Scanning Electron Microscopy of Cartilage*

5 Scanning Electron Microscopy of Cartilage* Alan Boyde Sheila J. Jones

I. Introduction II. Examination of Cartilage in the SEM: Previous Studies and Techniques A. Articular Surfaces B. Internal Surfaces C. Tissue Interfaces D. Chondrocytes E. Matrix Vesicles F. Cartilage Matrix G. The Mineral Phase of Cartilage III. Present Study A. Embryonic Cartilage B. Permanent Cartilages C. Skeletal Cartilages: Long Bone Epiphyses D. Cartilage and Intervertebral Joints: The Disc E. The Synchondroses F. Secondary Cartilages G. Dental Cartilage H. Chondrocytes in Culture I. Measurement of Shrinkage of Cartilage References

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I. INTRODUCTION Cartilage is a term used for a continuum of connective tissues whose basic and constant component is the cartilage cell surrounded by a territorial matrix that it has secreted. The extraterritorial matrix varies in origin, constitution, quantity, and arrangement and largely determines the difficulty or ease experienced by the scanning electron microscopist in his attempt to study the cartilage and the method used to prepare the tissue. Most biological specimens for SEM have to T h i s work was supported by grants from the Medical Research Council and the Science Research Council. Cartilage, Volume 1 Structure, Function, and Biochemistry

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Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-319501-2

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be fixed and dehydrated, and this process may substantially alter both their shape and their size. The component parts of a connective tissue probably shrink differently, distorting their relationships. Cartilage, with its high water content (up to 80%) and its variable and inhomogeneous structure, thus presents pitfalls both in specimen preparation and in subsequent interpretation of structure. Never­ theless, its study by SEM is particularly rewarding because of the ability to isolate chosen components and uncover internal as well as external surfaces. Cartilage has been classified historically according to the dominant component of its matrix—thus fibrocartilage, elastic cartilage, and hyaline cartilage (see Chapter 1 in this volume). Additionally, it has been considered either permanent and uncalcifying or temporary and calcifying. As in most classifications of biological structure these are near-truths, for some cartilages have matrices that are intermediate in composition and any permanent cartilage may mineralize. Therefore, the techniques enlisted by the scanning electron microscopist in the study of cartilage must be adequate to present or to isolate any or all of the possible components.

II. EXAMINATION OF CARTILAGE IN THE SEM: PREVIOUS STUDIES AND TECHNIQUES Most SEM studies on cartilage have aimed at characterizing the microscopic topography of the articular surfaces of synovial joints (Clarke, 1971a,b, 1973; McCall, 1968; Ghadially et al., 1978b; Gardner and Woodward, 1969) and the fibrillar architecture of articular cartilage (Bullough and Goodfellow, 1968; Clarke, 1971c, 1974; Mow et al., 1974a,b; Okuda, 1970; Redler et al., 1975). Fibrocartilage (Inoue, 1973; Cameron and MacNab, 1972, 1973) and elastic cartilage have not received the same attention, the latter by any method (Cox and Peacock, 1977). There has been an awareness that the methods of specimen preparation used may significantly alter the real and manufactured surfaces stud­ ied (contrast Inoue et al., 1969, Draenert et al., 1978, 1979, and Cameron et al, 1976). A. Articular Surfaces The only free natural surface of cartilage is the articular surface of a synovial joint. The structure and integrity of the tissue surface are of great clinical im­ portance, but determining its exact form is not easy. First, the articular surface is load-bearing and will change shape under different loads (Wright and Dowson, 1976; McCall, 1969; Frost, 1979). Second, not all the surface at the articular head of a bone will necessarily bear a load or an equal load during movement of the joint (see Chapter 3 in Volume 3). Most studies have concentrated on examining the surface of the cartilage when unloaded. The joint, dissected entire, is opened, the articular surface washed

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free of synovial fluid, and the articular cartilage is fixed in situ. In this way, the external surface of the cartilage is revealed and the cartilage remains supported by the underlying bone. Failure to retain this support may produce serious artifactual distortion (Gardner et al., 1981; Ghadially et al., 1976, 1977a,b). Specimens are then washed and dried using either air drying from a volatile solvent, critical point drying (CPD), or freeze-drying (FD). One drawback in such standard SEM preparative techniques for cartilaginous surfaces is that there is a large soluble matrix component which may be wholly or partially removed during washing or fixation. The amount of this component is variable in different cartilage types and ages (Maroudas et al., 1980). Fur­ thermore, the shrinkage that occurs on any dehydration of cartilage (Anderson and Sajdera, 1971; Gardner and McGillivray, 1971) cannot be prevented with either of these SEM drying methods. Shrinkage is greater with critical point drying than in freeze-drying (Boyde et al., 1977), and the greatest distortion might occur at a free surface. Arguably, such shrinkage may not matter for some studies where one wishes to contrast pathological or abnormal surface structure with the normal condition (Redler, 1974; Redler and Zimny, 1970; Puhl and Iyer, 1973). If, however, the goal is to characterize the form of the living unstressed normal joint surface, then improved preparative methods are essential (Draenert and Draenert, 1981). Considerable controversy exists as to whether this surface is perfectly smooth in healthy, young, normal individuals—it cer­ tainly is not in dried samples (Clarke, 1971b, 1972, 1973; Gardner, 1972; Ghad­ ially et al, 1976, 1977a,b, 1978b; Puhl, 1974; Zimny and Redler, 1974a). The examination of frozen samples eliminates several steps at which shrinkage and distortion might occur (Gardner et al, 1981; Draenert and Draenert, 1981). Joint surfaces in experimental animals frozen instantly upon exposure are perfectly smooth when examined frozen in the SEM (see Section II,C). Split-line patterns on articular cartilage surfaces have also been studied by SEM. The directions of the long axes of the oval slits made by piercing the surface with a round awl have been compared with the orientations of the collagen fibers in the superficial layers (Clarke, 1971c; Bullough and Goodfellow, 1968). However, Mow and his coworkers (1974a,b, and Roth et al, 1979) report that no good explanation has been found for the slits. Scanning electron microscopy has also been used to monitor the effects of experimentation on the articular surface (Puhl et al, 1971). Walker and his collaborators (1969, 1970) developed an interesting experimental approach for mimicking in the laboratory the conditions on the surface of the joint during loading and friction. The dried fluid film over the articular surface was examined by SEM, and the results analysed to show how the synovial aggregates might form a protective skin over the cartilage. Another aspect of the changes of cartilage in function has been examined by Ghadially et al. (1974). Cartilage was shaved from the articular surface of rabbits, and the remodeling of the defects after 6 months was studied with SEM. A new fine-textured material

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obliterated the exposed chondrocyte lacunae, and the topography of the surface observed was suggestive of cartilage flow during load bearing and joint move­ ment. The effects of in vivo short-term loading on the configuration of the joint surface have also been investigated with SEM (Gardner, 1972) but the description of the finer detail of the joint surface probably reflects drying artifacts. Replication of the bearing surface of human joints (Clarke, 1973) has been carried out but Clarke queried its value because the replica material might have altered the surface contours. The most extensive use of SEM to describe pathological changes in cartilage has been in the study of the degenerative changes that occur in articular cartilage in rheumatoid arthritis adjacent to the pannus (Inoue et al., 1969, 1971; Redler et al, 1970, 1972; Puhl et al, 1973; Richter, 1972). Observations have also been made of the integrity of the joint surface (Zimny and Redler, 1972a), the degree of breakdown in the fibrillar component of the matrix (Enna and Zimny, 1974), and the appearance of the cartilage and subsequent bone exposure in osteoarthrosis (Redler, 1974, 1975). SEM contributes to such studies because one can survey large surfaces, and it enables one to map the sites and degrees of degeneration (Gaucher et al, 1977; Ohnsorge et al, 1970). B. Internal Surfaces The internal surfaces of cartilage are the lacunar walls and the walls of vascular channels where these are present (Kugler et al, 1979; Lutfi, 1970). Unlike the situation in bone (Boyde and Jones, 1972), partly formed lacunae are not seen on the free surface of the undamaged tissue because the chondroblasts are not polarized in their matrix production in the same way as osteoblasts, although they usually show a gradient of differentiation. Hence, one would not expect to see, for example, a structural difference in the fibrillar architecture of the firstand last-formed wall like that present in the lacunae of lamellar bone (Boyde and Hobdell, 1969). Therefore, chondrocyte lacunar walls and the channels for blood vessels must be revealed by splitting, sectioning, or freeze fracturing the cartilage and viewing the ruptured lacunae and channels. C. Tissue Interfaces With the exception of the articular surface of cartilage in synovial joints, cartilage has an interface on all aspects. This is generally with another connective tissue, either the perichondrium or the subchondral bone. In mammals the only variation from a connective tissue interface is found in cavian teeth where the interlophal packing cementum is a vascular calcified cartilage laid down on enamel, an ectodermal secretion. The interface in this situation is readily identified, and the sequence of the buildup of cartilage on the enamel surface may be studied in the continuously growing molars (Jones, 1973; and see Section III,G). No other interface is so sharply demarcated. It is difficult to say histologically at what point during appositional growth the

