar surfaces has not furnished evidence of any erosion (Boyde, 1972; Jones and Boyde, 1977b); and SEM has indicated (Lindenfelser, Schmitt, and Haubert, 1973) that the lacunae in hyperparathyroidism, whether formative or resorptive, are not as large as when measured using light microscopy. Hence, decalcification, sectioning, and staining may artificially have increased the difference in size between large and small lacunae.
    2. It takes vital labeling or particular attention to the microarchitecture to be sure that bone present now was in existence, when the resorptive stimulus was introduced. Otherwise, only if the tissue is not usually remodeled or the time between the introduction of the stimulus and observation is short, can one cautiously assume that the bone in question was present throughout the experimental period. In many experiments using young rapidly growing animals the failure to use markers of growth means that, even after an interval of only one or two weeks, large-celled bone may be of recent origin and represent a formative response to the stimulus.
    One study, in particular, which seems to disregard its own signs of a new growth was that comparing active with hibernating bits (Whalen, Krook, and Nunez, 1972). The femoral shaft early in hibernation had an outer half of bone with flattened osteocytes, separated by a "cementing line" (their Figure 6), from an inner region of bone displaying "considerable enlargement of osteocytic lacunae." The authors ignored the implication of the cement line - that the larger-celled bone is a recent endosteal deposit - in reaching their conclusion that osteocytes of the inner half had destroyed the bone. They proposed that the erosion is of a kind that actually allows the bone to shrink and thereby cause the observed thinning of the cortex. Partly underlying this notion was their belief that the smooth surfaces - in light microscopy - of the shaft could not be the seat of erosive activities. Deeply scalloped surfaces are characteristic of a very active destruction of bone, but the corollary to this, i.e., a smooth surface means no resorption, has not been established (Weidenreich, 1930; Knese, 1978b).
    In their comparison of interradicular alveolar bone from hibernating and nonhibernating ground squirrels, Haller and Zimmy (1977) imply that the bone of their Figure 3, well populated with large cells, is the bone of their Figure 2 altered by osteocytic osteolysis. They make the comment that the number of lacunae in the "foamy-appearing" bone of hibernation is increased. In view of the "coalescence" of lacunae that they believed to be occurring, one would expect the hibernators to have fewer lacunae than the control animals. Their figures strongly suggest that they were comparing old with newly formed bone. 3. The amount of destruction that a single cell can cause is pertinent to making feasible a metaplasia of the bone around it. Volkmann (1863), while an early advocate of destructive osteocytes, astutely remarked that the largest lacunae in the ulcerating bone, believed by him to be experiencing such a lysis, were no larger than those in rapidly forming bone. Belanger, Choquette, and Cousineau (1967) noted that the large lacunae, supposed by them to be a sign of lysis, were to be seen in the period of the antler's fastest growth.

    Supposing that osteocytes are resorptive, how widely can they destroy matrix? Rasmussen and Tenenhouse (1967) believed the osteocyte's sphere of lytic influence to be small, although one would expect it also to influence pericanalicular matrix to some limited extent (Baylink et al., 1973). However, the shrinkage proposed by Whalen et al. (1972) would require each osteocyte to abstract something significant from the matrix as far away as the territories of adjacent osteocytes. Unless the foci of destruction were all confluent, the bone would not experience interstitial shrinkage: an osteoporotic bone does not become smaller overall merely because its interior is eaten away.

    Spokesmen for osteolysis have inferred a coalescence from the rare observations of two cells sharing a lacuna (Haller and Zimmy (1977) inter alios), but this is a feature of forming bone readily explained (Boyde, 1972) by the nature of osteogenesis where osteoblasts start out side-by-side. What is more significant is, first, the rarity of the sharing, and second, the overall upper limit on the size achieved by the vast majority of osteocytic lacunae, to which Volkmann and lately Baud and Boivin (1978) drew attention.
    4. The presence of granular crystalline material within the osteocytes' mitochondria is not necessarily a sign that the cells are destroying matrix (Bonucci and Gherardi, 1977), because, with special steps to preserve mineral, Gay and Schraer (1975) found large numbers of intramitochondrial crystals in the osteoblasts and osteocytes of forming avian medullary bone.
    5. The irregularity of the surfaces of the lacunar wall seen in TEM need not indicate an osteolysis, since the osteocytes of reptiles acting as controls in an experiment on mineral depletion have rough-textured lacunae (Anderson and Capen, 1976); and defective calcification during osteosynthesis can cause the lacunar form to be irregular (Bonucci, 1977; Bonucci and Gherardi, 1977).
    6. The resorption of bone by osteociasts requires a bone-facing ruffled border (Jones and Boyde, 1977b), although minor destruction may take place without one (Marks, 1978). No equivalent of the ruffled border has been seen in the osteocyte, thus casting some doubt on its ability to destroy bone.
    7. A dark line of demarcation seen around some lacunae in TEM - the lamina limitans of Scherft (1972) - was used by Tonna (1972b) as a guide for estimating whether more bone is made by the osteocyte, or the osteocyte has reversed its role and destroyed some of the matrix around it. Lipp (1954b) had followed the same line of reasoning as Tonna, but based his concept of a reversal of role on a doubled metachromatic Grenzscheide around some osteocytes viewed in LM. Lipp introduced evidence that the material of the Grenzscheide was mucopolysaccharide, but Scherft (1972) was unable to find such materials in the finer laminae seen in TEM.
    Although they wrote of a cycle of activities, Jande and Belanger (1973) outlined only a sequence, whereby osteoblasts became osteocytes which then went through three phases: formative, resorptive, and finally degenerative. In contradistinction to Tonna (1972b), they did not believe that resorptive osteocytes could switch back to bone formation (their osteoplasis). Tonna (1972b, 1973), on the other hand, placed heavy reliance on the number and position of the surrounding "laminae limitantes" or "osmiophilic laminae" as an indication of what the osteocyte had been doing. From the multiple osmiophilic laminae, he conjectured that the osteocyte was able to reverse its roles between osteogenesis and osteolysis. However, he, like Scherft (1972), was unsure of the nature of the lamina, and hence of exactly what it signified.
    Basing dynamic interpretations such as an osteocytic lysis on the osmiophilic lamina calls for caution for other reasons: its absence from sections decalcified after imbedding or obtained undecalcified (Bonucci and Gherardi, 1977); the inconstancy of its occurrence; and Scherft, Luk, Nopajaroonsri and Simon's (1974) different interpretation of doubled osmiophilic laminae as indicating an interruption in mineralization or apposition, rather than a reversal. This conclusion is strengthened by observation of the development of the lamina limitans in vitro (Scherft, 1978).
    8. Tonna (1972b, 1973, 1977), Jande and Belanger (1973) and others agree that, as osteocytes get older, they mostly become smaller with fewer organelles, and some show frank signs of degeneration. It seems reasonable to suppose that such older osteocytes are not capable of much activity of any kind. If they do have some small extracellular destructive action, it could be inadvertent, by a spillage of enzymes intended for osteocytic autolysis.
    9. The hydrolytic enzymatic content of osteocytes has been introduced as an argument for their having the ability to destroy matrix. Lipp (1959) cited their aminopeptidase in such a connection, and Baylink and Wergedal (1971), their acid phosphatase. The latter authors (Baylink et al., 1973) went on to confirm what seems to be a consistent finding after the administration of excess parathyroid hormone (Belanger and Drouin (1966) inter alios), or in human hyperparathyroidism (Bonucci et al., 1978), namely, a preferential distribution of large osteocytes in diaphyseal bone in the bone adjacent to the marrow cavity and close to sites of osteociastic activity.
    Baylink et al. (1973) noted that the large lacunae were close to endosteal surfaces and vascular canals undergoing resorption, and these lacunae also reacted positively for acid phosphatase. Their assessment of these findings was that osteocytes and osteoclasts both resorb bone, and their destructive activities are locally coupled in some way. One explanation they considered for the proximity of the large, acid phosphatase-positive lacunae to sites of osteoclastic activity was that "enzymes and other agents involved in bone resorption" could be transferred from the osteociasts to the osteocytes. They discarded this hypothesis as unlikely on the ground that there was no gradient of enzymatic activity away from the osteal surfaces. Their dismissal of an explanation by transport seems to have been hasty.
    In the first place, not enough is known of the dynamics of canalicular transport (Doty and Schofield, 1972; Piekarski and Munro, 1977) and of enzymatic storage and use to be certain that a gradient should be detectable. Second, having the superficial osteoclasts as the main source of the lytic agents would account for the observation of Baylink et al. (1973) that "these distal portions of canaliculi adjacent to vascular canals sometimes appeared to be more enlarged than the proximal portions adjacent to lacunae." If osteolysis commenced from the osteocytes, the wider regions of the canaliculi would be expected at their point of departure from the lacunae, as Lipp (1954b) remarked.
    The predilection of PTH-induced osteolytic-like osteocytes for the bone around the marrow cavity may be related to the observation of Rutishauser and Majno (1953) that an injection of parathyroid extract increased the level of proteolytic enzymes in the serum of dogs. Owen, Triffitt, and Melinck (1973) believed labeled albumin injected intravenously is taken up into the lacunar-canalicular system in the cortical bone of rabbits, so that it is conceivable that the canalicular system of transport picks up not only lytic enzymes of osteoclastic origin, but others probably abundant in the medullary fluids of parathyroid-treated animals.
    From this standpoint, the large lacunae seen within hours of giving PTH or an extract may indeed be enlarged, but more by the action of enzymes brought in from outside the bone than from ones produced locally by the osteocyte. The osteocyte would be actively lytic only in respect to its efforts in transporting the lytic agents to its lacuna.
    10. It has to be recognized that osteocytes may have the potential for some active destruction. Baylink et al. (1973) observed aminopeptidase in osteociasts, some osteocytes, and active osteoblasts, and Doty and Schofield (1972) found that, as the osteocytes lost their osteoblastic structure, they "lost their Golgi-associated acid phosphatase activity." The presence of these enzymes in osteoblasts and osteocytes suggests another way of looking at the lytic role of osteocytes, namely, to recognize the osteocyte as having a complement of destructive enzymes from the time of its first incorporation in matrix. The osteocyte, like other formative connective tissue cells, would then be able to rework its macromolecular products from the outset, but such a reworking might be intracellular, as may happen in the fibroblast (Bienkowski, Baum, and Crystal, 1978). Those enzymes found outside the osteoblasts (Poole, Hembry, and Dingle, 1973) may not be for future lysis but to reduce the osteoid's proteoglycans prior to its mineralization.

