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, Fi
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.