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cells of the perichondrium have reached a stage of differentiation warranting the title chondroblasts, and, hence, it is not possible to accurately prepare the in­ terface for examination by SEM or any other method. The interface with bone is also continuous and does not form a natural fracture line, although at sites of endochondral ossification where bone will be laid down directly on the calcified cartilage matrix, the template is open to view on a sequential basis rather like the situation in cavian teeth. There is no continuity of fibers across the carti­ lage-bone interface, and the extent of mineralization of the two tissues is dif­ ferent, the boundary being marked by a cement line. Cartilage is relatively poorly mineralized compared with bone, but to use these differences to define the interface with SEM requires their selection from the whole tissue by other meth­ ods (Hough etal., 1974). One interface within cartilage that has failed to attract attention from scanning electron microscopists is the irregular border or tidemark between calcified and uncalcified cartilage, particularly at the articular head of a bone. Collagen fibers pass from one layer to the other (Bullough, 1981). Unlike the boundary between the two calcified tissues, cartilage and bone, the junction between uncalcified and calcified cartilage is relatively weak mechanically. It may be exposed as a mineral surface following the removal of the unmineralized matrix. D.

Chondrocytes Cell density varies between cartilages of different types, within cartilage, and with age (Stockwell, 1971, 1979; Maroudas et al., 1975). Chondrocytes are exposed to view when cartilage is torn, cut, or fractured open and can be examined in situ using SEM. In some cases, the chondrocytes are lost from the plane of tear or fracture and may be damaged (Zimny and Redler, 1972b; Clarke, 1974). The advantage gained from better preservation of cells exposed imme­ diately to the fixative has to be weighed against the damage caused during their exposure and their subsequent change in shape. Cells that are fixed will suffer less distortion and traumatic change, but they may have been altered during the slower fixation time involved because the fixative will first have to pass through the intercellular matrix. The greatest problem with examining chondrocytes in situ is undoubtedly the shrinkage of the cell itself (Zimny and Redler, 1974b; Schneider et al., 1978) and the enlargement of the lacuna during shrinkage of the cartilage matrix (Laczko et al., 1975). Thus, it is difficult to be sure of the fit of the cell to the lacuna, and the existence or otherwise of a pericellular space cannot be assessed by these simple means. Soluble organic material within the lacuna may also have been lost (Clarke, 1974). In terms of practical SEM methods, the best fit of cells to lacunae has been obtained with freeze-fractured, freeze-dried, fresh cartilage (Draenert and Draenert, 1979, 1981). Better, in theory, would be to examine this tissue when freeze fractured but still fully hydrated. Retaining water as cold ice, however, presents several practical prob­ lems. There is little or no usable contrast to determine location in frozen hydrated

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specimens. Superficial freeze-drying must, therefore, be allowed to occur; it is, in any case, difficult to prevent or control. The water in the tissue must become ice, which, as it crystallizes, creates a new internal structure in the tissue. Solutes and delicate solid structures are pushed aside to occupy the spaces at the peri­ pheries of the ice crystals. Except in the superficial few micrometers of a tissue fragment frozen with the minimum amount of superficial water, fine structure may be drastically altered. In the case of cartilage, this certainly occurs within the chondrocytes and poor preservation of intracellular detail is well illustrated in the work of Draenert and Draenert (1979, 1981), but it may also occur within the extracellular matrix, and it is not certain that accurate portrayals of the fine structure have yet been produced. More positively, rapid freezing is not known to produce significant dimensional or shape changes in cartilage, and the most faithful representation of the original shape will follow from the examination of the frozen specimen. Chondrocytes for cell culture are generally isolated from cartilage by enzymatic digestion of their intercellular matrix with, for example, hyaluronidase and tryp­ sin followed by collagenase, or by trypsin and collagenase (von der Mark et al., 1977), or collagenase alone (Ali, 1979). Such a separation nets a large number of cells, but the chondrocytes originate from different levels and may be in different stages of development (Kincaid et al., 1972). There is an obvious advantage gained when the cells are retained in their own matrix, because their degree of differentiation can be assessed by their location. SEM studies of the cell surface changes of chondrocytes following isolation and culture are lacking. There has been a great deal of interest, highlighted by the meticulous studies by Simmons (1962, 1974, 1979, 1980), in the circadian rhythms of articular, epiphyseal, and elastic cartilages. Simmons has found that these rhythms differ, the articular cells responding to function with a rhythm like cortical bone, and the epiphyseal chondrocytes to growth hormones. It is obviously important to consider the alterations in cells that may occur over the day or night and to be aware that the appearance of the chondrocytes may differ simply because of the time of harvesting the specimen. As far as we know, no SEM studies have been conducted to look for morphological changes over a daily period. Although chondrocytes may be separated readily from their matrix and har­ vested, the separation procedures involve interfering with the cell surface chem­ istry and, hence, probably with the internal chemistry. Not only that, the selection of cells at different stages of development or differentiation must necessarily be inexact whether the selection be by sequential digestion or slicing the tissue. When live chondrocytes are not required, it is possible to dissect out individual chondrocytes from cartilage matrix from a known and recorded position using real-time 3-D SEM (Boyde, 1974). If fresh frozen cartilage is used, it is possible to collect individual (half) cells, characterize their vacated lacunae, and subject the cells and adjacent matrix to elemental analysis. The cells are speared on a tungsten needle attached to a micromanipulator fitted to the SEM and transferred to a collecting surface, such as a Formvar coat on a grid (Boyde and Shapiro,

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1980). Alternatively, each cell may be temporarily retained on the needle for further investigation. Scanning electron microscopy has not been used to any great extent to assess the effects of experimentation on chondrocytes. Chondrocytes are generally either isolated and cultured away from the matrix or cultured within small blocks of cartilage. Most experiments with cartilage cells have aimed at measuring bio­ chemical changes in the cells or the cell products or rate of production. The attention paid to the morphological appearance of the cell has been due to the likelihood that this might alter with modulation in the phenotype to a more fibroblastic cell producing type I collagen (von der Mark et al., 1977). In mixed cultures, Vertel (1976) found that cells dissociated from limb buds became stratified, with chondrocytes below myocytes, and a cartilage matrix with type II collagen was produced. The extent of intercellular contact and the degree of the dispersal of the cells within the medium is of great importance. Cultures of pure chondrocytes have not been studied by SEM. SEM should be an ideal tool to look for cell surface changes with alterations in hormonal and chemical levels in the environment because of its sensitivity. Any change in configuration of the surface can be seen, and the extent and shape of the cells can be measured either directly from SEM images by hand or with an automatic image analyzer such as the Quantimet 720. Cartilage cells are extremely sensitive to the amount and constitution of the matrix surrounding them (Miller, 1977), and Muir (1977) has suggested that chondrocytes in culture may be in a chronic state of repair, trying to surround themselves once again with normal matrix. The type of collagen made by chon­ drocytes in culture and, it is thought, in osteoarthritis is altered because of changes in or loss of the surrounding matrix (Miller, 1977). It also should be possible to assay the chondrocytes, whether isolated or retained on their own substrate, for receptors for, for example, somatomedin or parathyroid hormone, using techniques similar to those employed in searching for Fc receptors on osteoclasts using SEM (Hogg et al., 1980). Chondroclasts have rarely been visualized with SEM. Savostin-Asling and Asling (1975) examined the erosion front of calcified cartilage and observed some large cells spanning several chondrocyte lacunae. The main cell body was some distance from the erosion front to which a thin leading edge of cytoplasm extended. This is a good example of the usefulness of SEM in allowing the extent of whole cells to be known (a tedious and exacting task with TEM) and very thin cytoplasmic extensions to be resolved (an impossibility with light microscopy). E. Matrix Vesicles Matrix vesicles are small structures, about 100 nm across, bounded by a trilaminar membrane rich in alkaline phosphatase. They have been observed, using TEM, associated with chondroblasts in calcifying cartilages (Ali and Grif­ fiths, 1981) and with the formative cells of dentine, immature bone, fracture

