Cranio-Facial Growth in Man: Proceedings of a Conference on Genetics, Bone Biology, and Analysis of Growth Data Held May 1–3, 1967, Ann Arbor, Michigan

KROGMAN

PART I

PRELUDE

Outline

Chapter 1: Introduction to Conference

Chapter 2: Role of Growth Research at the National Institute of Dental Research

Introduction to Conference

DR. ROBERT E. MOYERS

It is my pleasure to welcome you on behalf of The University of Michigan, which is celebrating its 150th anniversary this year. This conference is one of a series of official sesquicentennial conferences.

Dr. Krogman and I have been planning this conference for two or three years, having been designated to do so by the Dental Study Section, National Institutes of Health. The conference is structured a bit differently than most such meetings, since it is intended to be a working, participating conference. Dr. Krogman will explain the modus operandi.

DR. WILTON M. KROGMAN

I would like to add a little to what Bob has said as to the genesis of this conference. To confer is, in a sense, to get together for the purpose of expressing a mutual need for clarification and an opportunity for further exposition. When you confer with somebody, you come to an attitude of both giving and receiving, the idea being that there will be a free and uncomplicated exchange of concepts, information, and goals. This suggests what Bob and I had in mind when we nodded to one another a couple of years ago. We were both on the Dental Study Section and, as we reviewed many of the applications, we realized that they were particulate, that they were isolate and, even if several were put together, there were lacunae in continuity. The idea of fragmentation caused us to turn to one another and say, in effect, Look, we must provide the opportunity for a free exchange of ideas, with a certain amount of structuring, that will given an opportunity for the presentation and discussion of information and problems at several levels.

In simplest terms, the rationale, the very spirit, of the present conference is this: that it brings together qualified men who are specialists, yet who are completely aware of the first need of careful planning, i.e. the apperception of the hypotheses to be tested; a knowledge, therefore, of an ultimate goal, the solution of which is to be sought. Secondly, thoughtful prosecution. This refers to the working out of the total program. There must be represented related steps that mesh, that integrate, that come together, so that we have a logical sequence and emergent interrelationships. There must be the cumulative development of ideas. Thirdly, we need conservative interpretation; i.e. we shall have emergent conclusions, ideas, goals, visions, that represent a careful appraisal and evaluation, so that the interpretation will be consistent with the data made available. Conservatism, therefore, in interpretation and, finally and ultimately, integrative analysis.

This explains to you the thought processes by which the conference was developed. It was emergent; it was an evolutionary process. There are three major types of papers. The first type may be termed essays, or reports, on specific areas. For example, we shall hear papers on both the ontogeny and phylogeny of cranio-facial growth, that there may be emergent patterns of development at several levels: first, a level with reference to our own kind; second, a perspective that will enable us to see in those forms of life that are antecedent to us, an emergent continuity. We shall observe the basic ground pattern that has been our phylogenetic inheritance and that through the selective mechanisms of hominid evolution have become peculiarly our own. Cranio-facial growth of Homo sapiens is, of course, the ultimate understanding that we hope to achieve; we seek the understanding of our own kind in terms of those forms of mammalian life that are peculiarly adaptable to the experimental approach, so that we may understand the phenomena that go into the total picture of human growth. We shall proceed from the ultimate structure of bone on to those much more gross phenomena of measuring, e.g. a roentgenographic cephalometric x-ray film, wherein we must be content to talk about ± half a millimeter or ± one degree of arc. We shall proceed from the microscopic by logical steps to the macroscopic, so that ultimately we may put our data together in such fashion that they relate to the problems of the clinician in the several fields or specialties of dentistry.

We then go on to what may be termed a seminar. A seminar is, or should be, a discussion between intellectual equals, grouped quite literally around a table; individuals who are not only able, but who are willing to participate, sharing of their own knowledge, pooling the knowledge already spread before them by the more formal seminarians who constitute the panels that have been set up.

We wish to emphasize that this is merely a structural dichotomy; it should not be divisive in practice or in its working-out. If a few names appear as being on a panel, this is for the priming of the pump—the lead comments, so to speak, which we hope and trust will be both provocative and evocative, i.e. an intellectual discourse between equals.

