J. Anat. (1978), 125, 2, pp. 349-360 With 13 figures Printed in Great Britain
349
A maturation change in the surface of cat articular cartilage detected by the scanning electron microscope F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Department of Pathology, University Hospital, University of Saskatchewan, Saskatoon, Canada
(Accepted 26 January 1977) INTRODUCTION
Since the advent of scanning electron microscopy a variety of surface irregularities such as (1) undulations and ridges, (2) oval depressions callea 'pits', and (3) occasional mound-like elevations referred to as 'humps', have been described on the surface of normal articular cartilage (McCall, 1968; Gardner & Woodward, 1969; Walker et al. 1969; Redler & Zimny, 1970; Clarke, 1971 a, b, c, 1973 a, b; Gardner, 1972; Ghadially, Ailsby & Oryschak, 1974; Mow, Lai & Redler, 1974). However, it has now been demonstrated that undulations and ridges are not a feature of the normal articular cartilage attached to bone but that such irregularities readily develop because of shrinkage and distortion when cartilage is detached from bone or is cut or injured in various ways, and that under such circumstances occasional humps are also seen (Ghadially, Ghadially, Oryschak & Yong, 1976, 1977a). Redler (1974) found some humps on human articular cartilage and thought that this feature was of more common occurrence on the articular cartilage of juveniles. However, the fact that this author considers ridges to be a constant feature of the normal articular surface raises the possibility that the humps, like the ridges, were artefactually produced by injury to cartilage or by detaching it from bone. The chance observation of humps, but no pits, on articular cartilage (attached to bone) of an 8 years old boy aroused our suspicion that a maturation change detectable by the scanning electron microscope perhaps occurs in the articular surface. We therefore studied the articular cartilage from young rabbits and found that there too humps abounded. Comparing these findings with the surface morphology of adult human and rabbit cartilage (where pits abound) described in the literature, we proposed that the surface of young and juvenile cartilage exhibits humps, but as the animal matures the humps are either transformed into, or replaced by, pits (Ghadially Moshurchak & Thomas, 1977b). Since this hypothesis is supported by comparatively few actual observations, and because one may argue that the preparative procedures adopted by us for the study of young and juvenile cartilage may not have been exactly the same as those used by us and others to study adult cartilage, it seemed desirable to carry out a more detailed and systematic study in another species to test the validity of our hypothesis. We now present results of such a study carried out on the cartilage of kittens and cats, using both air drying and critical point drying methods of tissue preparation. These validate our hypothesis, indicate how the cartilage changes occur, and show differences engendered by these two preparative techniques.
350
F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Fig. 1. Air dried right medial femoral condyle from a 3 weeks old kitten. Note shrunken appearance and distorted surface. x 13. Fig. 2. Critical point dried left medial femoral condyle from same kitten as in Fig. 1. Note smooth appearance of surface and an accidental cut inflected during specimen preparation (arrowhead). x 13. MATERIALS AND METHODS
A total of 12 kittens and cats from 2 days to 20 months old were used in this study. There were five kittens, aged 2 days, 2 weeks, 3 weeks, 1 month and 2 months; five cats between 6 and 8 months old; and two cats aged 12 months and 20 months. The animals were killed by an intracardiac injection of Nembutal, and the lower ends of the femora from both legs were collected from all animals. The articular surface was then washed thoroughly in normal saline to remove synovial fluid and any blood that might have contaminated it. At no stage of the procedure was the articular surface touched or allowed to become dry. All specimens were fixed in 3 % glutaraldehyde in cacodylate buffer (pH 7 3). The specimens from the cats (6 months to 20 months) were fixed for a period of 2 weeks, a time interval which we have found to be optimal for the larger specimens from more mature animals. In the case of the kittens a shorter fixation period of 1 week was employed. All specimens were then dehydrated in increasing concentrations of ethanol over the course of another week. One of the femoral condyles from each animal between 2 weeks and 8 months old was dissected off with a knife or a fine fret saw and processed by the critical point drying method, using substitution with amyl acetate and carbon dioxide. The other condyle from each animal in this age group (2 weeks to 8 months) and all condyles from 2 days, 12 months and 20 months old animals were air dried. Critical point drying was not attempted in the case of the 2 days specimen because of its small size and fragility, while the large condyles from older cats were difficult to accommodate in the equipment used and there were difficulties also with penetration and exchange of amyl acetate with carbon dioxide. Specimens were mounted with the aid of Electrodag on aluminium stubs. For small specimens standard stubs (12 mm in diameter and 3 mm thick) were employed, but the larger condyles from the older cats were mounted on specially prepared larger lower aluminium stubs (15 mm in diameter and 1 mm thick).