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callus cartilage (Ketenjian and Arsenis, 1975; see Chapter 12 in this volume for a discussion), and osteosarcoma. In these calcifying tissues matrix vesicles pre­ cede mineral deposition and are thought to initiate the process. It is possible to isolate the vesicles from cartilage for biochemical analysis and TEM (Ali, 1979; Kahn et al., 1978; Anderson, 1976), but the isolates have not been studied with SEM, perhaps because it would not appear to be useful. Some authors have used the terms matrix vesicles and calcifying globules synonymously (Ornoy et al., 1976a,b; Ornoy and Langer, 1978), but this causes confusion. It is difficult to be sure that small spherical blobs on the side of a fixed cell are matrix vesicles (Ornoy et al., 1976a, Ornoy et al., 1981) and not blebs due to the fixation process. Some cells will bleb when their surrounding fluid environment is changed or even agitated (Vesely, 1981), and the more active a cell, the more sensitive it is to disturbance. Extracellular vesicles have also been found in association with elastic cartilages (Nielsen, 1978; Cox and Peacock, 1977) but were not thought to be a focus for mineralization in this tissue. Membranous vesicles also occur in fibrocartilage, but these are linked with cell disintegration (Ghadially et al., 1978b) rather than the metabolically active process postulated for epiphyseal cartilage (Wuthier et al., 1977). We do not think they have been isolated from elastic and fibrocartilage or examined in association with the chondrocytes by SEM. It would be interesting to examine those elastic cartilages and fibrocartilages that normally mineralize (Beresford, 1981) or mineralize as an aging or pathological phenomenon (Shitama, 1979; Beresford, 1981; Chadwick and Dowham, 1978) to see if matrix vesicles of some kind are always present (Bonucci and Dearden, 1976) and associated with the mineralization, and whether this mineralization is spheritic or not. F. Cartilage Matrix Cartilage matrix varies with type, position, and age (Venn, 1979). The matrix component that has received the most attention using SEM is collagen. 1.

Collagen

The collagen fiber orientation can be seen on any manufactured surface of cartilage and is particularly well displayed where splits have been made parallel to the main fiber direction (Bozdech and Horn, 1975). The internal structure of cartilage matrix has been examined by cutting or splitting the tissue either before fixation (Inoue, 1973), after fixation (Cameron and MacNab, 1973) or after drying (Clarke, 1973). Because hyaline and elastic tissues are both elastic and tough and fibrocartilage is tough, neither wet nor dry fractures are possible without distortion of the tissue. Better, cleaner fracture planes in the complete tissue may be formed if the tissue is frozen. Ice crystal growth during freezing is limited by quenching in CC1 2F 2kept at its melting point of — 155°C. The plane of section may be selected

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by nicking the tissue when wet. The differences among the three ways of opening up the cartilage are that splits will follow the fibrous planes and may skirt lacunae, sections will cut through the tissue in any plane but damage the surface and cause rents (Inoue, 1973), and freeze-fracture planes will be propagated with the least respect for any of the structural components of cartilage, that is through cells, lacunae, and intercellular matrix (Draenert and Draenert, 1981). Even extensive disruption of the tissue may, at times, be advantageous and expose changes in directions of fiber bundles (Inoue, 1973; Kessel and Kardon, 1979). Minns and Steven ( 1977) found that the collagen could be examined in greater depth when the noncollagenous matrix was digested away with, for example, hydrogen peroxide and trypsin. In this case, it would be advantageous to take stereopairs of the specimen so that the true orientation and position of the collagen could be seen. SEM is a particularly useful tool for investigating fiber organi­ zation, and it has been applied to the question of whether there is a special order of collagen in the territorial matrix in hyaline cartilage. No distinctive pattern has been noted in articular cartilage (Chappard, 1979; Clarke, 1974). Addition­ ally, it had been thought that density of the collagen might be different in the two regions, although X-ray microprobe studies contradicted this (Maroudas, 1972). Clarke (1974) could not demonstrate such a difference by SEM. The collagen fiber structure in fibrocartilage and elastic cartilage has not received quite the same amount of interest. However, fibrocartilage has a very high collagen content, about 80% dry weight (Peters and Smillie, 1971), and the collagen is well ordered, lending itself readily to study by SEM using con­ ventional and primitive preparative techniques (Inoue et al., 1971; Inoue, 1973; Cameron and MacNab, 1972, 1973; Refior and Fischer, 1974). Experimental alternation in the type of collagen produced by chondrocytes cannot be detected by routine SEM unless it is accompanied by change in texture of the collagen. However, the fineness, order, and density of the collagen in different zones of the cartilage under different experimental conditions could easily be monitored by SEM. Equally, the texture of the collagen produced by chondrocytes in culture and the causes of any modification to that texture are also open to examination because the cells may be removed by microdissection or stripping. The distribution of different types of collagen in fibrous, elastic, and hyaline cartilages is of interest in a morphological context (Eyre et al., 1975). Hyaline and elastic cartilage contain only type II collagen, and this exists as fine fibrils. Fibrous cartilage generally contains both type I and type II collagen, and much coarser bundles predominate. Those fibrocartilages containing only or mainly type I collagen such as the meniscus, the temporomandibular disc, and fibrous annulus (Eyre, 1979) are, perhaps, merely honorary members of the cartilage club. Thus, monitoring the size of fibrils and fiber bundles by SEM can provide clues for the tentative characterization of the collagen produced under different experimental and pathological conditions. This aspect has not been properly exploited using SEM.

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The other main fiber-associated protein found in cartilage is almost exclusive to elastic cartilage. Elastin comprises about 20% dry weight of mature elastic cartilage (Stockwell, 1979), having increased in amount with the development of the tissue (Cox and Peacock, 1977). The latter authors reported that TEM study suggests its formation is localized to only part of the cell membrane at any one time and that it coalesceses into an irregular network around the cells. Because this occurs within the matrix containing collagen fibers, these are better removed with collagenase or by autoclaving to reveal the three-dimensional organization of the elastin (Gotte et al., 1972; Tsuji et al., 1979). The method of drying the elastic component of the tissue for SEM will alter its fine detail, shape, and size, freeze-drying being best (Grut et al., 1977) and critical point drying the worst (Tsuji et al., 1979). Elastin does occur in fibrocartilage (Ghadially et al., 1978a), but mainly as very fine, young, immature fibrils that would not be in quantity sufficient to be recovered from that tissue for SEM or identified within the bulk of the tissue. 3. Ground

Substance

The nonfibrous and nonelastin component of the matrix is seen as a fine dense network in stained transmission electron micrographs. With SEM it appears more as an obscuring film or matrix about the collagen fibers, and it is this component that suffers most loss during specimen preparation because the proteoglycans get washed out. The effect of this is most apparent in older cartilage where the quantity of proteoglycans is greatly reduced (Elliot and Gardner, 1979). They have been best preserved and exhibited in fresh, unfixed, freeze-dried, freezefractured preparations (Draenert and Draenert, 1979). Ruthenium red has been used to preserve more of the proteoglycans in fixed tissue (Gardner, 1972; Highton and O'Neill, 1975). Gardner (1972) has also suggested that staining proteoglycans with ruthenium red and the copper-containing alcian blue should offer means by which elemental analysis of cartilage could be conducted in combination with SEM. We can think of no method that would allow the removal of the cellular and fibrous components of the matrix and leave the remainder of the organic matrix intact. 4. Matrix

Degradation

The breakdown of the structure of cartilage, in particular articular cartilage, under the action of enzyme has also been described using SEM (Puhl, 1971a,b; Richter, 1971), and we have used hyaluronidase experimentally to reveal the collagen microarchitecture. There are, doubtless, other common proteases that will remove all cartilage matrix elements not enshrouded in mineral. For example,