    In summary, it is clear that Belanger and his followers, in trying to demonstrate how widespread osteocytic osteolysis is, drew in so many bony situations that they were mixing fish with fowl. Very similar appearances - large lacunae in a matrix unusually rich in proteoglycans for bone - could arise in two quite separate ways: formative, whereby the osteoblasts in burying themselves and their predecessors, for various reasons fail to narrow their lacunae and bring the matrix to full maturation; or resorptive, involving a breakdown of matrix around osteocytes which have run to full maturity, with either an unmasking of proteoglycans or an actual increase in these or related materials.

    Most of the examples put forward to underpin the concept of osteocytic osteolysis involve bone that for one reason or another is slow to mature. These situations will now be reviewed to assess how chondroid is the bone.

    AVIAN MEDULLARY BONE

    One of the treatments of Belanger et al. (1963) resulted in the formation of a kind of chondroid bone, although the authors did not apply the term to it. In the tibia of chicks given Norethandrolone, "a large number of hypertrophic osteocytes are present; some are surrounded by strongly neutral red-positive matrix and look altogether like chondrocytes" (in the legend to Figure 16). Staining with toluidine blue "revealed that some of the hypertrophic osteocytes actually acquired all the staining behavior of cartilage cells and were also apparently responsible for a large concentration of acid mucopolysaccharides in the surrounding matrix." Although seen in the diaphysis of three-week-old birds, these had been fed the drug for a week, so that the bone observed may be, at least in part, an evoked medullary bone.

    Medullary bone refers to trabecular bone that occupies what would otherwise be the marrow cavity in long bones of laying birds and of male birds and mice given excess estrogenic hormones (McLean and Urist, 1968). The material of medullary bone veers in the cartilaginous direction. Its osteocytes are large. (The size and other content of their lacunae have been taken for indications of an osteocytic osteolysis by Bonucci and Gherardi, 1975.) The bone's matrix stains intensely with the PAS technique (Hancox, 1972), and with alcian blue and colloidal iron (Bonucci and Gherardi, 1975), reflecting the greater content of proteoglycan and lesser amount of collagen than in the more typical avian cortical bone. In one respect, medullary bone is less like cartilage than ordinary bone - its degree of calcification is greater.

    In another avian experiment, Belanger and Narbaitz (1978) caused a bone to form which was chondroid by description and as illustrated (Figure 1). After eight days on a diet of Caestrum diurnum leaves, containing a vitamin D-mimicking substance, the chick's thick diaphyseal trabeculae had cores of a bone with large cells in a deeply thioninophil matrix. Again the authors claimed the cells to be "enlarged" and osteolytic, but noted that after the first week on the diet the cells became smaller. Their picture can be explained as well in terms of an osteogenesis with a delay in osteocytic maturation and an imbalance in the matrix macromolecules, as by an osteolysis. (The Caestrum material may have mimicked another steroid hormonal effect and evoked a precocious medullary bone.)

    "CHONDROID BONE" FROM EXCESS PTH AND VITAMIN-A DEFICIENCY

    When animals are given parathyroid extract, the matrix around some of the large osteocytes stains more with PAS (Heller-Steinberg, 1951) and alcian blue (Belanger et al., 1963), and reacts metachromatically with toluidine blue (Belanger and Drouin, 1966). One explanation might be that, just as degenerative cartilage loses proteoglycans and unmasks its collagen, a loss of mineral (and collagen) has increased the staining or visibility of the bone's polysaccharide-containing macromolecules (Lorber, 1951).
    However, Johnston, Smith, and Severson (1972) found that PTH promptly increases the uptake of labeled glucose or glucosamine by cells on rat's bone. Thus, while there may be an element of unmasking, it is possible that the alteration in the staining of bone's proteoglycans or glycoprotein after PTH reflects an increase in the synthesis of such materials, at least near the surfaces of the bone. (The authors, however, suspected the material whose synthesis increased not to be a normal macromolecule of matrix, but one linked to resorption.)
    Taken overall, bone responding to an excess of PTH has in its formative areas typical young large-celled bone, and in other places close to the endosteal surfaces older bone with slightly enlarged osteocytes and an altered matrix, with the cells perhaps victims as much as agents. High doses of PTH may lead to the death of osteocytes (Krempien and Ritz, 1978). These changes in the cells and matrix do not take the bone very far toward chondroid bone.

    Aside from parathyroid hormone, another agent shifts bone's nature slightly in the direction of cartilage. Harris and Navia (1977) report an increased uptake of 35-Sulfur for the formation of new bone in vitamin A-deficient guinea pigs, reflecting, they surmise, a greater content of sulfated glycosaminoglycans than in new bone of normal animals. Here, the alteration is in the character of the new bone formed. The original bone was unchanged.

    CHONDROID BONE IN OSTEOGENESIS IMPERFECTA CONGENITA

    In her introduction Zawisch-Ossenitz (1929a) mentioned Pommer's (1925) observation that the cells of bone in osteogenesis imperfecta fetalis were closely spaced and abnormally large, and some matrix was basophil. Pommer had cited many authors, from H. Muller on, who were struck by the large size of the osteocytes in the disease. Zawisch-Ossenitz suggested that the cells of periosteum and endosteum in such cases were in the unstable condition, otherwise seen only at certain places on the normally developing diaphysis, in which neither true osteoblasts nor chondroblasts formed, but the less well differentiated intermediate cells of chondroid bone. She thought that the skeleton in osteogenesis imperfecta might be made up of chondroid bone.

    Recent work on the disease supports her idea, if one is not rigid about what is meant by chondroid bone. Most chondroid bone so far considered has been based at least as much on morphology as on chemistry, thus chondroid bone I has large, closely spaced, vesicular cells in a bone-like matrix, staining strongly for collagen and sometimes for calcium, less well for proteoglycans.
    What is to be made of a tissue where the cells are large osteocytes in a mineralized, collagenous matrix, but by biochemical measures the tissue is three times richer in proteoglycans than normal bone, and some of the proteoglycan is abnormally disulfated? This, the chemical nature of bone in osteogenesis imperfecta (Engfeldt and Huerpe, 1976; and earlier authors cited by Munzenberg, 1977), justifies Zawisch-Ossenitz's description of it as chondroid bone, although the collagen is also chemically abnormal and the skin and cartilage may display chemical and structural abnormalities (Cetta et al., 1977). Johnson (1966) characterized the bone as the "primitive perichondrial chondro-osseous type."

    Robichon and Germain (1968) saw an unusual rim of metachromasia around the large osteocytes of osteogenesis imperfecta fetalis, when they stained with toluidine blue, and also that the cells were densely packed. The large size of the lacunae and the thinness of the bone between osteocytes are clearly visible in the scanning electron micrographs of Ornoy and On Ja Kim (1977). Both pairs of researchers assumed that the lacunae are large because of an osteocytic osteolysis. But since young bone is large-celled, and would remain so if the incorporated osteoblasts failed to continue synthesis, large lacunae per se are not evidence of resorption. Pommer (1925) and others before and since have suspected that the defect in osteogenesis imperfecta fetalis is an inadequate and deranged synthesis by the osteoblasts. The resulting tissue has a chemistry that requires it to be considered as a pathologically chondroid kind of bone, which needs to be better related to the several expressions of the disease(s).

    OSTEOPETROSIS

    Zawisch-Ossenitz (1929a,b) observed that the periosteum at the ends of the femur formed, for a brief period in fetal life, a tissue with much basophilic matrix and a variety of cells ranging from osteocytes to cells very like chondrocytes (Pseudoknorpelzellen). She (1947) believed that this chondroid bone was involved in another disease of osteogenesis - marble bone or Albers-Schonberg disease. In the thickened femoral diaphysis from an afflicted infant, she saw a wide layer of irregular bone very prominent in its basophilia, buried beneath another thick zone of denser, less abnormal and more lamellar bone, but with "patches of chondroid bone," the tissue predominant in the deeper layer. Normally,
    "it plays a merely transitory role and is soon superseded by the ongrowing diaphyseal cortex. Here it has been formed throughout the diaphysis and during the second period. Besides being out of place, it is also malformed. The most striking feature is the intense basophilia of its ground substance." Thus, the disease included a hyperplasia of chondroid bone - representing a disturbed differentiation - coupled with a failure to resorb the tissue once formed.

    The reduced resorption in osteopetrosis naturally leaves more residual cartilage at sites of endochondral ossification. After excluding this material, Zawisch, in reviewing several other osteopetrotic cases which had come to histopathology, proposed that a variety of puzzling reports of "cartilage rests" in cortical bone, "basophil formations" in the calvaria, and "metaplastic neoformations of cartilage", probably involved excessive or surviving chondroid bone in locations for which the tissue is normal, but sparse and fleeting, and of whose existence the observers were unaware.

    In the rib of the case she examined the thin cortex consisted "almost exclusively of second period chondroid bone of the same pathologic structure as that found in the femoral diaphysis." Unfortunately, her description of the tissue established only that it was primarily the basophilia of the matrix that made this bone chondroid, but she also mentioned a crowding of the osteocytes and a lack of fibrils stainable with a silver method.

    The Op/Orl mutant rat has "long bone modifications similar to those seen in humans suffering from Albers:Schonberg disease" (Moczar et al., 1978 ), and appears to have an abnormally high amount of structural glycoprotein in its bones.

    BONE-TO-CARTILAGE METAPLASIAS?

    The instances are very few, and may proceed only as far as chondroid bone. Haas (1914), in an experiment on the ribs of rabbits, removed a two-cm piece of cartilage from the chondro-costal junction towards the sternum. He wrote of the changes at the bone-stump in one rabbit 20 days later, "there appears to be a direct transformation of bone into cartilage. The bone at that place is arranged in islands which are beginning to take on a bluish tinge like cartilage. The nuclei of the bone cells are enlarging and appear to be changing directly into cartilage." He offered no illustration to support this claim.