Maturation change in articular cartilage
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Fig. 3. Numerous closely packed humps (H) are seen on the surface of an air dried condyle from a 2 days old kitten. x 1150. RESULTS
Two days to two months old specimens Naked eye inspection, as well as examination with a x 7 magnifying glass, of fresh and processed specimens from these kittens showed that marked shrinkage and irregularity of the surface developed in air dried specimens, but this was not evident in critical point dried specimens. Such shrinkage was more marked in younger members of this group. The difference between air dried and critical point dried specimens could also be readily demonstrated at low magnification in the scanning electron microscope. Figure 1 shows a markedly shrunken air dried right medial femoral condyle with a distorted surface from a 3 weeks old kitten, while Figure 2 illustrates the critical point dried left medial femoral condyle from the same animal. It is evident that the critical point dried specimen is much larger (i.e. less shrunken) and the surface is smoother. Besides the gross irregularities engendered by shrinkage, the air dried specimens showed a surface so densely covered with humps that the articular cartilage had a cobble-stoned appearance (Fig. 3). Once again this change was somewhat more marked in the younger than in the older (1 month and 2 months old) kittens, so that in the latter the humps were less pronounced and not quite so closely packed as in the former. At higher magnifications the surface of the humps appeared quite rough and wrinkled (Fig. 4). The difference between air dried and critical point dried specimens (at higher magnifications than in Figures 1 and 2) is illustrated in Figures 5 and 6, which show the right and left medial femoral condyle respectively from the 3 weeks old kitten. It will be noted that closely packed humps are seen on the air dried specimen, but on
352
F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
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Fig. 5. Air dried right medial femoral condyle from a 3 weeks old kitten showing numerous humps (H). For comparison with Fig. 6. x 1000.
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Fig. 7. Air dried femoral condyle from a 4 weeks old kitten showing humps (arrowheads) amongst parallel ridges (arrows). x 580. 23
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354 F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Fig. 8. Critical point dried condyle from a 4 weeks old kitten. Some of the humps appear to have ruptured and produced craters (arrowheads). x 650.
9! Fig. 9. Critical point dried condyle from a 2 weeks old kitten showing a crater presumably produced by the collapse of a hump. Note small shrouded mass (arrow) in the centre of the crater which probably represents a shrunken chondrocyte. The general surface of the specimen is beset by innumerable fine holes (arrowheads). x 2500
Maturation change in articular cartilage
355 the critical point dried specimen the humps appear fewer, less elevated, and separated by a relatively flat but finely wrinkled surface. In areas of the air dried specimens where considerable surface distortion and caving in or curling had occurred, the humps were seen to lie amongst a series of parallel ridges or undulations (Fig. 7). In some of the specimens dried by the critical point method a few of the humps seemed to have ruptured, producing a crater-like appearance (Fig. 8). In some instances a crater seemed to have been produced by a collapse of the superficial surface of a hump; in such instances a small shrouded mass, probably representing a shrunken chondrocyte, was seen in the centre of the crater (Fig. 9). A fairly constant and characteristic appearance of kitten cartilage dried by the critical point method was the presence of innumerable extremely fine holes which gave the surface (at high magnifications) a porous appearance. Six to eight months old specimens The air dried specimens from this age group showed humps, humps in pits, and pits. In some animals the humps predominated, in others there were more pits. A feature of interest detected in two of these animals was that, while the anterior part of the condyle showed humps and humps in pits, in the posterior part humps were absent and only quite shallow pits, difficult to record, were present (Figs 10, 11). Such features were even more difficult to discern in specimens dried by the critical point method, although at higher magnifications, and at high tilt angles, one could discern quite large shallow pits (Fig. 12).