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Gardner (1972) found in an SEM investigation that papain progressively degraded glutaraldehyde-fixed collagen matrix, exposing, then releasing, the cells in 6 hours, but that cathepsin D left the treated surface intact after 24 hours with loss of some of the deeper proteoglycans. Collagenases are believed to be very important in the erosion of cartilage in early rheumatoid lesions (Woolley et al., 1975a, 1977, 1979), and the character and rate of progression of the lesion produced by different collagenases on various cartilages could also be investi­ gated experimentally using SEM and may provide feedback information on the clinical situation. Synovial collagenase, for instance, acts differently on cartilage and tendon collagen (Woolley et al., 1975b), and type I collagen is degraded six times more rapidly than type II. Woolley and coworkers (1979) have also used SEM to illustrate the distinctive morphology of the dendritic cells dissociated from rheumatoid synovia that pro­ duce collagenase in culture. Growing such cells on cartilage in vitro and assaying by SEM the destruction caused by any release of collagenase should be a fruitful line of investigation. In a different approach than most SEM studies on cartilage matrix, a combination of SEM and X-ray elemental analysis has been used in in vitro studies on cartilage aimed at finding the diffusion rate of substances through the tissue (Omar et al., 1979). G. The Mineral Phase of Cartilage This phase of hyaline calcified cartilage is most appropriately studied by SEM, and the preparative technique is simple. Fresh cartilage is immersed in either cold 5-10% sodium hypochlorite or warm (50°C) 5% sodium peroxide to remove all unmineralized organic matrix and cells (Boyde and Jones, 1972; Lester and Ash, 1981). An earlier technique of refluxing with 1,2-ethanediamine at 110°C was fraught with health hazards and has been effectively replaced by the use of these solutions. The specimen is then carefully washed and either air dried or dehydrated in alcohol and dried by solvent evaporation or critical point drying. All mineralized parts that are linked even tenuously to one another will remain as a skeleton. Unfortunately, there will be a loss of those particles that were entirely discrete. These may be visualized if the matrix is only partially digested away so that they are still trapped in a little of their surrounding matrix. Washing and air drying will ensure that the matrix shrinks down on to them, revealing their position and approximate size through the veil of organic matrix, but dislodging them spatially with the shrinkage. The mineralization pattern in growth plate cartilage is spheritic (Boyde and Jones, 1972) and associated with matrix vesicles. Once the mineralization has exceeded the limits of the vesicle and spread spherically into the adjoining matrix, the mineral clusters may be seen through the thin walls surrounding the chondrocytes. They may also be gathered by digesting the matrix and collecting the mineralized particles on a filter in a manner similar to that used in bone (Sela and Boyde, 1977).

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III. PRESENT STUDY A. Embryonic Cartilage We have found that embryonic cartilage specimens are best prepared by cutting followed by a critical point drying procedure (Fig. 1) or by breaking critical point dried tissue; or by freeze-fracturing and freeze-drying (Fig. 2). If the tissue is cut or dry-fractured, the chondrocyte lacunae are either empty where the cells have been removed from the lacunae or the cell is retained in the lacuna and protrudes above the surrounding cartilage surface, displaying its own free surface. Chondrocytes vary in appearance according to their functional state. Young embryonic hyaline cartilage contains cells actively engaged in chondrogenesis, and these differ in shape, size, and surface features according to position within the cartilage organ and contribution to the matrix. The surface of the chondrocytes almost invariably presents a uniform coating of fine projections which presumably fit in the interstices of the fibrous cartilage matrix. In freeze-fractured specimens the fracture plane passes with equal ease through the cells and the matrix, a typical appearance being cells fractured through their centers. The nucleus of the chondrocyte tends to cleave out of the surrounding cytoplasm, and many chondrocytes show a large depression at the center where the nucleus has been removed. The meshwork of fibrils of the cartilage matrix is seen in the walls of the lacunae in cut or CPD dry-fractured samples, but is distorted at the torn surfaces of the matrix (i.e., those surfaces created by the preparative procedure). The fibril orientation within the matrix can better be studied in freeze-fractured spec­ imens because the distortion of the structure is much less. Freeze-fractured embryonic cartilage sometimes shows a rim around each chondrocyte lacuna; this is due to an edge-brightness effect at the border of the lacuna. The fine collagen fibrils are evenly distributed throughout the matrix. They show no preferred orientation where the cells are approximately spherical except that adjacent to the cell the fibrils lie parallel to its surface in the lacunar wall. Chondrocyte lacunae must be undergoing constant change during interstitial growth; any change in shape of the cell associated with matrix production will generally require a change in the outline of the lacuna. One would therefore expect a definition of pericellular matrix as a distinct part of the separated intercellular matrix only once most interstitial growth has ceased. B. Permanent Cartilages Permanent cartilages may be hyaline, elastic, or fibrocartilage. 1. Hyaline

Cartilage

A notable feature of mature hyaline cartilages is that the interstices of the fibrous reticulum in the tissue are much smaller than in embryonic cartilage, the

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tissue presenting a much more homogeneous appearance in freeze-fractured prep­ arations, which are of great value in studying this type of tissue. There is a welldefined capsule of territorial matrix. The structure of the cartilage matrix cannot be properly interpreted in speci­ mens that have been cut because of the distortion of the cartilage matrix by the cutting process. In material torn in the dry condition there is less deformation of the original arrangement of the matrix fibers (Fig. 3). The tissue is far less cellular than the embryonic type. Microvilli are less prominent and sometimes absent on older cartilage cells but these cells may be associated with blebs or vesicles of cellular origin sited close to the cell in the matrix. 2. Elastic

Cartilage

A good method of preparing elastic cartilage is by freeze-fracturing in ethanol followed by critical point drying (Figs. 4 and 5). The texture in the walls of the chondrocyte lacunae is remarkably different from that of hyaline cartilage, pre­ sumably reflecting the arrangement of the elastin in this matrix. In the adult mouse pinna, which we have studied, the lacunar boundaries have a fine texture devoid of fibrils and canalicular openings (Fig. 6). In the adult human ear, the elastic fibers are prominent and may form an irregular network or show some ordering into lines in the wall of the lacuna (Figs. 4 and 5). The elastin in such cartilage forms a shell around each cell which may separate from the rest of the matrix when the tissue is fractured. The cells are numerous and may be strikingly oriented. It is possible to remove all components of the matrix other than elastin by autoclaving, but it is impossible to retain or retrieve the true three-dimensional organization of the elastin for study by SEM even when the residue is freezedried (Fig. 7). 3.

Fibrocartilage

Fibrocartilage is supposed by most authorities to be a separate tissue entity. In our own experience, it represents the blend of frank fibrous tissue with adjacent hyaline cartilage such as the junction of the annulus fibrosus, with the cartilage covering the ends of vertebrae at intevertebral joints. The tissue covering the obvious hyaline secondary cartilage at, for example, the mandibular condyle may also be described as fibrocartilage where the tissues fuse. The degree of organization of collagen is greatest in fibrous cartilage. Two sets of collagen fibers are often present in this tissue: the main bundles and connecting fibrils interspersed between and linking them. Fiber groups may also run in different directions in layers rather as in lamellar bone. In the annulus fibrosus, for example, although the mean fiber direction is vertical, alternating layers spiral to the left or right. The lacunae in adult fibrocartilage are relatively sparse and always aligned in the same axis as the fibers in the immediate neighborhood.

