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
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
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
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.
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.
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..
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
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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 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).
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.
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.
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.
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 (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.
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.
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.
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).
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 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.
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."
Conclusions A survey of the information on secondary cartilages gained by histology, histochemistry, electron microscopy, radioautography, etc., brings one to the following conclusions:
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.
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.
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.
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.
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.
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.
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).
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 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).
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.
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.
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:
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.
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:
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.
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:
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).
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).
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:
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
What the term dedifferentiation fails to express openly is:
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.
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.
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).
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 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.
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.
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.
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.
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:
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.
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.
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.
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.
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.
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.
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.
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?
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 - osteoblastThe 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.
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
YoungBy 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.
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.
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:
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:
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.
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.
The diagrams can lead one's thinking into treacherous paths.
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.
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 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?
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.
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.
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.
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).
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.
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."
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."
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.
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.
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.
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.
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.
Schaffer distinguished four varieties of chondroid supporting tissue:
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."
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.
Other tendinous and ligamentous chondrifications are identified by Schaffer (1902a,b;
1930) and Knese (1978b) as:
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).
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.
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.
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."
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.
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.
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 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.
Fig.,, 8. Radial t.b (Fig. 176 Knese, 1978b)
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.
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.
Chondroid Bone II (Hyaline)
Chondroid Bone II (Fibro)
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.
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.
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.
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."
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.
Cellular Viability and Chondroid Bone
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).
Occurrence of Bone
Stimuli for Degeneration, Calcification, and Ossification
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.
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).
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.
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.
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.
"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.
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.
Haines (1969) reviewed his own (1941) and others' work on reptilian osteogenesis.
Among his points these are relevant here.
The Patella and Cartilage Canals
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.)
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.
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.
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
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.
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
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).
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.
In their conclusion Knese and Biermann brought together the results of all their
observations favoring a metaplasia:
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
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).
For chondroid bone II arising in unequivocal cartilage, e.g., costal or articular,
there are three issues to be kept apart.
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).
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.
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.
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.
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.
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.
Growth
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.
Mechanical Roles
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.
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.
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.
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."
Non-mechanical Roles
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.
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.
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).
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.
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.
Next, distinguishing bone from cartilage in the developing skull presents several
problems:
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).
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).
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)?
Thus, one may conclude that:
Angular and Coronoid Cartilages
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.
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
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.
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."
Cartilage on Alveolar Processes
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.
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."
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.
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.
Fig. 15
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 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.
Condylar Cartilaginous Tissues: A Stable Basis for Comparisons?
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:
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:
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
Periosteal Bone: Living Cartilage Boundary
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.
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.
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
Fuchs's Comparative Studies
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.)
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
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."
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."
Chondroid Bone in Rodent Fossa
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.
Temporal Chondroid Bone: Reactions and Remodeling
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.
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.
Pterygoid
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.
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:
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.
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.
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:
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,
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,
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,
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.
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 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:
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.
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.
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.
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,
He did waver in his estimate of the time of development of the intermediate tissue,
thus (Murray, 1963),
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.
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,
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.
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).
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).
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.
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,
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), 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.
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.
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.
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.
First, exactly how osteoid is to be defined is unsettled. Zawisch (1947) was
correct but a little overwrought when she wrote,
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.
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.
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.
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.
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.
The Anterior Fibrocartilaginous Process
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 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.
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).
Although there are a common morphology and process of ossification, several
differences exist among the four sites.
Chondroid Bone
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 Fig 23
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
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). Fig 25,
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.
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).
Fig 28
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.
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.
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.
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.
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).
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.
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.
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.
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.
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).
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.
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:
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,
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.
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.
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,
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
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)."
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.
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,
Altmann (1964) encountered chondroid bone in two situations in his experiments
on regeneration in the rat's hind-limb. He distinguished four tissues:
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.
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.
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 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
Fig 32
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.
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.
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.
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.
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.
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.
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,
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).
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.
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).
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.
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:
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:
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.
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.
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,
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."
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.
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.
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.
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).
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.
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.
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,
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."
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. . . ."
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.
What are the implications of these interpretations? And are there alternative
conclusions?
Lufti (1971) saw in the chick's tibia:
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:
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.)
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.
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.
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:
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:
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.
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.
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.
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).
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,
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:
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).
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).
"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 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).
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.
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.
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
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
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
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
Livingstone and Sandison (1962) observed in an osteogenic sarcoma of the thyroid,
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).
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.
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.
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.
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.
Kyle's (1927) introduction, while focused on fishes, is pertinent to all chondroid
bone:
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.
Fig 35
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.
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.
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:
The unusual tissue lining the cartilage of the claspers in Chimaera and
Selachii is considered by Orvig (1951) as not a
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
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
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.
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:
Fig 36
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.
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.
Below the fibrocartilaginous articular surface of the mandible of Trigla
capensis, the Cape gurnard,
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:
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.
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 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.
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.
The manner they described by which the "hypertrophic" osteocyte erodes the matrix
did not call for the cell to do other than continue synthesis:
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)."
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.
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.
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.
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.)
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.
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.
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).
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.
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.
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.
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.
Fig 38
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."
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.
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.
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,
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.
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.
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.
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.
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:
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:
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.
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."
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."
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."
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:
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 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:
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.
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.
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.
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.
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)).
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?
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.
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.
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.
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.
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.
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.
TableIV.B.* Lubosch's (1924) Division of Endochondral Ossification
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.
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.
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.
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(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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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?
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 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?
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.
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.
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.
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."
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 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).
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.
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.
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.
In addition, "tendon bone" begs to be confused with a sesamoid, and "articular
bone" with the subchondral bony plate.
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).
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.
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.