We are hoping that the final three papers will be more than a mere recapitulation: You have heard this, you have heard that: we hope for papers that not only call to our attention, in a fashion of putting together, the various strands of thought that have been woven into a pattern which we hope will eventuate in this conference. We hope to go even further and step, as it were, into the bounds of the future and say: Arising from what we have discussed in the preceding sessions are the goals and visions that we now feel are on the extended horizons, that we, all of us, will envision as a result of hearing and sharing with our colleagues. We are not going to lay before you anything, I trust, that is ivory tower, anything that is isolate, anything that is particulate, anything that is not capable of being implemented into the mainstream of thought concerning cranio-facial biology and development. If I speak with a bit of vigor about ivory towers, it is because I recall the following quatrain:

It has been ordained by higher Powers

And is a law sidereal

That those who dwell in ivory towers

Have heads of the same material.

I suggest that there are none of us who have cranial vaults of ivorine texture ! We are realists; we have worked in the field, and we realize the problems of relating our basic findings to the clinician. This is essential in bridging the gap between theory and practice, between statement and implementation, between concept and the working out of that concept in the cultural values of our world, with special reference to the healing arts of medicine and dentistry. We feel very sincerely that a fundamental integrative knowledge of cranio-facial development will be to the advantage of both dentistry and medicine, and we dedicate ourselves to that as our basic conference motivation.

DR. MOYERS

As Bill has said, this conference had its genesis in several sessions of the Dental Study Section. The Dental Study Section has sponsored several conferences which have been singularly successful, e.g. the conference on salivary gland physiology. The hope was that, by having a conference on cranio-facial growth, we, in a similar fashion, might recharge and redirect the energies in the several laboratories around the world working in the field to new lines of effort.

There are two kinds of people here: those who are already working in the cranio-facial area, and those who are not. In each category there are three subtypes: (1) bone biologists, (2) geneticists, and (3) those expert in the analysis and treatment of growth data. So we have group A, those already trying to solve the problems of the face (bone biologists, geneticists, and analysts); and group B (the biologists, geneticists, and analysts) who do not know much about the face yet, or at least haven’t yet had any academic interest in the face. The basic idea of the conference is to mix groups A and B.

Since the idea for the conference started in the Dental Study Section, it is appropriate that a person from the National Institute of Dental Research make the first presentation, though the conference is jointly sponsored by the Institute of Dental Research and the Institute for Child Health and Human Development. Now Dr. Kenneth Hisaoka, who is in charge of growth and development programs at the National Institute for Dental Research, will present his report on The role of growth research at the National Institute of Dental Research.

Role of Growth Research at the National Institute of Dental Research

K. KENNETH HISAOKA*

Publisher Summary

This chapter discusses the role of growth research at the National Institute of Dental Research (NIDR). Cleft lip/cleft palate make up 13% of all the reported birth anomalies and are among the most common defects at birth. The absence of an intact surface between the oral cavity and the nose in the cleft-palate child causes difficulty in chewing and swallowing, affects hearing and speaking, and influences the social behavior of the child. Successful rehabilitation of the cleft-palate child requires the solution of complicated clinical problems through the coordinated efforts of many specialists, including orthodontists, prosthodontists, growth researchers, plastic surgeons, otolaryngologists, psychologists, speech therapists, and social workers. Currently, a major focus for cleft-palate studies supported by NIDR is directed to the development and evaluation of surgical repair techniques, ranging from simple flap operations through autotransplants of bone and cartilage and the insertion of teflon implants. Some ongoing research programs relevant to cranio-facial growth and development are being supported by NIDR. Research in cranio-facial biology which contributes towards an accurate prediction of normal and abnormal incremental growth will be encouraged, because this type of study should aid greatly in advancing the practice of orthodontics.

IT IS indeed a pleasure to have the honor of addressing this distinguished group on the interest of the National Institute of Dental Research in cranio-facial growth. As a background for my report, I would like to refer briefly to the reorganization of the Extramural Programs of the NIDR, since I believe that it will have a definite impact on research in cranio-facial growth. Originally, the extramural programs of the NIDR were administered in two broad sections: namely, research grants and training grants. Under the leadership of Dr. Seymour Kreshover, Director, NIDR, these two components were dissolved and five major program areas were formed. Each program area was designed to encompass both research and research manpower support in a specified research category. I have been given the responsibility for administering research grants, training grants, and fellowships in the program area entitled Oral-facial growth and development.

The new organizational structure of the extramural programs of the NIDR allows the scientist administrators of each program area to give greater visibility to a circumscribed area of research and permits them to relate to a specific segment of the research community. It allows the scientist administrators of each program to analyze and review past support in a prescribed research area and to ascertain the state of the art from time to time. The program approach is useful from the standpoint of planning, developing, and catalyzing research, and it permits the program leaders to focus their attention on specific research areas. However, I would hasten to point out that the areas of research which are highlighted by NIDR are not selected in an arbitrary fashion. Scientific peers from the academic community are consulted from time to time so that proper guidance is made available to each program area. In this connection the NIDR anticipates that the deliberations during this conference will serve to advise NIDR of exciting new areas of research which should be encouraged.