    The quadrato-jugal is a principally membranous bone involved in articulation of the beak (Murray, 1957). When broken in the young chick, it tries to heal by a periosteal callus of bone and cartilage (Hall and Jacobson, 1975). Close to the fracture within the bone they found hypertrophic chondrocytes which "were surrounded by prominent extralacunar capsules which stained with alcian blue and which were metachromatic after toluidine blue (indicative of the acid mucopolysaccharide nature of the capsules)." No extralacunar cartilage matrix was present.
    The authors believed these cells to be osteocytes that had undergone metaplasia to hypertrophic chondrocytes, because they were separated from the periosteum by a layer of bone formed after removal of the periosteum, and were not preceded by immature chondrocytes. Against another challenge, that the enlarged cells were osteolytic osteocytes, Hall and Jacobson countered with the cells' positive reaction to stains for the proteoglycans typical of chondrocytes.

    Abdalla and Harrison (1966) obstructed the venous drainage of the legs of adult rats by ligating the inferior vena cava. From 10 days to two months later, certain areas of the cortex of the femur and tibio-fibula experienced a substitution of basophilia for the acidophilia of the matrix, and the cells looked like chondrocytes. Since these changes occurred neither just under the periosteum nor close to the vascular canals (Figure 22), Abdalla and Harrison concluded that cortical osteocytes had transformed directly into chondrocytes - a process which, after mentioning metaplasia and redifferentiation, they settled on calling a dedifferentiation.

    Abdalla and Harrison's Figure 15 of the cartilaginous tissue shows a matrix still more like bone than cartilage. From this, and the other descriptions by Haas (1914) and Hall and Jacobson (1975) of a chondrocytic metaplasia of osteocytes, one may judge that the metaplastic process, for as long as it has been followed, leads not to full-blown cartilage but to cartilage-like cells in a still bony matrix, that is, to chondroid bone.

    CONCLUSION

    Three categories of bone have entered into the discussion:
    1) the presumed substrate for osteocytic osteolysis - older bone with small osteocytes and little proteoglycan evident after appropriate staining, but with the occasional large cell (Lipp, 1954a);
    2) young bone, having large cells and plentiful proteoglycan around them, because of the newness of its formation, but believed by the "lysers" to be older bone that underwent peri-osteocytic resorption; and
    3) older bone displaying the same properties, e.g., after PTH treatment (Baylink et al., 1973), or venous ligation (Abdalla and Harrison, 1966), and interpreted by the former group as post-lytic, by the other as dedifferentiated.

    The osteolytic changes were supposed to occur in type 1), bringing the bone to the condition of 2) or 3). These latter states - in the instances put forward, e.g., avian medullary, hibernating, osteogenesis imperfecta, antler, PTH-treated bone - are more chondroid than other young (or old) bone, as far as non-immunohistochemical methods can show, and so it is rather surprising that the supposed changes were not also viewed as a metaplasia. However, bone in category 2) does not progress from small-celled to large-celled states, but is made and remains in a chondroid condition, so that neither osteocytic osteolysis - a switch to significant osteolysis - nor a chondroid metaplasia takes place in these instances.

    What is left are the very few instances of changes in mature bone 3). These are limited to the vicinity of the cells, and so, as a metaplasia of a tissue, the process is incomplete. On the cellular level the alterations could reflect: a) a passive effect of agents extrinsic to the bone; b) a new active, pericellular degradation by osteocytes; or c) an imbalance between active degradation and synthesis already under way as the cells' day-to-day routine. More needs to be known of the osteocyte's normal role in establishing and maintaining itself, the lacunar and canalicular sheath materials, and the bone matrix, before the minor experimentally provoked changes can be understood.

    Should the osteocyte always be mildly lytic for purposes of turning over materials, there would be no basis for the transformation proposed by some "lysers": formative-to-destructive (a cellular metaplasia), or formative-destructive-formative (a modulation 2 or reversible cellular metaplasia). But from another aspect, when cellular metaplasia is taken to include a changed ratio of activates (see Chapter 6, Biochemical and Morphological Indices of Differentiation,), an osteocyte performing both synthesis and destruction could change to favor certain syntheses of constructional macromolecules and hydrolytic enzymes to render its surroundings more chondroid, and thereby experience metaplasia. In certain new bone, e.g., avian medullary, some such disproportion in activities may manifest itself from the start, expressing itself in all the bone, thus representing more a deviant differentiation of the precursors of the formative cells than a metaplasia of osteocytes.


    Chapter22 TEMPORAL BONE

    Introduction
    Globulus osseous (Knochenkugeln)
    Interglobularraum
    Residual cartilage and chondroid bone
    Cartilaginous interglobular spaces
    Otosclerotic bone - how chondroid?
    Metaplasia in interglobular spaces?
    Secondary cartilages
    Gussen's observations, nomenclature and conclusions: a critique

    INTRODUCTION

    The phenomena to be unravelled here are briefly these:
    1) The hyaline cartilage that precedes the petrous bone is not completely resorbed during ossification and remnants of it can last throughout life, probably in a calcified state;
    2) larger regions of cartilage can survive the initial endochondral ossification and remain until birth or later:
    3) other cartilage may, according to some reports, form at the surface of the bone as a secondary kind of cartilage;
    4) for all the three sorts of cartilage just listed, the possibility has been offered that the cartilage transforms into bone by a direct metaplasia; and
    5) part of the evidence for a metaplasia is a morphologically striking microscopic formation of bone called the globulus osseous, a term now misapplied in various ways.

    GLOBULUS OSSEOUS (KNOCHENKUGEL)

    At sites of endochondral ossification, with certain planes of section and particularly where the erosion into the cartilage is deep and irregular (Muller, 1858), one may encounter small round profiles of bone surrounded by cartilage. Reckoning that such profiles are of bony spheroids, Brandt (1852) called them globuli ossei. Whereas Brandt was struck by their apparently total enclosure by cartilage, Muller (1858), from a large number of variously oriented sections, took the globuli ossei to be cross-sections of bony extensions filling eroded cartilage lacunae, and derived from marrow in the spaces between the metaphyseal trabeculae.
    Brandt had proposed two modes of bone formation: bone made by marrow cells and filling eaten-out cartilage lacunae; and a second, metaplastic, formation of bone by the transformation of a chondrocyte and its surrounding matrix into an osteocyte in bone matrix, which, by contrast with the surrounding cartilage, would appear as a globulus osseous.
    However, if the bony globulus osseous were merely a visual effect of a special cut through bone formed by cells from the marrow within an eroded cartilage lacuna, there would be no basis for Brandt's second, metaplastic, kind of ossification - the countervailing view of Muller, who admitted that when the bone filling an old chondrocytic lacuna held only one osteocyte, and appeared to be completely enclosed by cartilage, it was very easy to believe that the cartilage cell had undergone metaplasia.

    Fig 38
    If the bone is not metaplastic and does occupy an eroded lacuna, it should be possible by serial sectioning to show that the globulus osseous in the lacuna connects at some point with the marrow spaces between the trabeculae (Figure 38). Shin-izi Ziba (1911a) and Weidenreich (1930), who both made serial sections, reported completely isolated globuli ossei (Section F), but the occurrence of some truly separate globuli ossei does not detract substantially from the adequacy of the general explanation offered by Muller, namely, that at epiphyseal plates and other sites of endochondral osteogenesis most globuli ossei seen in random sections are but deeply eroded cartilage lacunae taken over by new non-metaplastic bone.

    INTERGLOBULARRAUM

    Another term that came into use soon after globulus osseous was Interglobularraum or interglobular area, attributed by Shin-izi Ziba (1911b) to Gegenbaur (1864). According to the Japanese, Gegenbaur did not directly relate the term to the cartilage between globuli ossei, but meant rather any residual cartilage between the round and half-moon-shaped deposits of new endochondral bone. These rests were generally narrow, and diplayed alternate dilations and constrictions where erosion had spared the matrix to greater or lesser extent.

    Thus, as first understood, globuli ossei stood out only by their enclosure in cartilage, and Interglobularraume were assumed to be cartilage. Recent usage of the two names in the context of the otic capsule has strayed from these meanings, as some examples show. Bast and Anson (1949) called the residual portions of cartilage within the capsule by Bottcher's (1869) name cartilage islands, but put forward globuli interossei [sic] as a synonym, a perhaps unintended hybrid of globuli ossei and Interglobularraume. Gussen (1968a) wrote: "More often, the new endochondral bone only partially filled the degenerated cartilage focus, with varying amounts of uncalcified cartilage matrix remaining. These areas have been termed globuli ossei."
    Hawke and Jahn (1974) remarked, "The interlaced tangle of calcified fibers envelops the globuli ossei, islands presumed to be calcified embryonic cartilage remnants and chondrocytes." In another paper, Hawke et al. (1974) gave excellent illustrations of residual cartilage stained with alcian blue in undecalcified sections of temporal bone, but in the legend to Figure 3 they named the cartilage as globuli interossei (see also Gussen (1967)).

    What Hawke et al.'s figures showed were globuli ossei (large erstwhile lacunae now holding bone-like matrix and a cell) lying encompassed by Interglobularraume occupied by alcian blue-positive residual cartilage, with the whole region itself enclosed in bone.

    RESIDUAL CARTILAGE AND CHONDROID BONE

    The criteria for chondroid bone type II are persistence, matrix calcification, and the presence of cells. Muller (1858) noted that some cartilage is quite often spared in endochondral ossification and referred to Tomes and de Morgan's (1853) already having made the same observation, using the temporal bone as an illustration. Of his own material Muller (1858) commented only on the marked extent of residual cartilage in the auditory ossicies.