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C. Skeletal Cartilages: Long Bone Epiphyses The greatest interest in scanning electron microscopy of cartilage lies in the study of articular and growth cartilages of the skeleton. Although articular car­ tilage is permanent and the growth plate is a temporary cartilage in most, but not all, mammals, they have similarities in structural organization. We discuss the various aspects of these cartilages in the order they present themselves when proceeding from the articular end of a long bone toward the shaft. 1. The Articular

Surface

The greatest controversy in scanning electron microscopy of cartilage has certainly been whether the articular end is smooth. Whether or not it is depends upon preparative technique. We have been able to demonstrate the smoothness of articular surfaces to a submicron level using three different preparative techniques. First, we exposed the ends of long bones and shock-froze them in dichlorodifluoromethane at — 155°C, transferred these samples to an SEM equipped with a cold stage, and examined the surfaces at - 114°C. As soon as any condensated contamination ice has sublimed from the surface of the sample it can be scanned at TV rates without application of a conductive coat. Viewed stereoscopically in real time in the SEM, it is seen to be perfectly smooth. Second, we have exposed the ends of long bones in anesthetized animals, washed the exposed surface with 0Λ M N H 4H C 0 3 buffer, and shock-frozen and freeze-dried as before. The point of using the ammonium bicarbonate buffer is that this salt is volatile and may be pumped off the sample if the temperature is raised to 50°C after completion of the freeze-drying process. We have found experimentally that a 0.1 M solution causes no dimensional change. If the wash­ ing procedure is not sufficiently vigorous, then a matrix of ice crystal artifacts is formed in the residual synovial fluid covering the articular surface. This does Fig. 1. Mouse embryonic limb, cut and critical point dried. Embryonic hyaline cartilage. Field width = 17 μπι. SE (secondary electron) image 10 kV. Fig. 2. Mouse embryonic limb, freeze-fractured in water and freeze-dried from water. Embryonic hyaline cartilage. Note loss of nuclei from centers of cells. Field width = 52 μπι. SE 10 kV. Fig. 3. Chipmunk (Tamias striatus) nasal septum, osmium perfusion fixed, critical point dried, and dry-fractured. Adult permanent hyaline cartilage. Field width = 90 μπι. SE 10 kV. Fig. 4a,b. Stereopair: human ear pinna elastic cartilage, freeze-fractured in ethanol and critical point dried. Fracture plane often passes between pericellular capsule and surrounding matrix. Field width = 84 μπι. SE 10 kV. Fig. 5. Higher magnification of Fig. 4 showing detail of lacunar wall. Field width = 18 μπι. SE 10 kV.

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not, however, obscure the fundamental smoothness of the articular surface (Fig. 8). A third way in which we have examined this surface involves injecting fixative into the joint surface. We used 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.25 and injected this into the joint space in anesthetized rats. After allowing several minutes for prefixation, the samples were removed with the supporting epiphyseal bone, rinsed with fixative, and fixation was continued by immersion. After a total of about 30 min of glutaraldehyde fixation, the joint surfaces were post-fixed in 1% O s 0 4, dehydrated in ethanol, and critical point dried. These joint surfaces appear to be perfectly smooth (Figs. 9-10). 2. 3-D Structure of Articular

Cartilage

Cut surfaces of cartilage, whether cut wet or after freeze-drying or critical point drying, serve mainly the purpose of revealing the positions of the large holes in the matrix continuum occupied by the chondrocytes (Fig. 11). Surfaces prepared by tearing or splitting the tissue in the wet or dried condition are also mostly deformed but give an impression of the complexity of the fiber orientation. Cartilage will split parallel to the main collagen direction, and this has been used to illustrate the general organization of the fibers in articular cartilage. The fibrils run more or less parallel to the articular surface superficially and arc into a direction vertical to the articular surface in the deeper tissue (Figs. 12-14). Such preparations of cartilage reveal that ill-defined vertical columns are to be discovered by dissecting the cartilage in either wet or dry conditions (Figs. 12-14). Whether torn wet or dry, the surface produced is characterized by an apparent paucity of cells compared with the true number density seen in cut surfaces. Fig. 6. Adult mouse ear pinna, glutaraldehyde fixation, freeze-fractured in CC1 F , and critical 33 point dried. Field width = 41 μπι. SE 10 kV. Fig. 7. Human ear pinna, autoclaved to remove all but elastin fibers and freeze-dried from water. Field width = 17 μπι. SE 10 kV. Fig. 8a,b. Stereopair: rat lower femoral head, frozen to — 155°C and freeze-dried, showing synovial fluid over smooth articular surface at left and cells in immediate subsurface zone which have produced no impressions in the articular surface. Field width = 146 μπι. SE 10 kV. Fig. 9. Rat lower femur. Joint space injected with glutaraldehyde in vivo, post-fixed in osmium, critical point dried, then torn dry. Surface at upper right is smooth articular surface; projections are extravasated red blood cells. Field width = 146 μπι. SE 10 kV. Fig. 10. Same preparation as in Fig. 9 showing detail of collagen orientation at joint surface to left and alternating collagen fiber orientation in immediate subsurface layers to center and right. Field width = 13 μπι. SE 10 kV.

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Plastic deformation is evident in specimens that are freeze-fractured in either water or ethanol. There is strong internal evidence that the fracture mechanism for the tissue frozen at liquid nitrogen temperatures is still a ductile one. The bundle-like units of collagen pull, narrow, and neck as in a classical tensile test specimen. Thus the fracture surface is very irregular, consisting of valleys be­ tween the narrowed-down, vertical, columnar units and flat portions of the frac­ tured surface generated by high velocity rupture at the final yielding. Although it is clear that the principal collagen orientation in articular cartilage below the surface zone is perpendicular to the articular surface, this is a satistical mean. A high proportion of the collagen fibers lie at angles of roughly 15-20° to either side of the perpendicular (Fig. 15), and collagen fibrils in all orientations may be found in all regions of the cartilage (Fig. 16). The apparent orientation or striation parallel to the surface of the articular cartilage in the deeper layers is also to be partly attributed to a deformation of the tissue during preparation (Fig. 17). It is less evident in dry-fractured samples than in wet-torn or freezefractured samples. In cartilage as in other connective tissues, elongated fiber-producing cells generally parallel the fibers closely associated with them. The chondrocytes are just like osteocytes or fibrocytes in this respect. Thus, in the immediate subarticular zone the cells are oval in outline and aligned with their longest axes parallel to the surface of the joint (Figs. 8 and 9). Deep to this zone they become perpendicular to the articular surface (Fig. 12). It is not always possible to see whether an apparent difference in density of the collagen around a lacuna in hyaline cartilage is a real feature or is produced by the different pullout of Fig. 11. Rat knee joint articular cartilage fixed by injection of glutaraldehyde into joint space as in Figs. 9 and 10. Osmium post-fixed and superficial tangential slice made to reveal this surface showing cell nests in immediate subsurface zone. Field width = 36 μπι. SE 10 kV. Fig. 12. Human femoral head articular cartilage, cleaved wet in ethanol and then critical point dried. Field width = 1460 μπι. BSE (backscattered electron) image 30 kV. Fig. 13. Higher magnification of preparation in Fig. 12. Vertical columns are generated by this wet dissection procedure except in the immediately subarticular zone where the orientation changes to tangential (visible at bottom). Field width = 980 μπι. BSE 30 kV. Fig. 14. Higher magnification of preparation in Figs. 12-13. The mean orientation of the collagen fibrils constituting one of the columnar units is parallel with the long axis of this unit. Field width = 22 μπι. SE 10 kV. Fig. 15. Human femoral head articular cartilage, critical point dried and then dry fractured, showing predominant vertical orientation of collagen fibrils in mid-cartilage thickness. Field width = 18 μπι. SE 10 kV. Fig. 16. Human femoral head articular cartilage, freeze-fractured in ethanol and then critical point dried, showing predominant vertical orientation of collagen fibrils. Field width = 45 μπι. SE 10 kV.