The Oral-facial Growth and Development program encompasses a rather broad area. It covers normal and abnormal growth and development of the cranio-facial region and of other related growth components. More specifically, this program includes research in normal or abnormal development relating to the cause, prevention, correction, and rehabilitative aspects of oral-facial malformations such as cleft palate or malocclusion. In addition, studies in physiology, including neurophysiology, relating to the oral-facial area, will be supported by this program.

The expenditures for research support will indicate the relative size of this program at the present time. In fiscal year 1967, approximately $3,000,000 will be expended in support of 70 research projects, including eight program projects, by the Oral-facial Growth and Development program. Included in this sum is approximately $1,000,000 in support of 35 projects relevant to cranio-facial growth.

NIDR’s Interest in Cranio-facial Research

Although the reason for NIDR’s interest in cranio-facial research is apparent to the majority of readers, I shall try to express some viewpoints on this subject since I have been specifically requested to do so by Dr. Moyers.

Cleft lip/cleft palate make up 13 percent of all reported birth anomalies and are among the most common defects at birth. Today, over a quarter of a million persons in the United States have some form of oral cleft and 6000 babies will be born with this abnormality during 1967. The absence of an intact surface between the oral cavity and the nose in the cleft-palate child causes difficulty in chewing and swallowing, affects hearing and speaking, and influences the social behavior of the child. Successful rehabilitation of the cleft-palate child requires the solution of complicated clinical problems through the coordinated efforts of many specialists, including orthodontists, prosthodontists, growth researchers, plastic surgeons, otolaryngologists, psychologists, speech therapists, and social workers. Currently, a major focus for cleft-palate studies supported by NIDR is directed to the development and evaluation of surgical repair techniques, ranging from simple flap operations through autotransplants of bone and cartilage and the insertion of teflon implants. How-ever, since surgery does not always insure adequate function, the NIDR is supporting studies which seek to develop appropriate diagnostic criteria which will enable the cleft-palate team to choose the most effective treatment procedures for an individual patient.

Also, studies into the etiology and prevention of congenital oral-facial anomalies comprise an important part of the Dental Institute’s program in this area. Improper growth and development of oral-facial structures result in defects, ranging from the grotesque deformities of cleft lip/cleft palate, through gross malrelationships of the teeth and jaws, to malocclusions which are a major predisposing factor to dental caries and periodontal disease. Malposed teeth and jaws range from relatively insignificant deviations to severe and disfiguring malrelations of the teeth, jaws, and face. Malocclusion may be due to hereditary factors. However, poor tooth position and failure of the upper and lower teeth to meet properly upon closing of the jaws may be caused by other factors, such as missing teeth, extra teeth, abnormal frenum, tongue thrust, or thumb sucking.

Actually, little is known regarding the relative role of heredity and environment in regard to malocclusion. We recognize that current research in malocclusion has not yet produced findings which will bring about less costly orthodontic procedures and establish simple methods whereby malocclusion in children of lower income groups may be treated at low cost. In initiating a new thrust in the programming of new research grants in the etiology of cleft palate and malocclusion, particular interest will be given to research grant applications which propose the use of appropriate sub-human primates as the experimental animal so that a more meaningful extrapolation of data may be made to man.

Research Projects related to Cranio-facial Growth

At this time, I will describe briefly some ongoing research programs relevant to craniofacial growth and development which are being supported by NIDR. At present, major attention is directed to the postnatal factors which contribute to malocclusion, such as oral muscle forces, oral habits, and nutrition. A clinical study involving Indian children is evaluating a treatment procedure whereby a developing malocclusion resulting from crowding of the teeth is halted by selective extractions to provide proper spacing. Epidemiological studies are under way to derive a direct and reproducible method for determining the size and shape of dental arches, to develop criteria to assess the extent and severity of occlusal anomalies, and to elucidate possible relationships between the factors under study.

Other investigations seek to clarify the relative role of genetic and environmental factors in malocclusion and to determine the effect of malocclusion on facial growth. Another laboratory program has been engaged in studies of facial and dental growth in twins and their siblings. This investigation should contribute valuable information concerning the influence of genetic and environmental factors on individual growth patterns. In another study, the family-line determinants of dental development in a complete family-line context are being investigated. This study is also expected to yield significant data concerning genetic mechanisms involved in cranio-facial development.