Twelve and twenty months old specimens In the air dried specimens from these animals innumerable pits were easily detected. This gave the surface a beaten-copper, or golf ball-like, appearance, characteristic of adult cartilage viewed with the scanning electron microscope. Only a rare small flat hump in a pit was seen in the 12 months old cat, but we could not find this feature in the 20 months old animal. However, in this animal occasional long straight fibres were seen traversing the surface in some areas of the specimen (Fig. 13). DISCUSSION
Undulations and ridges The present study provides further evidence in support of the idea that ridges and undulations are not a constant feature of the normal articular surface as has been suggested by some authors (McCall, 1968; Gardner & Woodward, 1969; Walker et al. 1969; Redler & Zimny, 1970; Gardner, 1972; Mow et al. 1974; Redler, 1974), for such features were only seen in some areas of air dried kitten cartilage, where marked shrinkage and distortion of the specimen has occurred, but not in specimens dried by the critical point method, where such changes were absent or minimal. This observation is in keeping with our past study, where we found ridges on the surface of air dried young rabbit cartilage, but not on specimens dried by the critical point method (Ghadially et al. 1977b). That shrinkage and distortion can produce ridges and undulations is also evidenced by the fact that such features are not present on mature rabbit or dog cartilage attached to bone, but they are readily produced when cartilage is detached from bone or injured in various ways (Ghadially et al. 1976, 1977 a). The observations of 23-2
356 F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Fig. 10. Surface of anterior portion of an air dried femoral condyle from a 6-8 months old cat. Note humps (arrows) and pits (arrowheads). x 580.
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Fig. 11. Posterior end of condyle shown in Fig. 10. Note that only pits (P) are present. x 580.
Maturation change in articular cartilage
Fig. 12. Critical point dried condyle from a 6-8 months old cat. Note quite large but shallow pits (P) on the surface. x 1100.
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Fig. 13. Pits (P) and fibres (arrowhead) seen on the surface of an air dried femoral condyle from a 20 months old cat. Also seen are altered erythrocytes or cellular debris (arrow) acquired during specimen collection and preparation. x 1000.
357
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F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Clarke (1971, 1973 a) on adult human cartilage are essentially similar, for he too failed to find ridges on cartilage attached to bone, but found some adjacent to the fractured edges of the specimen. It seems to us that the artefactual nature of ridges and undulations has now been unequivocally established, and that this phenomenon when seen on normal cartilage is largely the result of tissue shrinkage and distortion.
Humps and pits These features are not quite as distinct and different as their names suggest, for small humps can at times be seen in the floor of pits, and large humps at times have a narrow moat around them suggesting that they too have originated from the floor of the pit. Both pits and humps are thought to reflect the presence of underlying chondrocytes and lacunae. The former feature is thought to be due to a caving in of the surface over a shrunken chondrocyte and lacuna (Clarke, 1973 a; Ghadially et al. 1976) while it has been suggested that the latter feature indicates a chondrocyte elevated above the general articular surface (Ghadially et al. 1977b). Support for this idea comes from our present study which shows that ruptured humps (Fig. 8) have a hollow interior, which one could interpret as a lacuna from which the cell had been lost. Even more intriguing is the appearance seen when the surface of a hump collapses (Fig. 9), for then a small central elevated mass is seen which can be interpreted as a shrunken chondrocyte lying under a shroud-like veil derived from the collapsed surface of the hump. The question now arises as to whether such craters occur in vivo, reflecting the way chondrocytes are lost from the surface as cartilage matures, or whether this is a preparative artefact. It is well known that the cellularity of the superficial zone of articular cartilage diminishes markedly as animals mature, but there is probably no further significant decrease in cellularity of cartilage as the animal becomes older (Stockwell & Meachim, 1972). It is thought that such reduction in cellularity is achieved by in situ necrosis of chondrocytes, and not by a shedding of cells from the surface (for references and review see Ghadially & Roy, 1969). Such contentions, however, are based on a study of articular cartilage from more mature animals, and not from very young animals such as were used in this study. Thus no firm answer can be given to the question posed by the craters found in this study. Whether pits and humps occur in vivo is debatable, but the results of our present study clearly show that tissue