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'.
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.
[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.]
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?
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.
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?
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?
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.
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:
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).
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.
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.
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.
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 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).
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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).
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
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,
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.
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.
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.
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.
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.
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.
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.
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.
FUNCTIONS OF CB II
Several purposes to be served by the mineralized regions within cartilages have
been put forward.
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.
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.
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).
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).
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.
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?
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).
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.
But recent evidence based on a hydrogen-washout method suggests that this route
is blocked when the cartilage mineralizes (Ogata, Whiteside, and Lesker, 1978).
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).
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.
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.
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).
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 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.
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.
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.)
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.
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.
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.
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.
However, Youssef (1969)
expressly distinguished an anterior secondary cartilage from the anterior ends
of Meckel's cartilages in the rat.
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.
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.
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."
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.
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.
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.
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.
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.
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 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 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.
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.
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.
"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."
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).
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.
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.
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.
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.
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 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.
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."
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.
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.
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.
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.
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.
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.
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.
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.
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.
"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.
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.
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.
"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".
"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."
"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."
"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.
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."
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.
(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."
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.
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).
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).
"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).
"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."
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.
"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.
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 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.
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.
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:
The modes of ossification proposed for the antler can be summarized thus:
(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.
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.
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):
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.
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.
"Banks and Neal (1970) however, have demonstrated from ultrastructural studies,
that the differences are minimal."
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.
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.
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.
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).
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.
"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.
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.
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).
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.
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.
Fig 18
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.
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 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).
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.
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.
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.
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.
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.
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.
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.
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 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.
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 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.
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.
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 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.
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).
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.
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).
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".
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."
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.
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'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.
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.
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.
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).
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 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.
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.
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.
"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.
"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).
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).
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.
"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. . . ."
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.
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.
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.)
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.
"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.
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.
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.
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.
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.
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.
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.
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.
"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.
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. . . ."
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.
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.
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.
"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.
"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.
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.
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.
"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.
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."
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.
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.
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.
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.
"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.
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.
"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.
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."
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.
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."
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.
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.
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.
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.
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.
hyaline cartilage cell--> mesenchymal-like cell--> fibrocartilage cell, which
could constitute an indirect metaplasia.
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.
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.
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,
"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".
"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."
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.
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:
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).)
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.
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:
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)?
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.
"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."
"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."
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."
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.
"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.
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.
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).
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).
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.
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).
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.
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.
"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.
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.
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.
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.
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.
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).
"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".
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.
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.
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.
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.
"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."
"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.
"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.
"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.)
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.
"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."
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.
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.
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.
"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."
"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.
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.
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.
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.
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 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.
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.
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.
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.
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.
"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.
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.
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."
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.
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 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.
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.
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.
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.
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)).
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.
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.
"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.
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."
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."
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.
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.
"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."
"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."
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.
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).
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.
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).
"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."
"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 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."
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.
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.
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."
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."
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.
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."
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.
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.
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.
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.
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.)
Table IV.A. Circumstances of Ossification
. intramembranous, e.g., skull vault
. endoblastemal, e.g., penile bone (Ruth), clavicle (Zavisch)
. intramesenchymal, e.g., mandible, cementum
. in dermal connective tissue, e.g., reptiles (Moss)
. in ligament, and tendons
. on diaphysyal calcified cartilage
. on diaphyseal uncalcified cartilage
. at periphery of epiphyseal cartilage
. endochondral in diaphyseal cartilage
. endochondral of secondary ossification center
. endochondral in articular cartilage
. endochondral in growth plate
. endochondral in cartilage canals
. endochondral in symphyses
. endochondral in synchondroses
. endochondral in Meckel's cartilage
. endochondral in airway "permanent" cartilages (Lubosch)
. intrachondrial in otic capsule (Bast and Anson)
. perichondral sIeeve on secondary (accessory) cartilage
. endochondral in antler
. endochondral in clavicular, mandibular and other accessories
. endochondral in fracture callus
. endochondral in cartilaginous tumors
. endochondral in induced ectopic cartilage
. endochondral in elastic cartilage
. endochondral in tendon-insertion chondrifications
. endochondral in sesamoid cartilages
. endochondral in other extraskeletal fibrocartilages
. from chondroid, e.g., fish sk.ull (Moss)
. from antler chondroid bone?
. from secondary cartilages, e.g., cranial, callus, tumorous?
. from primary cartilage, e.g., reptilian epiphysis (Haines)?
. from normal dermal and ligamentous fibrous tissue
. from disturbed connective tissue (ectopic ossifications)
. by normal growth and turnover
. in response to trauma or irritation
. in response to "osteoblastic" metastases
. in response to female hormone (medullary bone)
. disturbed by hypocalcemia, vitamin A, parathyroid hormone, etc.
. by periosteum
. by endosteum
. by perichondrium
. by marrow
. by chondrocytes in situ?
. by liberated chondrocytes?
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.
* The table is derived from the text of his article.
1. Normal, and typical in its course
(a) Widespread in reptiles and amphibia, but occurring also in birds
(b) Mammalian examples:
- - in sternal ribs of carnivores and ungulates
- - where the equine vertebral rib meets the intermediate piece
- - in the end cartilage of the horse's vertebral processes
- - in the small bones of the hands and feet of mammals in general
- - in cartilage canals
- - in some apophyses, e.g., tibial tuberosity
- - in Meckel's cartilage
- - in the labyrinthine capsule
2. Normal, but not running a typical course
(a) in laryngeal and tracheal cartilages
3. Pathological
(a) in chondrodystrophy
(b) in osteogenesis imperfecta
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:
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.
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.
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.
REFERENCES
A supplementary list follows the main bibliogaphy.