Research Areas of Interest to NIDR

New imaginative studies utilizing the specialties of genetics, physical anthropology, and orthodontia are needed to provide information concerning the normal longitudinal growth and development patterns of the cranio-facial region. Such knowledge is requisite to the early diagnosis of developing malocclusion. When this base of information is determined, clinical orthodontic research programs will be needed to develop those practical techniques which will intercept the malocclusion and thereby significantly reduce, or hopefully eliminate, the need for corrective therapy. At the same time, we recognize that progress has been made in studies utilizing cephalometrics to indicate a potential application of currently available skills and knowledge which would result in the development of the needed diagnostic indices for the treatment of malocclusion. A number of research areas have been highlighted for program relevance. For example, longitudinal and comparative investigations in the area of physical anthropology to study cranio-facial growth and development will be initiated to provide the necessary correlation between dental age, chronological age, and maturity. Support will be given to basic studies on growth and development which are relevant to the treatment of malocclusion in otherwise normal and malformed individuals such as those with cleft palate.

Studies on the little understood interaction of genetic and environmental factors on the growth of cranio-facial structures will be expanded. Special emphasis will be given to studies of the growth and development of the cranio-facial region from conception to 3 years of age. Epidemiological studies in search of causative factors will also be programmed, and research which will lead to the standardization of clinical diagnostic criteria for malocclusion will be encouraged. These criteria will be useful in appraising the need for orthodontic treatment as well as in developing population studies designed to identify causative factors. Research in cranio-facial biology which will contribute towards an accurate prediction of normal and abnormal incremental growth will be encouraged, since this type of study should aid greatly in advancing the practice of orthodontics.

Research Manpower Needs

The ultimate success of any comprehensive research program in oral-facial malformations, such as cleft palate or malocclusion, is dependent upon the availability of highly trained manpower in these areas. Currently, the Oral-facial Growth and Development program is supporting 36 training programs at a level of $1,900,000, ranging from biochemistry to speech pathology. This program is also supporting 19 research fellowships at a level of $179,000 and 11 research career development awards totaling $197,000. We recognize the need to develop more training programs, especially in genetics, embryology, physical anthropology, and research orthodontics. We hope that a continuing increase in support of training programs will be possible in the next fiscal year.

Conclusion

In closing, I would like to say that we anticipate that this conference will produce insights into future needs in research in genetics, bone physiology, and statistical analysis as they relate to cranio-facial growth. We are confident that this meeting will yield a critical assessment of the limitations of current studies of normal subjects as well as those with facial deformities. A reevaluation of present concepts and the assessment of methods currently used in the study of cranio-facial growth should provide a guide for future research. Further, this conference will serve to focus the attention of leading investigators in disciplines pertinent to cranio-facial growth on those complex problems which are timely for productive investigation. The definition of the most fruitful areas for future growth research of the head and face will help to give new guidance to the NIDR in terms of growth and development research.

*Chief, Oral-facial Growth and Development Program, National Institute of Dental Research, Bethesda, Md.

PART II

THE BIOLOGY OF BONE

Outline

Chapter 3: Introduction

Chapter 4: The Mechanisms of Cartilage Growth and Replacement in Endochondral Ossification

Chapter 5: Bone Morphology and Mineral Homeostasis

Chapter 6: Load and Electric Charge Relationships for Cortical Bone

Chapter 7: Comparative Ontogeny and Cranio-facial Growth

Chapter 8: The Modes of Growth of the Neurocranium: The Growth of the Sphenoid Bone in Animals

Chapter 9: Vertical Development of the Face — A Summary

Chapter 10: Ontogenetic Aspects of Cranio-facial Growth

Chapter 11: Some Characteristics of Cranio-facial Growth Cartilages

Introduction

THIS section consists of two parts: basic bone biology and the biology of cranio-facial bone and bones. In the papers of Anderson, Dullemeijer, McElhaney, and Yaeger we look at certain basic aspects of bone biology, both phylo- and ontogenetically. Here we come to grips with underlying processes at cellular level and onwards. This is the working-out of what we later measure in the growing head and face. The cranio-facial complex has bone and bones as its basic structure. Muscles, vessels, nerves, and organs (as the eyes) are, in a sense, an overlay of soft tissues, whose relation to the subjacent bony skeleton makes possible function. The two—structure and function—give form. The reports and discussions point out the important role of cartilage in bone growth and lead directly into one of the liveliest topics of the conference—the determinants of the form of bone. The relative roles of genetic controls and function in deciding form were strongly debated. The mode and site of gene action, electrical phenomena in growing bone, and the lessons from comparative and evolutionary biology were other recurrent themes. The papers of Hoyte, Bjork, Moss, and Koski relate basic bone biology to the specifics of cranio-facial growth. The reports and conference deliberations on bone biology constitute Part II, and while interesting and fruitful in themselves, also set a good stage for what follows. Our knowledge of bone growth in the cranio-facial complex is firm and sure and well related to the exciting advances going on in the general field of bone biology