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collagen fibrils ruptured at the edge of a lacuna. However, occasionally, the tissue ruptures at a tangent to the pericellular matrix, and there is a cocooning of the cell in fibers of a different orientation. In preparations where the collagen component has not been exposed by loss of the proteoglycans the fibrous texture is not obvious, and the lacuna partitions appear remarkably smooth and thin. 3. The Mineralizing

Front in Articular

Cartilage

The principal vertical orientation of the collagen fibrils of the articular cartilage matrix is also well demonstrated in the vertical orientation of the mineral particle clusters seen at the mineralizing front when this is exposed by wet-, dry-, or freeze-fracturing perpendicular to the cartilage surface (Fig. 18). In such prep­ arations it appears that mineralization progresses along the long axis of the collagen fibrils, much as occurs in the mineralization of Sharpey fibers at the surfaces of bone or cementum. However, this is not the impression gained when the mineralizing front is prepared as such by dissolving away all the nonmineralized cartilage matrix. The tidemark mineralizing front can be exposed by treatment of the cartilage with sodium hypochlorite, sodium peroxide (Fig. 19), or plasma ashing. The unevenness of this surface on a gross scale reflects the different levels of progression of the mineralizing front surrounding the different chondrocytes, and, on a finer scale, it shows the mineral particle clusters which appear to have a more or less spherical shape. Around each cell, calcification is in advance of the general background level of the mineral, and these regions form rough protuberances on the surface (Figs. 19-21). The adult human femoral head tidemark mineralizing front is grossly much rougher than that in the small growing mammal (Figs. 22 and 23). In the adult human tidemark case, there appears to be a much greater separation between the cartilage cells in the matrix, but the same basic features are present, namely, a large variation in the level of mineralization in local microscopic regions and a fine structure consisting of a very large number of very small mineral particle Fig. 17. Freeze-fractured in ethanol, critical point dried human femoral head articular cartilage showing apparent banding parallel to articular surface (horizontal) as well as vertical orientation of collagen fibrils. Field width = 17 μπι. SE 10 kV. Fig. 18. Vertical (radial) fracture through tidemark mineralizing front in adult human femoral head articular cartilage. Unmineralized cartilage is seen in the lower portion of the field. Field width = 200 μπι. BSE 30 kV. Fig. 19. Mineralizing front of rat humerus articular head exposed by treatment with N a 0 solution. 22 Field width = 470 μπι. SE 10 kV. Fig. 20a,b. Stereopair: mineralizing front of rat lower femoral articular head exposed by oxygen plasma ashing. Field width = 200 μπι. SE 20 kV. Fig. 21. Rat lower femoral articular head, mineralizing front exposed by plasma ashing, showing a region in which microcalcospherites are the most obvious features. Field width = 100 μπι. SE 20 kV.

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clusters. The numerical density of these fine clusters is higher than the highest frequency of matrix vesicles described or figured in the literature. Thus, we presume they are not related specifically to matrix vesicles but to the proteoglycan or collagen portions of the matrix. 4. The Osseochondral

Junction between Articular Cartilage and Bone

We know of no method to separate mineralized cartilage from bone because the latter is always deposited upon a resorbed surface of the former, and the junction is therefore extremely intimate. It may, however, be viewed on edge in a section (Fig. 24) or fractured preparation (Fig. 25). In polished samples sectioned perpendicularly to the surface and imaged by backscattered electrons at 30 kV, topographic contrasts can be eliminated; residual contrast shows density variations as in a microradiograph. The osseochondral junction line can then be recognized as an electron-dense reversal (cement) line (Fig. 24). Osteocyte la­ cunae are recognized by their characteristic size range in the bone layers deposited deep to the mineralized cartilage. The thickness of the mineralized cartilage varies considerably from one microscopic location to another (Fig. 24). 5. The Mineralizing

Front at the Epiphyseal Side of a Growth Plate

Growth plate cartilage may mineralize on both surfaces. A part of the attach­ ment of epiphyseal bone to the epiphyseal growth plate is via an osseochondral junction of bone to a resorbed mineralized calcified cartilage surface. Regions may also be found in which bone matrix is in direct continuity with uncalcified cartilage matrix. In the peripheral regions of the growth plate cartilage, it is common to find an active mineralizing front which may be continuous with that deep to the

Fig. 22. Human femoral head tidemark articular cartilage, mineralizing front exposed by N a 0 22 treatment showing protuberances associated with pericellular mineralization at the gross scale. Note small size of the mineral particle clusters or microcalcospherites. Field width = 760 μπι. SE 10 kV. Fig. 23a,b.

Stereopair of preparation in Fig. 22. Field width = 30 μπι. SE 10 kV.

Fig. 24. Vertical polished section through epoxy-embedded human femoral head. Tissue shown is mineralized cartilage below and bone above. Note that junction is marked by more electron-dense (white) line. Field width = 515 μπι. BSE 30 kV. Fig. 25. Vertical wet fracture through osseochondral and tidemark mineralizing front of human adult femoral head articular cartilage, critical point dried. Tissue layers are bone at top, mineralized cartilage at center, and unmineralized cartilage at bottom. Field width = 420 μπι. BSE 30 kV. Fig. 26. Mineralizing front (exposed by Na 0 ) at peripheral portion of deep surface of mouse 22 lower femoral epiphysis. Field width = 39 μπι. SE 10 kV.

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articular cartilage. Thus the appearance of the growth plate side of the epiphysis may be quite similar to that found in a growing articular cartilage, with prominent calcospherites (Fig. 26) and pericellular mineralization zones (Fig. 27). More centrally, the mineralization front may be much less active and present a much smoother (Fig. 29) or even planar (Figs. 27 and 28) surface. Centrally, the most common appearance is of a grossly irregular topography due to resorption cavities extending through to the unmineralized cartilage from the epiphyseal bone, with that bone covered by a relatively smooth dense varnish which is probably mineralized cartilage (Fig. 30).

6. The Polarized Structure of Typical Growth Plate

Cartilage

The structure of epiphyseal growth plate cartilage is well described in both the light microscopic and transmission electron microscopic literature and need not be reviewed here. The organization of the cells into columns in a typical active growth plate can be seen in Fig. 3 1 , where the cells are exposed by freezefracturing tissue that has been processed through to ethanol. Such preparations demonstrate the great difficulty in any kind of microscopy of fixing cartilage adequately. We have taken perfusion-fixed limb material, removed longitudinal slices for post-fixation in osmium, and then freeze-fractured the slices to study the depth of penetration of the osmium (usually limited to approximately 50 μπι after 1 h). If observations are confined to this very thin peripheral shell of wellfixed material, it is possible to study cytoplasmic detail in the chondrocytes (Fig. 32). If the tissue is dissected (i.e., torn in the live, wet condition), tubes of chondrocytes-are easily recognized (Fig. 33). Although the morphological preservation of the cells is grossly inferior, the use of freeze-fractured, unfixed, rapidly frozen growth plate cartilage has con­ siderable merit, because such preparations may be freeze-dried and the chon­ drocytes removed by dissection for microanalysis as described by Boyde and Shapiro (1980). Whether freeze-fractured in water or ethanol, SEM demonstrates the predominant longitudinal orientation of collagen fibers in the growth plate matrix (i.e., they are parallel with the columnar axis; Figs. 31 and 34).

Fig. 27a,b. Stereopair: rat humerus, mineralizing front (Na 0 preparation). Peripheral portion 22 of deep surface of upper epiphysis seen at right, smoother inactive front centrally, and partly resorbed central portion at left. Field width = 390 μπι. SE 10 kV. Fig. 28a,b. Stereopair: same preparation as in Fig. 27. Higher magnification of center of field. Field width = 78 μπι. SE 10 kV. Fig. 29a,b. Stereopair: anorganic preparation of mouse epiphysis (Na 0 ). Smooth, almost in­ 22 active mineralizing front of deep surface adjacent to growth plate cartilage. Field width = 78 μπι. SE 10 V.

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Scanning Electron Microscopy of Cartilage 7. The Growth Plate Mineralizing

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Front

Mineralization in active growth plate cartilage occurs principally in relation to the so-called matrix vesicles. The resulting spherical mineral particle clusters grow and become confluent but never extend throughout the whole of the matrix (Fig. 34). Their arrangement is particularly easy to visualize in back-scattered electron (BSE) images of longitudinal freeze-fractured specimens (Fig. 34) or in partly digested cut surfaces (Fig. 35). Microcalcospherites appear to amal­ gamate like pearls in a row vertically throughout the calcifying region of the cartilage, and this strongly suggests that the mineralization is collagen-ordered. This longitudinal row-like organization of the fusing calcospherites is often obscured when fusion has occurred in the central regions of the plate, but it is seen to great advantage at the most peripheral limit of the mineralizing growth plate (Fig. 37). The mineral skeleton forms tubes surrounding and isolating the cells comprising a row in growth cartilage but only partly separates cells within a row. Thus, in growth plate cartilage in which the cells are organized into columns, the intracolumnar transverse bars of cartilage fail to mineralize; this has been attributed to a difference between territorial and interterritorial matrix by some authors, or to a difference in the incidence of matrix vesicles in the two regions by others. This lack of mineralization in the transverse intracolumnar bars of cartilage can be seen in preparations from which the cells are removed by simple ultrasonication, leaving the matrix intact; the calcospherites can be seen through the walls of the chondrocyte lacunae (Fig. 38). That the external surface of the mineral tubes surrounding each column is so regular and straight is another feature that argues against the initiation and siting of mineralization in cartilage being solely dependent on matrix vesicles. Our observations support