    The early investigators of the temporal bone did not make a clear distinction between small areas and trabeculae of cartilage spared destruction and certain larger masses occurring with some consistency at set places, the "occasional persistence of large cartilage rests" of Eckert-Mobius (1926). He illustrated one rest in the middle layer of the bone by the Fissura ante fenestram, studied by others before him, and persisting late in life in many individuals. Other major but smaller rests lie in the vicinity of the Fissura post fenestram, the ampulla of the posterior semicircular canal and the Fossa subarcuata. The latter are resorbed and replaced by bone in the first years of life, but although the cartilage by the Fissura ante fenestram likewise undergoes endochondral replacement in infancy, the process does not usually run to completion.
    Bast (1940) undertook a thorough study of such residual cartilages and, taking in the whole temporal bone, listed them thus: Fissula ante fenestram; Fossula (fissura) post fenestram; intracochlear region; region of the semicircular canals, base of the styloid bone; and region of the petrosquamosal suture and the capsule below it. Bast stigmatized the residual cartilages as a defective ossification. Whether there is a defect, or rather a natural variety of tissues and times of completion of ossification that is of no particular consequence, is yet to be established.

    Do such major rests give any sign of a metaplasia into bone? Shin-izi Ziba (1911b), a strong proponent of metaplasia on the part of minor residual cartilage, maintained that the major cartilage below the ampulla of the posterior canal had different chondrocytes and underwent only a typical substitution by bone. According to Eckert-Mobius, Kosokabe (1922) was the only observer to suggest any metaplasia, and that accompanied an osteoblastic bone formation in the cartilage by the anterior fissure.

    It has been the assumption of most authors, e.g., Muller, Eckert-Mobius, Bast and Anson, Hawke and Jahn, that the cartilage that persists for years in the peri-otic bone has a calcified matrix (and may include calcified cells; Eckert-Mobius, 1926). The sole questioner of the assumption (Gussen, 1968b) can be faulted on argument and technique. She wrote,
    "it is emphasized that at no time in the normal cartilage is any massive type of calcification of cartilage matrix seen, neither in the original cartilage focus nor in the matrix of the interglobular spaces ... Calcified cartilage matrix cannot maintain itself in the body and is resorbed following loss of its chondrocytes. In addition, calcified cartilage matrix is readily identifiable, even in decalcified sections, by its deeper affinity for hematoxylin and its more sharply accentuated lacunar margins."
    First, calcified cartilage can persist in the body. One cannot argue from the presence of cells that the matrix must be uncalcified. Second, she used only decalcified tissue, in which hematoxylin is not a reliable indicator of mineralization (Bloom and Bloom, 1940), except perhaps at tidemarks, where the degree of mineralization changes abruptly. Nevertheless, she raised a question needing an answer, which can only be forthcoming from undecalcified temporal bone such as that used and described briefly by Hawke et al. (1974). Their Figures I and 4 show von Kossa-stained undecalcified sections, in which only the vascular channels appear not to be calcified. They did not directly address the problem of how calcified is the interglobular cartilage, and their better magnified figure is of mastoid, not otic, bone.

    CARTILAGINOUS INTERGLOBULAR SPACES

    Most of the long-standing cartilage in the capsule of the labyrinth is in the form of small islands and trabeculae discussed by Eckert-Mobius under the title "Dauernde Persistenz von knorpelhaltigen Interglobularraume." Their considerable extent in this site is generally attributed to the absence of erosion and reshaping of the deeper labyrinthine bone, although Gussen (1968b) cited some work suggesting minor remodeling; Hawke and Jahn (1975) found a modest uptake of tetracycline on surfaces within the middle layer of human otic bone; and Roberto (1978) saw a slow internal turnover in the dog's auditory ossicles. There are many reports that chondrocytes are present within the wider trabeculae and islands. Therefore, this interglobular cartilage appears to meet the three specifications for being chondroid bone II (hyaline) - it persists, holds cells, and is calcified.

    In what other senses are the otic capsule and the auditory ossicles examples of chondroid bone? Bone containing persisting islands of cartilage matrix, with or without cells, has a more cartilaginous nature than bone without them. Perhaps the continued presence of the cartilaginous material plays a role in restraining the remodeling of the labyrinthine bone; but, of course, some earlier influence must have already hindered erosion so that the cartilage islands were themselves left behind.
    The former suggestion is not new. Bast and Anson (1949) passed on remarks made at a conference by Siebenmann (1912) that the cartilage remnants reduce the blood supply and maintain the size of the capsule. Bast and Anson (1949) also offered a name for the cartilage-bearing bone, namely, intrachondrial bone, based on this reasoning:
    "Although the original cartilage matrix remains to lend the appearance of cartilage, the tissue may be regarded as bone, since the lacunae are filled with true bone and bone cells. The term intrachondrial bone seems to be appropriate, since, among all bones, it is the only example of true osseous tissue formed within cartilage."

    Another more far-fetched application of chondroid bone is to the globuli ossei, assuming that these comprise bone made by osteoblasts within an opened chondrocytic lacuna. The cartilage imposes the shape of its lacuna on the bone and, if the bone is not resorbed, this shape endures. This idea of a transfer of form alone from cartilage to replacing bone was expressed by Muller's (1858) Pseudomorphose, to contrast with a metamorphosis of chondrocyte to osteocyte, which he believed did not occur.

    OTOSCLEROTIC NEW BONE - HOW CHONDROID?

    In the otosclerotic labyrinth, bone resorption and deposition increase. Some of the new bone laid down in eroded spaces and vascular canals is abnormally basophil - Manasse's (1922) "blue mantles." The partly chondroid character of the bone is otherwise evident in the numerous large cells, sometimes in still-confluent lacunae (Weber, 1933): "The cells were similar to those of a primitive bone tissue. Now and then cells could be observed which morphologically looked like cartilage cells." The matrix had a fine plexus of fibrils, and its mineralization appeared to be not homogeneous, but "granular crumbly."

    The similarity between this bone and that of osteogenesis imperfecta fetalis (o.i.f.) caused Weber to propose a common genetic origin for the two conditions. Meyer's (1930) skepticism of this idea was based on seeing small amounts of Weber's "primitive bone" in normal labyrinths, and his failure to find Weber's "intermediate between osteoid and chondroid" in the labyrinths of victims of osteogenesis imperfecta. Weber (1930) had brought this second criticism upon himself by mistakenly introducing, as evidence of the nature of o.i.f. bone, typical chondroid bone I in the fracture callus of o.i.f. patients. Thus Weber's (1930) Figure 4, of the largely unreworked callus of an old radial fracture, has CB I described as "An intermediate between fibroblastic osteoid and chondroid. Cartilage-like cells." This fracture callus tissue is more consistently chondroid in its cells than are the tissues of o.i.f. and otosclerotic foci.

    METAPLASIA IN INTERGLOBULAR SPACES?

    While usually acknowledging the major role of endochondral ossification in the formation of the petrous bone, many microscopists have held that some of the cartilage remnants and their cells could turn into bone. The temporal bone has long been a stronghold of the adherents of metaplasia, with each sally in its favor meeting a prompt rebuttal. So Bottcher's (1869) proposal that the labyrinthine capsule forms by a metamorphosis of hyaline cartilage elicited Gottstein's (1872) description of the capsule as developing, as for other bones preformed in cartilage, by the destruction of cartilage, not only without any metaplasia, but with some doubt as to the contribution of periosteal osteogenesis.

    Shin-izi Ziba (1911a) collaborated with Manasse to continue the study reported earlier by Manasse (1897). They found in the interglobular cartilage rests a variety of cartilage cells. Some were small, osteocyte-like and appeared to send out processes. These, and some larger, more typical chondrocytes, were enclosed in complete or partial capsules of a fine fibrous matrix staining with eosin or fuchsin. They interpreted these various forms of cell and adjacent matrix to be participants in a "chondrometaplastic osteogenesis," whereby cartilage cells and their matrix turned into the globuli ossei of Brandt. Shin-izi Ziba claimed that serial sections showed some globuli ossei to be isolated from sites of erosion and marrow. Shin-izi Ziba's second paper (1911b) described the extensive distribution of interglobular cartilage and the near-constant occurrence of "metaplasia" in man's otic bone from six months post-conception to 76 years.

    His first article (1911a) reviewed at some length the literature on metaplasia and also discussed the junction between residual cartilage and both osteoblastic bone and the supposedly metaplastic globuli ossei. He believed that the pale zone often present at the boundary was a result of a dissolution of calcium salts: one step in a process of gradual dissolution of cartilage matrix to make way for bone. However, he admitted the problem that many islands of cartilage showed no tendency to disappear. I have come across no direct evidence on events at this cartilage-bone junction. In Hawke and Jahn's (1975) illustrations of tetracycline-labeled otic capsules, there is no marked fluorescence at the periphery of the interglobular areas that might indicate a redeposition of labile calcium, but interestingly some nuclei within the lacunae do fluoresce.

    Zawisch-Ossenitz's (1929b) discussion of her work on long bones included a critical interpretation of Shin-izi Ziba's work. Her points were the following.
    He drew no conclusions concerning the variety that he saw among the so-called young cartilage cells.
    Second, the cartilage cells that he drew were not convincing and were at a low magnification. It appeared to her that the interglobular material was not true cartilage, but was the same kind of basophilic island with pseudocartilage cells as she saw in animal bone, i.e., it was a form of bone.
    Third, his first specimen, at six months of fetal life, showed the islands already formed, implying that he lacked earlier specimens able to yield evidence on the origin of the interglobular tissue.
    Fourth, he had not adequately explained his observation that the interglobular areas widened towards birth, narrowed in the first three years of life, and then again became wider.
    Zawisch-Ossenitz's (1929a) own proposition that the islands are bone and not cartilage is contrary to the conclusion of all other workers, although it did find an echo in Ruth's (1961) belief that basophil material within metaphyseal trabeculae was bone undergoing resorption rather than residual cartilage.

    Zawisch-Ossenitz did not cite Eckert-Mobius (1926), whose description of the interglobular areas is close to Shin-izi Ziba's and Manasse's, but whose summing up is more cautious:
    "For the definiteIy peripherally located globuli ossei there is no reason to prefer a metaplastic origin (MANASSE, ZIBA) to one based on their forming during fetal development from the closest osteoblasts. But, as KOSAKABE emphasized, within the matrix of the interglobular areas closer study often reveals globuli ossei with included cells whose staining, size and form correspond with unopened chondrocytes, whereas the lacunae of the peripheral globuli ossei mostly appear empty. Here we may scarcely completely reject the possibility of a direct chondrometaplastic osteogenesis, which would also account for the quite apparent reduction with age of chondrocytes within the interglobular areas, supposing that we still (compare Weidenreich's most recent work) hold firm to a basic difference between calcification and ossification."