The Mechanisms of Cartilage Growth and Replacement in Endochondral Ossification

CARL ANDERSON

Publisher Summary

This chapter reviews the process of cartilage tissue preparation for its invasion and eventual replacement, and the immediate subsequent events, mentioning some of the modifying factors. The processes of endochondral ossification at the electron microscope level are discussed in the chapter from the morphological standpoint. These processes are dependent upon the complex interactions of many genetic, cellular, and environmental factors. Understanding these processes and the factors that modify them makes it possible, within certain limits, to control and deliberately alter the skeletal growth process. In the orderly process of endochondral ossification, the invasion process moves along as a straight-line advancing front; therefore, both sides of a cartilage wall are frequently covered with bone simultaneously. This forms a bone and cartilage sandwich known as the primary bone trabecula. In the normal process of endochondral ossification in mammals, we do not see fully developed chondrocytes becoming osteoblasts, or anything else. They invariably mature, degenerate, and die. In tissue culture and in other areas, sometimes in wounds of various sorts, one can see cells that have apparently moved into a chondroblast stage. But under the influences of different oxygen tension and other environmental factors, they revert to other forms. However, there is a critical point in differentiation beyond which one cannot change the path along which the individual cell is going.

ABOUT 10 or 15 years ago, veterinarians at the California Agricultural College, now a liberal arts college at Davis, were interested in some dwarf cattle and brought down to our orthopedic pathology laboratory at the University in San Francisco sections of sphenoccipital synchrondroses from these dwarf cattle that were apparently closing prematurely, and asked us to do histochemical and other studies on them. The first thing we found out when we looked at these synchondroses was that we didn’t know anything about cartilage growth, maturation, or endochondral ossification. This was the beginning of a study that we conducted for a number of years.

With the exception of some of the membrane bones of the head, the bony skeleton is preformed in cartilage. Skeletal cartilage enlarges, generally, in accordance with genetically determined plans. The growth in size of the skeleton is due to the production of extracellular matrix, by chondrocytes, and by increasing numbers of chondrocytes as a result of mitotic division and a differentiation of the primitive mesenchymal cells into cartilage. As the chondrocytes complete their life cycle, which may be as brief as 10 days in some instances, they undergo degeneration and the matrix surrounding them becomes partially calcified. At this particular point in skeletal development, this tissue is invaded by a complex of capillary endothelial cell projections, mononuclear macrophages, and partially differentiated cells destined to become either bone-forming osteoblasts or bone- and cartilage-destroying osteoclasts. These processes of chondrocyte maturation, matrix production, mineralization, invasion, bone formation, and remodeling are subject to alteration by a variety of factors which may retard, may accelerate, or may distort any of these processes, resulting in various types of deformity. Such factors may be genetic, nutritional, hormonal, toxic, or mechanical. As a result of understanding the processes of endochondral ossification and the factors which modify it, it is possible, within certain limits, to control and deliberately alter the skeletal growth process. In this presentation I shall trace briefly the process of cartilage tissue preparation for its invasion and eventual replacement, and the immediate subsequent events, mentioning some of the modifying factors.