Fig. 30a,b. Stereopair: mineralizing front in upper head of rat humerus (Na 0 preparation). Field 22 is typical of areas where resorption cavities from the epiphyseal ossification center intrude upon nonmineralized cartilage of growth plate. Field width = 450 μπι. SE 10 kV. Fig. 31. Rat lower femoral growth plate (glutaraldehyde fixation, ethanol freeze-fractured, critical point dried) showing division of chondrocytes (paired daughter cells) in longitudinal columns. Field width = 41 μπι. SE 10 kV. Fig. 32. Rat lower femoral growth plate fixed in glutaraldehyde and osmium, freeze-fractured in ethanol, and CPD, showing details of cytoplasmic organization in a chondrocyte. Field width = 26 μπι. SE 10kV. Fig. 33. Rat lower femoral growth plate cut longitudinally and cultured 5 h in standard medium. Wet tear occurred through matrix between columns of cells, which can be recognized as tubular structures (hypertrophic zone). Field width = 124 μπι. SE 10 kV. Fig. 34. Rat upper femoral growth plate mineralizing front (NaOCl preparation), viewed obliquely down the long axis of tubes formed by the cell columns. Note that mineralization does not spread to the centers of thick intercolumnar matrix. Field width = 167 μπι. SE 10 kV.

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those of Barckhaus and Hohling (1978) in this regard. Some longitudinal fibrous restriction and marshalling or mineralization must be occurring. The fate of the calcified cartilage at the growth plate is, of course, to be partly resorbed immediately, the remaining portions functioning as a skeleton on which appositional bone growth occurs. Resorbed cartilage surfaces present an ex­ tremely smooth appearance which is then obscured by the deposition of the collagenous bone matrix. D. Cartilage and Intervertebral 1. Nucleus

Joints: The Disc

Pulposus

The nucleus pulposus tissue shrinks as a consequence of the ethanol dehy­ dration and critical point drying regimes, but its internal structure can be ex­ amined in the more continuous areas of ethanol freeze-fractured samples. Figure 39 is probably typical of the younger stages of development of this tissue. It contains some large smooth-walled channels presumably filled with liquid in life, and it is difficult to distinguish the individual fibers. Adult human nucleus pulposus (Fig. 40) shrinks less on drying, the high density of oriented fibers reflecting its lower water content. 2. Annulus

Fibrosus

In the young condition the nucleus pulposus and the annulus fibrosus are well discriminated one from the other, but in the adult human condition they merge almost imperceptibly. The annulus region is composed of very densely packed collagen organized as large bundles decussated in alternate layers (Fig. 41). The Fig. 35. Longitudinal freeze-fracture through glutaraldehyde and osmium fixed rat growth plate cartilage, showing the mineralizing region. The white spherical structures are large mineral particle aggregates or calcospherites originating from matrix vesicles, at least in part. They appear to be in a specific relationship to the longitudinal collagen fibers of the intercolumnar matrix. Field width = 18 μπι. SE 10 kV.

, of rib in hereditary multiple exostosis Fig. 36. Mineralizing cartilage from abnormal outgrowths in the horse. Cut surface treated with 0.5% trypsin for 4/2 hours, washed, and dried from ethanol. Field width = 11 μπι. SE 10 kV. Fig. 37a,b. Stereopair: anorganic preparation at margin of rat femoral growth plate, showing sides of longitudinal tubes formed by mineralization in intercolumnar matrix. Field width = 134 μπι. SE 10kV. Fig. 38. Rat femoral growth plate cartilage, cut longitudinally and cleaned by ultrasonication in detergent before critical point drying. Note calcospherites seen in lateral walls of the chondrocyte lacunae but not in the transverse intracolumnar matrix. Field width = 112 μπι. SE 10 kV. Fig. 39. Nucleus pulposus of rat lumbar intervertebral joint. Karnovsky perfusion fixation, freezefractured in ethanol, and critical point dried. Field width = 145 μπι. SE 10 kV.

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bundles merge into the hyaline cartilage covering the ends of the vertebral bodies and their epiphyses (if present) and more peripherally into the fibrous layer of the periosteum of the vertebral bodies (Fig. 42). Many SEM preparations of rat vertebrae suggest the large diameter fiber bundles continue from the annulus to lie parallel to the surface of the cartilage opposite the nucleus pulposus, pre­ sumably pointing towards a focus in the center of the vertebral body. 3. The Mineralizing

Front of Vertebral Body

Cartilage

The mineralizing front under the articular cartilage of a vertebra may be exposed by any of the routine procedures (hypochlorite, peroxide, plasma ashing, or enzyme detergent). The mineralizing fronts opposite the nucleus pulposus or annulus fibrosus in the growing youth and in the mature adult, respectively, are different. In the rat the vertebrae have epiphyses, the calcified portions of which may be entirely calcified cartilage, or they may be fully developed ossification centers as is usual in the caudal vertebrae. The mineralizing front in rat caudal vertebrae exposed by extraction with N a 20 2 solution is shown in Figs. 4 3 - 4 5 . The mineralizing front adjacent to the annulus fibrosus can be seen in Fig. 43. Examination of the stereoscopic image will show circumferentially arranged rings of pits that have alternating directions of entry and lie between projections corresponding with the inserting annulus fibrosus collagen fiber bundles. The pits represent, in some instances, the centers of large-diameter collagen fiber bundles that, as yet, have failed to mineralize, but, in most cases, the pits show the location of the chondrocytes that are constrained between the collagen fiber bundles. Figure 44 shows the region of junction between the annulus fibrosus and the nucleus pulposus. There are annulus fibers entering this mineralizing front whose orientation can be determined by stereoscopic examination. The chondrocyte lacunae are aligned in rows between the circumferential groups of annulus fibers. In the dead center of the nucleus pulposus zone (Fig. 45) an irregular deposit

Fig. 40. Human adult nucleus pulposus, freeze-fractured in ethanol and critical point dried. Field width = 78 μπι. SE 10 kV. Fig. 41a,b. Stereopair: adult human annulus fibrosus, freeze-fractured in ethanol and critical point dried, showing very dense fibrous nature of this tissue in the adult. Field width = 157 μπι. SE 10 kV. Fig. 42. Junction of epiphyseal cartilage, body, periosteum, and annulus fibrosus of rat lumbar vertebra. Karnowsky perfusion fixation, freeze-fractured in ethanol, and critical point dried. Field width = 750 μπι. BSE 30 kV. Fig. 43a, b. Stereopair: rat caudal vertebra mineralizing front in peripheral annulus fibrosus region of epiphyseal cartilage (Na 0 preparation). Field width = 178 μπι. SE 10 kV.

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of calcospherites protrudes above an otherwise slightly concave surrounding. This deposit is composed of rather larger calcospherites and is apparently devoid of features that could be interpreted as chondrocyte lacunae. It may thus represent mineralization of nucleus pulposus tissue proper. Surrounding this can be seen the typical feature of the mineralizing front adjacent to the nucleus pulposus. The most prominent portions are mineralized pericellular capsules, and calcos­ pherites are clearly seen if the magnification is high enough (Fig. 45). The mature condition of the human annulus fibrosus illustrated (Figs. 46 and 47) differs from the young (rat) condition in the more rugged topography. How­ ever, there are similarities. Longitudinal rows of microcalcospherites evidently situated in the peripheral portions of bundles of collagen fibers can be seen (Figs. 46 and 47). Pits in the mineralizing front show the location of the chondrocytes (Fig. 46). The mineralizing front of the adult human vertebral body opposite the nucleus pulposus is also extremely rugged. It shows an irregular pattern of gross pro­ tuberances due to irregular progression of mineralization across the entire surface. Lesser protuberances show mineralization more advanced in the areas surround­ ing individual chondrocytes, and calcospherites can usually be distinguished (Fig. 48). 4. Mineral Surface Beneath Vertebral

Epiphyses

The mineral surface beneath rat vertebral epiphyses is not commonly smooth, with large confluent calcospherites representing an inactive mineralizing front in which no new matrix vesicles are being formed. If bone is found in the center of the epiphyses, it is also common to find areas where resorption cavities have extended up the unmineralized cartilage, as in long bone epiphyses (Fig. 49). Fig. 44a,b. Stereopair: rat caudal vertebra epiphysis mineralizing front (Na 0 preparation) at 22 border of annulus fibrosus (to left) and nucleus pulposus (to right). Field width = 185 μιη. BSE 30 kV. Fig. 45. Same specimen as in Fig. 44 showing center of nucleus pulposus region (inorganic, N a 0 22 preparation). Dead center is region where no chondrocyte lacunae can be recognized, and an irregular mound of calcospherites protrudes above surrounding concavity. Field width = 400 μπι. SE 30 kV. Fig. 46. Subarticular tidemark mineralizing front in a human lumbar vertebra (Na 0 preparation). 22 The various directions of the annulus fibrosus fiber groups can be discerned. Field width = 325 μπι. SE 10 kV. Fig. 47. Same preparation as in Fig. 46. At higher magnification the alignment of microcalco­ spherites parallel with the fiber groups can be seen. Field width 67 μπι. SE 10 kV. Fig. 48. Same preparation as in Figs. 46-47, region opposite the nucleus pulposus where the mineralization is calcospheritic in nature. Field width = 76 μπι. BSE 30 kV.