shrinkage is at least a factor in their production, for these features were more prominent in air dried specimens where tissue shrinkage is quite marked, than in critical point dried specimens where such changes are minimal. The results of light microscopic studies aimed at resolving this dilemma are singularly difficult to interpret. The most extensive studies on this point are those by Gardner and his colleagues who at first (see review by Gardner, 1972) described systems of undulations of various dimensions on the surface of human articular cartilage, but later (Longmore & Gardner, 1975), talked about 'quaternary ridges' and 'tertiary hollows' (presumably the same as our pits). Nevertheless one remains unconvinced that such features occur in vivo. In their recent report Longmore & Gardner (1975) deal with human articular cartilage collected at autopsy from persons 0-47 years old. Pieces of cartilage detached from bone were examined by scanning electron microscopy, and cartilage attached to condyles was studied by reflected light interference microscopy. It seems remarkable that no mention of humps is made in the text, even though humps are
359 Maturation change in articular cartilage quite clearly seen in Figure 14 in their paper which depicts the scanning electron microscopic appearance of the articular surface from the femoral condyle of a 10 months old infant. Pits were detected by interference microscopy, but the significance of this observation is limited by the use of autopsy material in which shrinkage of chondrocytes and other post-mortem changes would be expected to occur. It may be argued that whether pits or humps develop depends upon variations in preparative procedures. However, such a contention appears to be untenable, for in the present study we found that in 6 to 8 months old cat cartilage, humps, humps in pits, and pits occurred in one and the same specimen. This clearly absolves the preparative procedures from responsibility and indicates that one must seek for an explanation of these differences (humps and pits) in the tissue itself. But this must not be construed to mean that pits and humps occur in vivo, or that they are not engendered by preparative procedures, only that artefacts though they may be, they are meaningful artefacts which show a difference between immature young and mature adult cartilage. Speculations regarding the nature of such differences have been discussed in a previous paper (Ghadially et al. 1977b) and will not be repeated here. In a previous study (Ghadially et al. 1977b) we showed that the surface of young rabbit and juvenile human cartilage shows humps but published reports on adult cartilage from these species indicate that it is covered by pits. The present study clearly establishes that the change of surface topography from humps to pits is a maturation phenomenon. In the cartilage of kittens up to 2 months old, innumerable humps are seen but no pits are found. At the age of 6-8 months humps begin to be transformed into or replaced by pits. This change seems to commence on the posterior part of the condyle, for here pits are found while humps and humps in pits are still present on the anterior part of the same condyle. One may well envisage that progression of such changes ultimately (12-20 months old specimens) produces the adult-type articular surface where the surface is covered with innumerable pits and only a rare hump in a pit, or no humps at all, are seen. Another probably significant change revealed by our study is the occurrence of fibres on the surface of the articular cartilage of the 20 months old cat. Since this feature was witnessed in only one animal no categorical statement can be made, but it seems likely that this is not a maturation change but a regressive age change involving loss of surface matrix and laying bare of superficial fibres. SUMMARY
The articular surface of the femoral condyle of kittens (2 days to 2 months old) and cats (6 months to 20 months old) was studied by examining air dried and critical point dried specimens in the scanning electron microscope. The surface of kitten cartilage was found to be populated by innumerable humps which were more prominent in air dried than in critical point dried specimens. Undulations and ridges were seen on air dried kitten cartilage, which was markedly shrunken and distorted, but undulations and ridges were absent from critical point dried specimens where shrinkage was more modest or even undetectable. The surface of the articular cartilage of 12 months and 20 months old cats was populated by innumerable pits. A rare hump in a pit was seen in specimens from the 12 months old cat, but not from the 20 months old animal. In 6-8 months old cats an intermediate situation prevails, for in some specimens pits were present on the
360 F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
posterior part of the condyle but humps and humps in pits were present on the anterior aspect. This study shows that the surface of young articular cartilage is populated by humps, but as the cartilage matures these formations are either transformed into pits or replaced by pits.