Many of the chondrocytes of cartilage, destined to be replaced by bone, persist throughout the growth period in a resting or equilibrium state until some stimulus, as yet poorly understood, causes them to undergo a rapid, yet orderly increase in activity and growth which proceeds within a few days to their degeneration and death. The stages in the chondrocyte’s life cycle are designated as resting, proliferating, hypertrophic, degenerating, and then the calcified state. This picture (Fig. 1), I am sure, is familiar to all of you. The resting, or quiescent, chondrocyte may persist for many years, exerting just enough cellular metabolic activity to maintain a balance between matrix synthesis and matrix degradation. This relatively inactive chondrocyte with a small amount of cytoplasm to nuclear size indicates its relatively quiescent state. In the proliferating or multiplication phase, individual cells undergo division in a spatially oriented manner to form columns of flattened cells. Matrix synthesis, as indicated by greater cytoplasmic activity, is increased during this stage, and the entire mass of cartilage enlarges. In the maturing or hypertrophic phase, formation of cartilage matrix proceeds at a rapid rate. This is indicated by cytoplasmic enlargement with large numbers of very active organelles. The length growth of the cartilaginous skeleton is most rapid in this phase, due to the large amount of matrix synthesized and also to enlargement or hypertrophy of the cells themselves. The final stage is that of degeneration and death of the swollen chondrocytes. During this stage (Fig. 2) the matrix surrounding the cells normally undergoes partial mineralization to form the zone of provisional calcification. These stages of chondrocyte maturation and matrix change occur in any situation where cartilage is replaced by bone. They are most readily identified in the epiphyseal cartilage plates of the long bones. It might be of interest to consider briefly some of the conditions which interfere with normal chondrocyte maturation and result in growth disturbances. A few samples only will be mentioned.

FIG. 1 Resting chondrocyte.

FIG. 2 Degenerated chondrocytes and calcifying matrix.

Achondroplasia

This is a genetic disorder in which chondrocytes proliferate, forming columns of flattened cells, but appear to be incapable of maturation and hypertrophy in an orderly fashion. The matrix calcifies very irregularly, resists invasion by the elements from the metaphysis, and thus cannot be invaded and replaced by bone.

Rickets

The nutritional disturbance in rickets causes no interference in chondrocyte maturation. It goes along in a perfectly logical, normal way, but, because of the unavailability of adequate calcium or phosphate, mineralization cannot occur, the invasion of chondrocyte columns is prevented, and large numbers of hypertrophic chondrocytes accumulate in greatly elongated columns. Cartilage mineralization is a prerequisite to vascular invasion and cartilage replacement.

Cortisol administered to growing animals markedly depresses matrix formation, resulting in thin cartilage plates and marked reduction in the number of hypertrophic cells. Continuation of Cortisol administration can result in severe dwarfism in addition to other better-known abnormalities.

The matrix, which the chondrocytes produce and in which they are protectively embedded, consists of 85–90 percent water, bound tightly, in the form of a firm gel, by complexes of mucopolysaccharides, usually chondroitin sulfate complexed with non-collagenous protein. These complexes are aggregated in macromolecules with molecular weights of from 1 to 10 million, producing a tremendously hydrophylic, firm gel. Embedded within this gel are many long thin collagen fibrils. In young animals and in human infants, these collagen fibers range from 50 to 200 Ångstrom units in diameter, much smaller than the classical collagen fiber we see in bone or tendon. In the resting zone of epiphyseal cartilage, the collagen fibers are arranged in a more or less random orientation forming an interlacing lattice, sometimes referred to as feltwork. However, in the walls of cartilage matrix separating the columns of chondrocytes in the zone of hypertrophy and degeneration, the fibers assume a discernible orientation with their long axes parallel to the direction of growth.

It is within these longitudinally oriented walls of cartilage matrix, and to a lesser extent in the transverse partitions between individual chondrocytes, that calcification or mineralization occurs. The zone of calcified cartilage is normally about 100 micra in width, so the mineralized matrix surrounds just the last two or three rows of generated chondrocytes.

As seen in the electron microscope, using standard techniques of fixation and embedding, the first indication of cartilage mineralization (Fig. 3) occurs as a dense, formless cloud surrounding some of the collagen fibers. Shortly thereafter, apatite crystals measuring approximately 20 × 200 Å in size appear within this cloud, on or immediately adjacent to the collagen fiber. The nature of the amorphous cloud which precedes the appearance of crystals has been a matter of concern and interest to investigators of the calcification mechanism. Urist and others have postulated that a non-crystalline calcium phosphate phase precedes apatite crystal formation in the mineralization process. There is much experimental evidence to support this two-phase concept.

FIG. 3 Initial stages of mineralization of cartilage matrix.

After the first appearance of crystals in or near the collagen fibril, other crystals soon appear in an orientation resembling spokes radiating out from the fibril, to form clusters or rosettes (Fig. 4).

FIG. 4 Mineral clusters in calcifying cartilage matrix.

These crystals are added peripherally until much of the space between the collagen fibers becomes densely mineralized. It should be noted that many centers of mineralization are present in a given septum or wall of cartilage matrix and, while they tend to coalesce, it is infrequent that the entire septum becomes solidly calcified. In the transverse partitions between individual chondrocytes, mineralization is virtually always spotty and