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Scanning Electron Microscopy of Cartilage 5. Mineralizing

Front of Main Vertebral Body Beneath Vertebral

139

Epiphyses

The mineralizing fronts beneath rat vertebral epiphyses are similar to beneath epiphyses of a long bone, but the columns of cells are not so long because of less active growth at such sites. The amount of intercolumnar matrix is also less, and it mineralizes more completely. Thus in anorganic preparations viewed obliquely from above the plane of the mineralizing front, it is more common to be able to see through obliquely from one longitudinal tunnel to the next (Fig. 50). E. The Synchondroses The synchondroses (e.g., in the cranial base and between sternebrae) are part of the primary cartilaginous skeleton. They are basically symmetrical growth plate cartilages with similar features found on either side of an equitorial trans­ verse plane. The details of the mineralizing front depend upon the rate of growth of the cartilage and vary from clearly columnar arrangements of cells with longitudinally oriented rows of calcospherites to irregularly stacked chondrocytes with mineralization occurring in the chondrocyte lacunar walls. F. Secondary

Cartilages

1. The Mandibular

Condyle

The secondary hyaline cartilage in the head of the mandibular condyle in a mammal is covered with a tissue layer variously described as fibrocartilage or dense fibrous connective tissue. A rupture through this layer is shown in Fig. 51. Large collagen fiber bundles are interspersed with nearly spherical cells mimicking the shape of chondroblasts rather than fibroblasts. Indeed, they closely resemble the quite spherical cells seen deeper within the frank hyaline cartilage that constitutes the growth cartilage at the head of the condyle (Fig. 52).

Fig. 49a,b. Stereopair: mineralizing front of deep surface of rat caudal vertebra epiphysis (Na 0 22 preparation). Field width = 390 μπι. BSE 20 kV. Fig. 50. Mineralizing front (Na 0 preparation) at cephalad end of rat lumbar vertebral body 22 showing little matrix, all of which is mineralized, between the columns of cells. Field width = 1 4 5 μπι. BSE 20 kV. Fig. 51. Rat mandibular condyle, cleavage through fibrous zone above hyaline cartilage. Glutar­ aldehyde and osmium fixation, critical point dried. Field width = 157 μπι. SE 10 kV. Fig. 52. Rat mandibular condyle, cut vertically and cultured for 5 hours, chondrocytes remain adherent to lacunar walls. Field width = 100 μπι. SE 10 kV. Fig. 53. Rat mandibular condyle, mineralizing front (Na 0 preparation). Field width = 84 μπι. 22 BSE 30 kV.

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Chondrocytes in the mandibular condyle are not organized into columns but remain in an irregular three-dimensional array. This is reflected in anorganic preparations made to examine the mineralizing front in this tissue. Mineralization occurs close to the cells, the pericellular capsules being prominent portions of the mineralizing front together with the finer relief of calcospherites (Fig. 53). There is comparatively little matrix between adjacent hypertrophic cells. 2. The Rat Os Penis The appearances of the mandibular condylar secondary cartilage just described and figured are characteristic of slowly growing cartilages in which a vertical columnar polarization does not develop. Very similar appearances characterize secondary cartilages generally (e.g., the secondary cartilage of the rat os penis, Fig. 54). Here it will be again noticed that mineralization occurs first in cal­ cospherites very close to the chondrocyte capsules. G. Dental Cartilage In the suborder Caviidae of the hystricomorph rodents, calcified cartilage forms a portion of the crown of the molar teeth. This vascular cartilage is attached to enamel and is a permanent cartilage; it is not resorbed. It constitutes part of the functional exposed tooth structure and is lost by attrition at the occlusal surface. The chondrocytes in this tissue are irregularly arranged, not stacked in columns. The kind of mineralization observed is that of larger calcospherites of the type usually and probably correctly associated with matrix vesicle mineral­ ization (Fig. 55). The structure of this tissue, which is an infilling or packing cementum, is completely different from the acellular attachment cementum found over the enamel on the outer surface of the tooth. Sharpey fibers are included in the cartilage cementum in the narrow bands where it extends to the periphery of the tooth. H. Chondrocytes in Culture Chondrocytes have been studied by SEM following isolation by total disso­ lution of the matrix by enzymatic digestion. Such preparations suffer from the disadvantages that the cells may have been significantly damaged by the enzyme treatment and that differences related to their functional state or position of origin cannot be studied. We have investigated the possibility of studying chondrocytes in cut and torn cartilage. The responsiveness of the chondrocyte cell surface to culture conditions appears to be dependent on its functional state. We have found, for example, that cells closer to the joint in the condylar head of the mandible react more to a change in the environment and develop prolific ruffles. (Fig. 56). There is a change to a less responsive cell surface reaction in deeper parts of the cartilage.

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Fig. 54. Mineralizing front of secondary carti­ lage in rat os penis (Na 20 2 preparation). Field width = 157 μπι. BSE 30 kV. Fig. 55. Mineralizing cartilage on guinea pig molar dental enamel (1,2-ethanediamine extrac­ tion). Field width = 41 μπι. SE 10 kV.

Fig. 56. Cut surface of rat mandibular condyle, cultured for 5 hours in standard medium (same preparation as in Fig. 51). Cells closer to the joint surface engage in prolific ruffling activity while remaining attached to adjacent matrix. Field width = 17 μπι. SE 10 kV.

It is interesting that cells exposed on the broken surface of cartilage remain attached to the surface during culture (Fig. 52), suggesting a high degree of adhesion. They do not migrate within the first 24 hours of culture. Studying cultured cells retained on their own matrix has the added advantage that cells are held apart from one another rather than grown together, the latter situation being an abnormal condition for chondrocytes. Thus, for short-term experimentation on the effects of, say, the alteration of levels of calcium or growth hormone, culturing chondrocytes exposed but still seated within their lacunae and monitoring their response by SEM would seem to offer advantages unobtainable by other microscopic techniques. The position of each cell within the tissue would be known, and its response compared to that of its neighbors could be seen.

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Fig. 57. Tracings of photographs of the face of a block of human femoral head articular cartilage in water (continuous line), after substitution with ethanol (dashed line), and further after critical point drying via CC1 F and 33 C 0 (dashed and dotted line). The articular surface of the cartilage is to the 2 right. Note that radius of curvature of this surface decreases with specimen shrinkage.

/. Measurement of Shrinkage of Cartilage We have measured the shrinkage and distortion of adult human femoral head articular cartilage using the techniques described by Boyde and Maconnachie (1979). Assuming the volume of a cartilage block in water is 100%, a typical result was shrinkage to 75% in 100% ethanol, to 70% immediately after critical point drying from Freon 113 through C 0 2, and further shrinkage to 6 1 % 2 days after critical point drying. Such shrinkage after critical point drying is a normal phenomenon for all tissues we have studied. Examining the tracings of articular cartilage blocks during processing (Fig. 57) shows that deep layers of cartilage shrink more than superficial layers. This can be interpreted to mean that cartilage shrinks more across the principal collagen fiber axis. This results in bending of the articular cartilage surface.

Acknowledgments We would like to thank Elaine Maconnachie for technical assistance and Penny Hand for typing the manuscript.

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