    The last histologists to give serious attention to the implication of globuli ossei for metaplasia seem to have been Weidenreich (1930) and Haines (1938a). Weidenreich cut serial sections and established the presence of giobuli ossei without any communication with vascular spaces or canals in the hammer and anvil of the ox, and also in the long bones of older amphibia. In all these bones, the isolated globule ossei were at the join of the bone with the persisting articular cartilage. Weidenreich commented:
    "Depite all assertions to the contrary, I consider the question of how globuli ossei arise as not yet decided. Indeed, I do not believe that cartilage matrix is able to transform directly into bone matrix, since that would require an apparently impossible re-orientation of the fibrils within the calcified cartilage. However, where there is a slow ossification the cartilage cells survive. I now consider it as quite possible that a chondrocyte on the bone-cartilage boundary can become an 'osteoblast' and lay down bone on its lacunar walls."

    He offered an illustration of such a cell at the ossification line in the femoral epiphysis of a grown guinea pig, and another (his Figure 41) of globuli ossei arising in this way in the anvil of ox. He went on, "The absence of canaliculi and corresponding cell processes is no reason to deny this interpretation because these are also missing from globuli ossei (in the femur) and also are often not present in the outermost parts of Haversian systems...."

    It might be objected that bone cannot be formed by cartilage cells isolated from a blood supply by their own matrix. Tissue culture shows that a blood supply is not essential for osteogenesis. Weidenreich's contribution was written just before the observation of osteogenesis in vitro by avian limb rudiment tissues (Fell 1928, 1931a) and mammalian tissue (Niven, 1931).

    Haines (1938a) illustrated (Figure 8D) numerous globuli ossei in the young crocodile's tibia, and remarked on the resemblance of the contained cells to undegenerated hypertrophic chondrocytes, and the doubt concerning their origin from marrow or cartilage.

    The Interglobularraume and the globuli ossei within them lie in the middle, endochondral, layer of the labyrinthine capsule's three layers. The metaplasia claimed more recently by Gussen (1968a,b) took place not in this middle layer, but in the inner endosteal zone, in cartilage claimed by her to be secondary.

    SECONDARY CARTILAGES

    Since the petrous, mastoid, and styloid parts of the temporal bone develop endochondrally from primary cartilage (Bast and Anson, 1949), the suggestion that any of them should be the site of secondary cartilage is surprising.

    Bast (1940) clearly regarded the cartilage found on the petrous bone at the petrosquamosal suture in 29 per cent of human infants as a residuum of the primary cartilage in which the petrous bone formed. If true, the cartilage is not a secondary formation of the kind observed at the sutural margins of bones of the growing cranial vault.

    Another cartilage has a more ambiguous status. This is the cartilage bordering, and sometimes filling, the Fissula ante fenestram. Eckert-Mobius (1926) and earlier workers described this cartilage only as a persistent remnant of the primary cartilage. Bast (1933) and his co-workers started and elaborated the idea that some of the cartilage lining the Fissula a.f. was a later development. Bast's (1933) first indication of such an event was only to mention that after about 30 weeks in utero, "The connective tissue of the fissula bordering the fissula next to the bony capsule changes to cartilage; this cartilage normally remains as cartilage throughout life."
    Anson and Martin (1935) described this cartilage in infants and children as immature or young, noting, "At no point does the mature cartilage or the intrachondrial bone pass by an intermediate transitional tissue into the young cartilage which occupies the fistula." In a footnote, they comment that "within the newly formed immature cartilage the lacunae are large, are not arranged in definite patterns and lie within a matrix which is light staining and continuous with the more cellular perichondrium from which it was derived by appositional growth." It will be remembered that large cells and a pale matrix are characteristic of many secondary cartilages (Schaffer, 1888, 1930).

    Bast (1936) devoted a paper to this late-forming cartilage in the fissular region, but tempered his 1933 conclusion as to its origin in connective tissue - "Such a mass of active cartilage apparently arises in and from the dormant fissular cartilages, which may be augmented by new cartilage derived from the fissular tissue by metamorphosis of its connective cells to cartilage cells."
    He noted so much variety in the fissular tissue that he hesitated to speak of a normal histological picture. Clearly, he (1936) was uncertain as to how the new cartilage formed. While it may have its origin in primary cartilage, it is tardy in forming and has some of the morphology of secondary cartilages, and hence falls into the same category of questionable secondary cartilages, as new cartilage in cartilage canals and cartilage within perichondrium.

    Having already dealt with the fissural cartilages, Bast (1940) gave them no further attention in his review of residual cartilages. In the canal region some of the masses of cartilage lay bordered by, or extended into, connective tissue of the subarcuate fossa. Again, he was unable to determine "whether they are remnants of the original capsular cartilage or whether they are products of chondrification of some of the vascular connective tissue ... no direct transformation of connective tissue into cartilage was observed."
    Although Bast and Anson (1949) referred to this as a "secondary cartilage formation," they also called it "neoplastic" and appear not to have used secondary with the intent of placing residual otic cartilages in general in the category of secondary cartilages. This, however, was the use of secondary by Gussen (1968a,b).

    GUSSEN'S OBSERVATIONS, NOMENCLATURE, AND CONCLUSIONS: A CRITIQUE

    Gussen's (1968a,b) two papers are hard to follow because of her idiosyncratic use of chondroid, secondary cartilage, chondroid bone, and globuli ossei (see Chapter 2, Chondroid Membrane Bone, p. 20); also see her diagram of the relationship (1968b).

    Gussen's (1968a) first use of the term, chondroid bone, was in connection with the remnants of cartilage within the middle endochondral layer of the otic capsule - remnants that she termed cartilage foci or globuli ossei, and believed to be uncalcified:
    "Occasionally, cartilage foci appeared to transform into bone without prior resorption. The chondrocytes became more stellate in shape, and the thin, uncalcified matrix bars took on an eosinophilic stain. The focus was then indistinguishble from bone, although retaining the honeycomb structure of the previous cartilage. Cartilage matrix with large empty lacunae was also seen undergoing transformation to bone with small numbers of individual bone cells entering some of the empty lacunae. In the younger specimens, this new chondroid bone was often very cellular ... At times, the cartilage matrix and cells transform directly to a chondroid type of bone."

    From these passages and her discussion, it is evident that Gussen (1968) was following the practice of Moss (1961) and calling bone, e.g., her Figures 7B and 7C (1968a), "chondroid bone" to call attention to how it supposedly had formed, rather than to a persisting difference from the normal run of bone.

    Moreover, her interpretation of the supposed transformation of the cartilage islands hints obscurely at two kinds of metaplasia: the classic way, whereby chondrocytes and matrix together become bony elements, and a second in which only matrix is transformed, while bone also is deposited by osteoblasts that have migrated into the lacunae. It does not appear that Gussen saw anything in the interglobular areas other than the events described and variously interpreted by Shin-izi Ziba, Eckert-Mobius, Bast and others. To call the bone of the globuli ossei, or even bone resulting from a metaplasia of cartilage, by the name chondrold bone is misleading for anyone wishing to reserve the term for a tissue still with bony and cartilaginous features.

    Aside from the tissue thought to arise from the cartilage islands of the endochondral layer, Gussen also believed that a chondroid bone was present at the endosteal surfaces of parts of the cochlea and the semicircular canals -
    "in some specimens, a continuous uncalcified cartilage lining could be demonstrated along portions of the bony cochlea which appeared to be undergoing transition to chondroid bone ... At times, the cartilage was separated from the soft tissues by a thin rim of chondroid bone - (Figure 8B)." In the legend to Figure 8B the surface of the 69-year-old cartilage is described as being "covered by a rim of acellular chondroid bone," and in the legend to what seems to be the same figure designated 4B in Gussen (1968b) as a rim of "acellular chondroid membrane bone." From her lower-power Figure 4A of the latter publication, it seems that she was reporting a major residual cartilage, but what precisely is occurring at its "endosteal" surface cannot be made out.

    In an addendum to the first article (1968a), she retracted the proposal that a direct transformation to bone of "chondrold cartilage" took place, offering instead alternating changes in the state of polymerization of the marginal matrix - "an unusual form of 'remodeling'". In her second paper (1968b) she confined the retraction to the deeper chondroid bone, as the following two quotations show:
    "In a previous study (1968a), the author described the presence of secondary cartilage (or chondroid) and chondroid membrane bone in the labyrinthine capsule. At that time, the normally occurring processes of depolymerization and repolymerization occurring about the blood vessels were not recognized as such, and were described as areas of chondroid cartilage because of the occasional resemblance of the depolymerized matrix to cartilage matrix."
    She went on, "Chondroid, or secondary cartilage, transforming directly to chondroid bone, appears to occur only in the surface of the cartilage of the inner layer of the labyrinth. As has been shown, the deeper portions of this cartilage (away from the surface) may be replaced by endochondral globuli ossei. Once the surface of the inner layer of the labyrinth has formed as chondroid bone, this chondroid bone appears to undergo the processes of depolymerization and repolymerization, as described."

    To conclude, the tissue overlying the cartilage at the labyrinthine surfaces might be bone and might be formed by metaplasia, but the cartilage itself appears to be residual and hence primary rather than secondary, although Gussen claimed that labyrinthine mesenchymal cells contributed to it and explicitly equated it with typical secondary cartilages. It may be that she made too much of the variety of staining reactions at the margins of the bone and cartilage, and the seeming widespread absence of osteocytes. That her fixative, formalin, is feeble, and the long time needed for decalcification in EDTA, together may have resulted in the "ghost-like" osteocytes; hence some osteocytes may have escaped detection, leading to an underestimation of the cells' number.