This work was supported by grants from the Medical Research Council of Canada and the Canadian Arthritis and Rheumatism Society. REFERENCE S CLARKE, I. C. (1971a). Human articular surface contours and related surface depression frequency studies. Annals of the Rheumatic Diseases 30, 15-23. CLARKE, f. C. (1971 b). A method for the replication of articular cartilage surfaces suitable for the scanning electron microscope. Journal of Microscopy 93, 67-71. CLARKE, I. C. (1971 c). Surface characteristics of human articular cartilage - a scanning electron microscope study. Journal of Anatomy 108, 23-30. CLARKE, I. C. (1973a). Correlation of S.E.M. replication and light microscopy studies of the bearing surfaces in human joints. Scanning Electron Microscopy (Part 3). Proceedings of the Workshop on Scanning Electron Microscopy in Pathology (ed. 0. Johari & I. Corvin), pp. 659-666. Chicago: IH Research Institute. CLARKE, I. C. (1973 b). Quantitative measurement of human articular surface topography 'in vitro' by profile recorder and stereomicroscopy techniques. Journal of Microscopy 97, 309-314. GARDNER, D. L. (1972). The influence of microscope technology on knowledge of cartilage surface structure. Annals of the Rheumatic Diseases 31, 235-258. GARDNER, D. L. & WOODWARD, D. (1969). Scanning electron microscopy and replica studies of articular surface of guinea-pig synovial joints. Annals of the Rheumatic Diseases 28, 379-391. GHADIALLY, F. N., AILSBY, R. L. & ORYSCHAK, A. F. (1974). Scanning electron microscopy of superficial defects in articular cartilage. Annals of the Rheumatic Diseases 33, 327-332. GHADIALLY, F. N., GHADIALLY, J. A., ORYSCHAK, A. F. & YONG, N. K. (1976). Experimental production of ridges on rabbit articular cartilage: a scanning electron microscope study. Journal of Anatomy 121, 119-132. GHADIALLY, F. N., GHADIALLY, J. A., ORYSCHAK, A. F. & YONG, N. K. (1977a). The surface of dog articular cartilage: A scanning electron microscopic study. Journal of Anatomy 123, 527-526. GHADIALLY, F. N., MOSHURCHAK, E. M. & THOMAS, I. (1977b). Humps on young human and rabbit articular cartilage. Journal of Anatomy 124, 425-435. GHADIALLY, F. N. & Roy, S. (1969). Ultrastructure of Synovial Joints in Health and Disease. London: Butterworths. LONGMORE, R. B. & GARDNER, D. L. (1975). Development with age of human articular cartilage surface structure. A survey by interference microscopy of the lateral femoral condyle. Annals of the Rheumatic Diseases 34, 26-37. MCCALL, J. G. (1968). Scanning electron microscopy of articular surfaces. Lancet ii, 1194. MOW, VAN C., LAI, W. M. & REDLER, I. (1974). Some surface characteristics of articular cartilage. 1. A scanning electron microscopy study and a theoretical model for the dynamic interaction of synovial fluid and articular cartilage. Journal of Biomechanics 7, 449-456. REDLER, I. (1974). A scanning electron microscopic study of human normal and osteoarthritic articular cartilage. Clinical Orthopaedics and Related Research 103, 262-268. REDLER, I. & ZIMNY, M. L. (1970). Scanning electron microscopy of normal and abnormal articular cartilage and synovium. Journal of Bone and Joint Surgery 52-A, 1395-1404. STOCKWELL, R. A. & MEACHIM, G. (1972). The chondrocyte. In Adult Articular Cartilage (ed. M. A. R. Freeman). New York: Grune & Stratton. WALKER, P. S., SIKORSKI, J., DOWSON, D., LONGFIELD, M. D., WRIGHT, V. & BUCKLEY, T. (1969). Behaviour of synovial fluid on surfaces of articular cartilage. A scanning electron microscope study. Annals of the Rheumatic Diseases 28, 1-14.