    Gussen's (1968b) notion of her "cell-free" bone that "the mucopolysaccharides and mineral matrix of this bone apparently alter to a degree that allows the penetration of new bone cells from adjacent perivascular bone or adjacent soft tissue" need not be given much heed, until it has been ruled out that the variations in the pallor and cellularity of the matrix and in the perivascular basophilia are not artifacts.

    Those variations do not demand consideration as a possible instance of tissue transformation or metaplasia for a second reason, namely, that if indeed cells are absent, the material is not viable, is not a tissue and cannot experience metaplasia.


    Chapter 23 RICKETS

    Introduction to rachitic "chondroid bone" and the metaplastic question
    Metaplastic interpretations: Virchow's
    Non-metaplastic assessments: Muller's
    Nature and origin of the fibrous cartilage
    Similar "chondroid bone" in congenital syphilis
    Rachitic cartilage as secondary?

    INTRODUCTION TO RACHITIC "CHONDROID BONE" AND THE METAPLASTIC QUESTION

    Rickets comes about variously from too little dietary vitamin D and no exposure to sunlight, from renal disease interfering with the metabolism of vitamin D to its active form, or because of insufficient minerals in the diet or enteric illness that disturbs their absorption. The syndrome of rickets includes an overgrowth of cartilage at the ends of long bones, a failure of that cartilage to mineralize and be resorbed, and the formation of much poorly mineralized organic bone matrix or osteoid. Intramembranous bones and other tissues are also involved.

    In rickets the epiphyseal growth cartilages are large and partly tunnelled through by canals. Much of the cartilage is spared destruction, and some is also abnormally acidophil, fibrous, and small-celled. These conditions, coupled with the abundant production of osteoid, lead to the intimate and disorderly juxtaposition of hyaline cartilage, abnormal and unresorbed hyaline cartilage, osteoid and bone. The tissues do not lie in this sequence, in distinct layers, which would draw one strongly to the conclusion that they are participating in a direct metaplasia of cartilage to osteoid and then to bone.
    Rather, the tissues are intermingled, so that the above order of the tissues is arbitrary; nonetheless their presence has been given a metaplastic interpretation, with the abnormal cartilage playing the key role as evidence of a transformation, because of its somewhat bony characteristics, as evinced in many descriptions, and in figures such as Dodds and Cameron's (1938a) Figure 13. Virchow (1853) called the tissue with both cartilaginous and bony characteristics in rachitic long bones and ribs knorpeligosteoides Gewebe. He believed that cartilage was transforming into bone, in which circumstances the region overall had to include cartilage, bone, and something in-between.

    Others, who have been doubters or outright disbelievers in metaplasia, have introduced the potentially misleading names "chondro-osteoid trabeculae" (Park, 1939), or "cartilage-osteoid trabeculae" (Dodds and Cameron, 1938b), to signify that osteoblasts have been obliged by the lack of calcium to lay down osteoid in place of bone on the largely unresorbed cartilage. The resulting trabeculae comprise a composite of tissues, as expressed by the names, which were not intended to convey that there is a tissue present of a kind half-way between cartilage and bone.

    Both reports referred, in addition, to abnormalities within an unresorbed cartilage, but either used another term for its alteration, pseudoosteoid transformation (Park, 1939), or simply referred to it as uncalcified cartilage (Dodds and Cameron, 1938a). All observers of rachitic histology acknowledge that some of the epiphyseal and costal cartilage is abnormal. What is the nature of this atypical cartilage? How does it form? Should it be called chondroid bone and, if so, what relation does it have with the types I and II? Is the unusual cartilage evidence of a direct metaplasia to bone, or at least to osteoid?

    Muller (1858) probably has been the author most concerned with finding answers for these questions. More recent writers on rickets have explicitly set aside the question of metaplasia (Pappenheimer, 1922), admitted an inability to answer it on the evidence available (Schmorl, 1909), or, from identical materials, asserted metaplasia to be incontrovertible fact (Kassowitz, 1911), or lately have ignored the topic (Durkin, Heeley, and Irving (1971) inter alios).

    Muller's controversial report on normal and rachitic ossification stood against a rising tide of microscopic observations interpreted as showing that bone forms by a transformation of connective tissue or of cartilage into bone, or into marrow and then bone. Thrown into the metaplastic pan of the balance were the histological findings in rachitic bone, first reported by Kolliker (1847), followed by another brief report by Meyer (1849b). Virchow's more comprehensive description of the histology and the disease followed in 1853.

    METAPLASTIC INTERPRETATIONS: VIRCHOW'S

    In his task of discerning the nature of ossification in general, Virchow was handicapped by a glut of pathological materials from the widely prevalent rachitic cases and rarer conditions such as congenital syphilis and osseous and cartilaginous tumors. For him, the theory that accounted best for the rather cartilaginous appearance of some new subperiosteal bone, ossification in some fibrocartilage at the knee, for the events of normal enchondral growth, the mixed tissues in some tumors of bone, and the variety of tissues in rachitic epiphyses, was a metaplastic one whereby bone, marrow, cartilage, and periosteum could turn into one another.
    Virchow (1853) recognized that at the normal epiphyseal disk many cartilage cells clearly did not become osteocytes, raising the issue of whether any did: a question that he could not resolve in the healthy growth plate. He believed that the rachitic condition held the answer, because the absence of calcium there allowed changes in the cartilage to manifest themselves -
    "in normal endochondral ossification calcification always precedes ossification; the osteoid transformation of the cartilage occurs in the already calcified part and is obscured by the calcification. Only in advanced rickets is the osteoid metamorphosis truly shown, because here the calcification is retarded, and precisely because of this is the disease so suited to make known an otherwise thoroughly cryptic event."

    What was Virchow describing as an osteoid transformation in the ribs and long bones? Numerous channels penetrated the rachitic cartilage, but were occupied by fatty or fibrous tissue rather than typical small-celled marrow. Although he noted the channels' similarity to "perichondral buds" (cartilage canals?), he believed that cartilage turned into marrow and hence called them marrow spaces. The cartilage close to these spaces appeared to undergo these changes: the cellular capsules became thicker, making the lacunae smaller with a serrated outline, and the capsular material became fibrous, as did the inter-territorial matrix. In an adjacent but somewhat different tissue, he could see communicating canaliculi running from stellate cells.
    He accordingly distinguished three tissues: an osteoid or fibrous kind of cartilage; a more homogeneous osteoid material with stellate, indented cells; and regular calcified bone holding stellate bone cells. He was able to find some trabeculae having a central core of bone on which lay osteoid. Virchow deduced from this arrangement that calcification spread from the interior of the trabecula outwards into the osteoid, and itself had been preceded by a transformation of cartilage into osteoid. The existence of the three sorts of tissue in rachitic epiphyses and ribs has been confirmed time and again, but the evidence for a transformation remains only what it was then, viz., the proximity of the tissues and the regions of transition between them.

    NON-METAPLASTIC ASSESSMENTS: MULLER'S

    Muller's (1858) conclusions on rickets have been confirmed but not bettered:
    "Intracartilaginous ossification in rickets differs from the normal in these points. (1) The preparatory calcification stops or is insufficient. (2) The marrow canals penetrate the uncalcified cartilage, but are abnormal in their extent and form and in how they develop. In general their development is less. The cartilage thereby suffers a fibrous or osteoid transformation. (3) Within the marrow spaces of the cartilage an osteogenic material forms in many places in a position dependent on the orientation of the spaces that often gives the appearance as though bone cells had formed within enclosed cartilage lacunae. In other places it is not osteogenic material that is built, but the marrow spaces hold only soft tissue or incompletely osteoid masses. Various intermediate tissues also are present. (4) The cartilage persisting abnormally between the marrow spaces contains appositional layers (perilacunar) whereby the lacunae become somewhat like bone cells. (5) In the interior of bone formed a marked deposition of uncalcified osteogenic material occurs, along with a partial resorption of the hard bone.
    In connection with normal intracartilaginous ossification, there is evidence that true bone forms in marrow cavities from soft tissue, on the other hand the metamorphosis of the cartilage, which is absent from normal bone growth, allows no direct conclusion. But this finding does show well how various are the intermediaries of the different forms of connective tissue, and warns one not too readily to extrapolate from one situation to another, since almost every kind of tissue conceivable seems to occur. I will on this account not conclude that the process (metaplasia) that until now was taken to be usual for intra-cartilaginous osteogenesis cannot occur somewhere."

    Muller (1858) also noted that the general risk of mistaking old chondrocyte lacunae filled with bone as evidence for metaplasia was increased, because in rickets more cartilage was spared erosion than normal and hence could experience more of this partial replacement by small spherical masses of bone - globuli ossei (see Chapter 22, Globulus Osseous (Knochenkugel)).

    NATURE AND ORIGIN OF THE FIBROUS CARTILAGE

    Muller remarked that in rickets the pattern of irregular penetration of vessels, and the extent of spared cartilage between them, resemble the circumstances of normal endochondral ossification he had seen in lower vertebrates. Second, the rachitic cartilage appearing to become osteoid is seen close to the canals. Now, around the vascular canals in healthy cartilage, the matrix is cosinophil and has given some observers, e.g., Carey and Zeit (1927), the erroneous impression that it was becoming osteoid or bone (see Chapter 10, Patella and Cartilage Canals). Hence, the possibility exists that the perivascular cartilage in rickets is not of itself abnormal, but is typical of what lies around cartilage canals, and the disturbance lies primarily with the distribution of vessels and the availability of calcium.

    This view conflicts with the expressed or underlying assumption of some writers on rickets that normal hyaline cartilage forms, and then, as a result of actions by penetrating vessels, undergoes a transformation, in that it loses proteoglycan, acquires more fibrous collagen and its cells become smaller; thus Park's (1939) pseudo-osteoid transformation. It is conceivable that, instead, some of the cartilage of the epiphyses forms in the fibrous condition in which it comes to microscopic observation. Why should this occur?
    The problem is allied with that of whether cartilage canals in normal epiphyses form by inclusion with the growth of cartilage or by a penetrating erosion of cartilage (see Chapter 10, Patella and Cartilage Canals). Virchow (1853), Muller (1858), Schmorl (1909) and others referred to the perichondral nature of the canals within rachitic cartilage. Schmorl took the point further:
    "The osteoid growing in the rachitic cartilage region is, as Schmidt rightly claimed, histogenetically to be distinguished from that forming in the marrow cavity. The latter is a product of the marrow, the former is derived from the cartilage marrow and is, in the first instance, a derivative of the perichondrium."

    If the circumstances within the canals are close to those of the perichondrium, and vessels and cells from the perichondral-metaphyseal junction are "included", any cartilage forming in their vicinity would be ex ected to be like that under the perichondrium: eosinophil, with fairly small cells and a collagen of type I (Miller, 1973). Thus, much of what is seen could be typical "subperichondral" cartilage, abnormal only by its endochondral location, rather than a degraded or transformed hyaline cartilage.

    The fibrous, small-celled cartilage, whether formed thus or changed by ingrowing vessels, is not true osteoid, and has not been shown to progress to bone when the rickets heals. This latter event has not been absolutely excluded, but most investigators report a rapid resorption of the bulky mixed trabeculae of cartilage and true osteoid, and an absence of any metaplasia of cartilage through osteoid to bone, e.g., Wolbach and Bessey (1942).

    Thus, while rachitic cartilage is rather bone-like around canals and where it merges with osteoid (see Figure 3 of Sheldon and Robinson (1961) and of Rohr (1965)), this characteristic brings it into the category neither of blastemal CB I, nor mineralized enduring CB II. It would have to be considered as an atypical variety of chondroid bone, were it to be included in the category.

    SIMILAR "CHONDROID BONE" IN CONGENITAL SYPHILIS

    Vitamin D may not play a specific role in the development of the abnormal rachitic epiphyseal cartilage, since Schmidt (1906) reported a similar fibrous tissue around the abnormally oriented and deeply penetrating canals in the epiphyses of children with congenital syphilis. He viewed the disease's effect on the epiphyses as a disturbance of the perichondral connective tissues, including, he believed, a metaplasia of connective tissue and cartilage into bone; but, in commenting on Schmidt's paper, Pommer and Schmorl disputed his metaplastic interpretation of the areas of bone-like cartilage. Wislocki (1942) was struck by the similarity of the chondroid bone of the growing antler to the new bone in a case of congenital syphilis, but here the syphilitic "chondroid bone" came from the periosteum and was unrelated to the cartilage of the epiphyses.

    RACHITIC CARTILAGE AS SECONDARY?

    Much rachitic cartilage is large-celled cartilage (Durkin, Heeley, and Irving, 1971), whether it is in the growth plate of long bones, their articular cartilage, or in the mandibular condyle. A scantiness of matrix and large chondrocytes are also characteristics of secondary cartilages. Durkin et al. commented that "in rickets the tibial growth plate cells revert to a more primitive type of arrangement similar to that in the condylar and articular cartilages, which we already feel to be primitive in nature, and as a result are probably more aptly classified as 'secondary cartilages'." Although in rickets the epiphyseal cartilages take on more of the morphological nature of secondary cartilages, they are not, of course, anything other than elements of the primary cartilaginous skeleton. Furthermore, the idea that some cartilages are more primitive than others is likely to prove a distraction both troublesome and untrue.

    The concept of secondary cartilage was extended in an interesting way by Muller (1858). He saw what looked to be new cartilage among the marrow within the eroded cartilage of the head of the rachitic humerus. In a footnote (p. 218), he likened this new, i.e., late, building of cartilage to the accessory or secondary cartilages reported shortly before by Kolliker and Bruch on the mandible and other facial bones. Muller assigned such new cartilage a secondary character, although it formed deep within a primary cartilage. Since then, others have reported the occasional formation of new cartilage within canals of non-rachitic cartilage (Hintzsche and Schmid, 1933; Haines, 1937) and within the marrow spaces of older cartilage (Bohmig, 1929; and Fig. 255 of Schaffer, 1930). Other examples of late-forming "secondary" cartilage of primary origin are given in Chapter 3, this volume.


    Chapter 24 CONCLUSIONS

    To what chondroid bone, metaplasia, and secondary cartilage refer and what they exclude are unclear, since for over a century each term has been applied to a variety of phenomena. Attempts to confine the meaning of chondroid bone and metaplasia have been unfruitful, but overly successful with secondary cartilage.

    SECONDARY CARTILAGE

    This was first observed on long bones as a constituent of fracture callus, but today it signifies cartilage formed in development on cranial membrane bones. What had been the strictly defined secondary cartilages (Schaffer, 1930) - any cartilage appearing after and separate from the primordial cartilaginous skeleton - thus have fallen into an undeservedly nameless category, unless secondary cartilage will again be accepted as:
    1) sensu lato, replacing the earlier accessory cartilage for formations on membrane bones; and
    2) sensu stricto, with the scope of Schaffer's definition.
    That the strict usage is much broader in its application than the laxer one is not a contradiction, but the redressing of an overrefinement of usage that overlooks the many instances of late chondrogenesis sharing etiological circumstances with the cartilages on membrane bones.
    There is a degree of unity among Schaffer's secondaries:
    as latecomers in already established tissues, they all must arise from residual stem cells or by a metaplasia of differentiated cells.
    Second, Schaffer's hypothesis of a common mechanical stimulus to late chondrogenic differentiation seems to apply to more secondaries than just the accessory ones, although the induction of other secondary cartilages by hormones and other non-mechanical agents rules out a universal mechanical etiology.
    Third, many secondary cartilages share a form characterized by large, haphazardly strewn cells in very little matrix.

    METAPLASIA

    This signified a transformation of one mature tissue into another. When the transforming tissue's cells are considered, these can:
    1) lose their specialized character, divide and execute a differentiation into a new type - an indirect metaplasia, or
    2) without prior loss of their first character, and without division, start turning into another specialized cell type - direct metaplasia.
    Alternatively, although the tissue is mature, some of its cells, as stem cells, may not be. Should these stem cells be responsible for the change in the tissue, by a novel differentiation, say, to squamous epithelium instead of columnar, the transformation is commonly held to be a tissue metaplasia but not a cellular metaplasia.
    But metaplasia was defined and is still used by pathologists with reference to the tissue, so that metaplasia sensu stricto encompasses direct and indirect metaplasias and the novel stem-cell differentiations: in each, the tissue changes. All three are tissue metaplasias, but since cellular activities must underlie each transformation, all three are also cellular metaplasias.

    One approach to a clearer terminology is to let metaplasia continue to refer to a metamorphosis of a mature tissue. Adding to it indirect or direct tells one what is believed to be happening to its cells: in the former, the iridial cells or fibroblasts dedifferentiate before becoming lenticular cells or osteoblasts, respectively; in the latter, the chromatophores, tendon fibroblasts, or smooth muscle cells turn directly into melanocytes, chondrocytes, or juxtaglomerular cells, to use three transformations for which there is good evidence.

    What if the metaplasia is one involving an epithelium or hemopoietic tissue where stem cells seem to be responsible? These can be designated novel-stem-cell differentiations, but also made part of the basis for a broad definition of cellular metaplasia. Sooner or later the latter term is unavoidable, given that changes in the specialization of cells effect the transformation in the tissue.
    In the narrow sense these transformations can be construed as cellular metaplasias only if they start with one kind of mature cell and end with another, i.e., are indirect or direct metaplasias. However, the start and end for some tissues' cells are not clear-cut, because of the many organelles and molecules common to them. Also, the stem cells, where they exist, are not as undifferentiated as once was thought, so that their differentiation, novel or normal, is a change of specialization.
    Hence, cellular metaplasia sensu lato has some utility for bringing into consideration any change in specialization by the cells of a mature tissue comprising the making of new products or a durable change in the ratio of products. Thereby included are novel-stem-cell differentiations, dedifferentiations, direct metaplasias, transformations among subtypes of a tissue, e.g., types of skeletal muscle (thus giving the fiber types the status of tissues), changes from purportedly immature tissues, e.g., hyaline cartilage to fibrocartilage, cell-initiated calcifications, and changes with aging.
    This nomenclature in effect gives cellular metaplasia one current meaning of modulation, but does not require that the change be reversible, so that modulation can then be restricted to the physiologist's one sense of reversible short-term fluctuations in a cell's activities not involving an alteration to another cell type.

    Also, by focusing on the alteration to the cells, the broad cellular metaplasia, for comparison and study, can categorize many changes in the adult connective tissues as metaplastic, although the tissue is not transformed to a kind exactly like one formed de novo, e.g., the character of the fibers may be little changed.

    CHONDROID BONE

    If bone and cartilage are broadly defined to the limit of each other, chondroid bone can be eliminated from the nomenclature. What cannot be done away with are the many occurrences of a tissue with mostly chondrocyte-like cells in a bony matrix, and of bone and cartilage that are out of the ordinary in their resemblance to the other tissue. If chondroid bone is unavailable as a category, the scopes of bone and cartilage have to be broadened to take in these instances, with various consequences.
    The practice detracts from the precision of bone and cartilage as meaningful terms. Even when narrowed by keeping separate the various kinds of chondroid bone, bone and cartilage still are diverse in their forms and behavior, even early in development (Hall, 1978). Moreover, there is a psychological penalty: if unaware of the category of chondroid bone, the observer will, by the operation of visual constancy, tend to see only bone or cartilage, even when chondroid bone is present.

    Bone may be thought to be more uniform than it is, because it is often regarded as the product of one kind of cell acting in just two situations: endochondral and intramembranous. Tables IV.A. and B. list most of the many circumstances in which bone can form. While different routes need not lead to different kinds of bone, the situations in which osteoblasts work are so varied that it is perhaps more surprising that the product has any typical forms than that bone is sometimes chondroid.
    The divisions arise out of the sites and processes discussed earlier, and are not mutually exclusive. For example, A's "ossification" of dense connective tissue involves a metaplasia (D), if the end-result is reckoned to be bone.
    Thejustification for listing the items is that, for each, something special has at one time or another been pointed out concerning ossification in that situation. Table IV.A illustrates the ways in which the various kinds of chondroid bone arise. The letters below refer to those marking divisions in Table IV.A. (Table IV.B being more of historical interest is placed after the chondroid-bone discussion.)

    A . When a population of young cells deposits bone, chondroid bone I may accompany it, e.g., at margins of the skull vault, at the penile bone's tip, and (E) in fracture healing.


    Table IV.A. Circumstances of Ossification
    A. When a population of young cells deposits bone, chondroid bone I may accompany it, e.g., at margins of the skull vault, at the penile bone's tip, and (E) in fracture healing.
    C. When a germinal population switches from osteogenesis to chondrogenesis to make an accessory secondary cartilage, the cells often make CB I during the switch-over.
    D. An intermediate tissue indistinguishable from CB I occurs when chondroid or cartilage turns into bone, as in some piscine cranial osteogenesis, and far less certainly in the antler, fracture callus, accessory cartilages, and tumors. In the latter group, the recent existence of a germinal cellular population provides another, nonmetaplastic, explanation for the CB I seen.
    B, C. Endochondral ossification in any cartilage - primary or secondary, hyaline, fibro- or elastic - eventually halts in birds and mammals, leaving some calcified cartilage to endure as chondroid bone II. Chondroid bone II differs from CB I by having a still recognizable affinity with the cartilage before mineralization.
    C. If the natural turnover spares the bone in which rests of calcified cartilage matrix remain, as in the otic capsule and the penile bone's shaft, the resulting bone is not truly chondroid. It is just bone with non-vital inclusions of residual cartilage matrix. Likewise, in rickets where the resorption of cartilage fails, the marginal, often small-celled, proteoglycan-deficient cartilage is not so much a form of chondroid bone as an abnormal cartilage.
    D. Some CB II has been reported to turn into bone in fishes, reptiles, and amphibia but the evidence is only static and elementary morphological.
    C. Although the tissue of the antler which true bone replaces is here classified as a secondary cartilage, this tissu preosseux has a more bone-like matrix than, say, the mandibular condylar or clavicular cartilages. The antler's first firm tissue appears to be a formation de novo of a chondroid bone I on an exceptionally large scale.
    A. By contrast, the first firm tissue of the clavicle is a bone brought a little closer to cartilage by the close packing of its cells. It merits electron microscopic study.
    Among the intra-mesenchymal, better "ectomesenchymal" (Ten Cate, 1975), formations in the jaws is cavian cementum, peculiar for cementum in its closer resemblance to cartilage than bone.
    E. Some of the many agents controlling and influencing osteogenesis after the establishment of the skeleton result in either the formation of somewhat cartilaginously atypical bone, e.g., avian medullary bone, or chondroid alterations in existing bone, e.g., after venous stasis or an excess of parathyroid hormone (the alterations are accompanied by the deposition of new bone, most of which is large-celled but not unusual for its young age).
    The failure of osteoclasia in osteopetrosis leaves much young bone in the skeleton, but whether this bone is any more cartilaginous than the bone normally first laid down is uncertain. However, in osteogenesis imperfecta fetalis the bone, by its larger cells and greater content of proteoglycans, is closer to cartilage than normal human bone of the same age.
    None of the kinds of chondroid bone mentioned in section E. above falls as close to midway between bone and cartilage as CB I does, and none is exactly like any other. Keeping this in mind, one will see that they have been arbitrarily assigned to a miscellaneous third category.
    Chondroid bone I and II themselves are by no means homogeneous entities. For example, some CB I may reflect a process of metaplasia, particularly in fishes, rather than the action of blastemal cells. Second, in reptiles and fishes some longstanding mineralized primary cartilage (CB II) can take on more the appearance of mammalian CB I or an entity even closer to bone. The tripartite division of chondroid bone is therefore only a tentative working one to bring some initial order to the topic.
    F. Chondroid bone I is a common occurrence when skeletal germinal tissues are grown in culture or grafted elsewhere in the body. The frequency with which it is seen and the greater or lesser degree of artificiality in the grafts' circumstances are the reasons for a separate listing (F.) in Table IV.
    For example, some chondroid bone may form by default because the cells did not have all the materials needed for making bone.
    Second, the cells, deprived of the clear stimuli to chondrogenesis or osteogenesis present in their normal site, may nevertheless react to form an intermediate.
    Third, with fewer blood-derived clastic cells to destroy the cartilage held in vitro, the cartilage cells may be freer than usual to do one of two things. As they go about turning over the cartilage matrix, they may have time to shift toward osteogenesis and execute the metaplasia reported by Fell (1933). Alternatively, the reworking of, or failure to maintain, the matrix might result in chondrocytes being liberated into the medium to assume the role of osteoblasts. Both events find stronger support in avian cultures than those of mammalian tissues; piscine and reptilian cultures await study.

    TableIV.B.* Lubosch's (1924) Division of Endochondral Ossification

    * The table is derived from the text of his article.
    Im mammals II generally follows I; while in non-mammalian cases I and II can co-exist, but with a spatial separation.
    Note that several, but not all, sites of his Markverknocherung are sites of CB II.

    SOME QUESTIONS

    The chondroid bones are provocative tissues. Among the many questions outstanding are the following:

    1. How do the genetic controls operate in cells making CB I? This form of chondroid bone is frequently present in small, short-lived amounts at sites where a large germinal population changes from osteo- to chondrogenesis or vice versa. Here, the chondroid bone might be merely an epiphenomenon of the switch by the germinal cells, reflecting only that the syntheses of proteoglycan and collagen are under separate control, and some disjunction in timing. Perhaps the synthesis of cartilaginous proteoglycans runs on, requiring a chondrocytic morphology after the deposition of bone-like collagen has started, resulting in the typical chondrocyte in an osteoid matrix. This staggered timing could apply to chondroblastic cells turning to osteogenesis, but would have to be reversed when the CB I occurs where osteogenesis changes over to chondrogenesis.
    Next, to what extent the calcification of a matrix is under separate genetic control is unknown for any connective tissue. If CB I is mineralized, this event would give more credence to naming it in a bony way, but, from the customary circumstances of its observation, the extent of its mineralization is usually unknown.

    On the other hand, the evidence that germinal cells can deliberately set out to produce just chondroid bone comes from cavian cementum, the antler's preosseous tissue, and the chondroid bone in placoderms and certain extant Selachii. These chondroid bones cannot be denied the status of a tissue. They are appreciable in amount; in fishes and the tooth, they endure; they have a specific location, and in antlers there is a precise relation with blood vessels. In mammalian and avian skeletal grafts the cells also sometimes seem to settle down to the stable production of chondroid bone.
    In the initial formation of the bony sleeve on primary and secondary cartilages, the clarity of the line of demarcation, with little or no CB I present, indicates that the germinal cells' change-over need not be slow. This suggests that even the fleeting CB I of other sites is specified by the cells to meet a need.

    2. In chondroid bone II, the cell's role in controlling mineralization again prompts inquiry, as well as the particular query, how do some chondrocytes survive a substantial degree of mineralization in the surrounding matrix. The vitality of the cells, how they inhibit vascular invasion, the nature of the mineralization, the rates of diffusion, and the role of canaliculi in the viability of osteocytes, all call for re-examination.

    3. Connected with the last point is the question of what degrees of metaplasia occur among, to and from the firm connective tissues and by what mechanisms. For example, the very limited scope of osteocytic osteolysis, if it even occurs in normal bone, limits bone's potential for metaplasia. The unlikelihood of chondrocytes' extending processes through a mineralizing matrix to become osteocytes has made many skeptical of a chondro-osseous metaplasia, supposing such cell processes are needed to keep the tissue alive. But it could be argued that, were the chondrocytes to make collagen of the bony type with a fibril thickness and packing density appropriate for bone and followed by a mineralization, they would have achieved the best part of an osseous metaplasia. Their lack of processes might have no more significance than that these provide the means for osteoblasts to communicate on what fibrillar and trabecular patterns the bone should have, and are not essential to their later life as osteocytes. The tissue, otherwise confirmed in its metaplasia, presumably would lack the fibrillar orientations of true bone. Similarly, the chondrocytic transformation of tendon fibroblasts does not result in the tendon's acquiring the fibrous architecture of a symphysis or annulus fibrosus.

    Whether these late, at least partially metaplastic changes in the connective tissues are in response to particular recent stimuli or are programmed from the period of initial histogenesis may vary with the particular tissue and place.

    4. What is the biomechanical significance of chondroid bone? Chondroid bone II is a junctional tissue serving to unite with bone the most heavily loaded tissue of the body (articular cartilage) and the most tensed (tendons and ligaments), but its own physical properties and why it occurs where it does are obscure. Its occurrence in other sites such as the tracheal and laryngeal cartilages and enchondromas cannot readily be related to a mechanical role.
    In general, CB II participates in the puzzling sequence - chondrification, calcification, erosion, ossification and myelogenesis - widely employed in skeletal development, but apt to occur in any cartilage except the elastic ones. Outside the mammalian skeleton the slower pace of events leaves more CB II. The fact that one happening in the sequence can trigger its successor is established (Hall, 1978), but how the tissues of the sequence change their nature and form so drastically, while keeping account of the mechanical demands, is unexplained.
    Chondroid bone I certainly acts to fuse together new bone and cartilage, but it is most often seen early in life when the bones have light loads or, like the antler and penile bone, have no discernible loading except for gravity and vascular motions, unless the periosteum places them under some restraint. (That fishes have some persisting CB I may be related to the reduction in skeletal loading from the water's support.) The mechanical purpose of chondroid bone I and the members of class III is a mystery.

    5. Is there a stem cell in adult connective tissues? Leaving aside the 'clasts, some cells on the surfaces of mature bone and cartilage and among the soft connective tissues are relatively inactive. Do such cells differ from more active ones in both their activity and their state of differentiation? If the fibroblast can maintain its matrix, divide, and reactivate itself to repair wounds, and occasionally to transform into osteoblasts, a separate soft connective stem cell - the undifferentiated mesenchymal cell - is redundant. The same reasoning can be applied to cells on the surface of bone: either they are osteociasts (mono- or multinucleated) or they are osteoblasts, unless they can be shown to have the surface and other markers of immature cells. If the surface cells are all differentiated, then the secondary accessory cartilages form by an indirect metaplasia of osteoblasts.


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