349
A maturation change in the surface of cat articular cartilage detected by the scanning electron microscope F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Department of Pathology, University Hospital, University of Saskatchewan, Saskatoon, Canada
(Accepted 26 January 1977) INTRODUCTION
Since the advent of scanning electron microscopy a variety of surface irregularities such as (1) undulations and ridges, (2) oval depressions callea 'pits', and (3) occasional mound-like elevations referred to as 'humps', have been described on the surface of normal articular cartilage (McCall, 1968; Gardner & Woodward, 1969; Walker et al. 1969; Redler & Zimny, 1970; Clarke, 1971 a, b, c, 1973 a, b; Gardner, 1972; Ghadially, Ailsby & Oryschak, 1974; Mow, Lai & Redler, 1974). However, it has now been demonstrated that undulations and ridges are not a feature of the normal articular cartilage attached to bone but that such irregularities readily develop because of shrinkage and distortion when cartilage is detached from bone or is cut or injured in various ways, and that under such circumstances occasional humps are also seen (Ghadially, Ghadially, Oryschak & Yong, 1976, 1977a). Redler (1974) found some humps on human articular cartilage and thought that this feature was of more common occurrence on the articular cartilage of juveniles. However, the fact that this author considers ridges to be a constant feature of the normal articular surface raises the possibility that the humps, like the ridges, were artefactually produced by injury to cartilage or by detaching it from bone. The chance observation of humps, but no pits, on articular cartilage (attached to bone) of an 8 years old boy aroused our suspicion that a maturation change detectable by the scanning electron microscope perhaps occurs in the articular surface. We therefore studied the articular cartilage from young rabbits and found that there too humps abounded. Comparing these findings with the surface morphology of adult human and rabbit cartilage (where pits abound) described in the literature, we proposed that the surface of young and juvenile cartilage exhibits humps, but as the animal matures the humps are either transformed into, or replaced by, pits (Ghadially Moshurchak & Thomas, 1977b). Since this hypothesis is supported by comparatively few actual observations, and because one may argue that the preparative procedures adopted by us for the study of young and juvenile cartilage may not have been exactly the same as those used by us and others to study adult cartilage, it seemed desirable to carry out a more detailed and systematic study in another species to test the validity of our hypothesis. We now present results of such a study carried out on the cartilage of kittens and cats, using both air drying and critical point drying methods of tissue preparation. These validate our hypothesis, indicate how the cartilage changes occur, and show differences engendered by these two preparative techniques.
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F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Fig. 1. Air dried right medial femoral condyle from a 3 weeks old kitten. Note shrunken appearance and distorted surface. x 13. Fig. 2. Critical point dried left medial femoral condyle from same kitten as in Fig. 1. Note smooth appearance of surface and an accidental cut inflected during specimen preparation (arrowhead). x 13. MATERIALS AND METHODS
A total of 12 kittens and cats from 2 days to 20 months old were used in this study. There were five kittens, aged 2 days, 2 weeks, 3 weeks, 1 month and 2 months; five cats between 6 and 8 months old; and two cats aged 12 months and 20 months. The animals were killed by an intracardiac injection of Nembutal, and the lower ends of the femora from both legs were collected from all animals. The articular surface was then washed thoroughly in normal saline to remove synovial fluid and any blood that might have contaminated it. At no stage of the procedure was the articular surface touched or allowed to become dry. All specimens were fixed in 3 % glutaraldehyde in cacodylate buffer (pH 7 3). The specimens from the cats (6 months to 20 months) were fixed for a period of 2 weeks, a time interval which we have found to be optimal for the larger specimens from more mature animals. In the case of the kittens a shorter fixation period of 1 week was employed. All specimens were then dehydrated in increasing concentrations of ethanol over the course of another week. One of the femoral condyles from each animal between 2 weeks and 8 months old was dissected off with a knife or a fine fret saw and processed by the critical point drying method, using substitution with amyl acetate and carbon dioxide. The other condyle from each animal in this age group (2 weeks to 8 months) and all condyles from 2 days, 12 months and 20 months old animals were air dried. Critical point drying was not attempted in the case of the 2 days specimen because of its small size and fragility, while the large condyles from older cats were difficult to accommodate in the equipment used and there were difficulties also with penetration and exchange of amyl acetate with carbon dioxide. Specimens were mounted with the aid of Electrodag on aluminium stubs. For small specimens standard stubs (12 mm in diameter and 3 mm thick) were employed, but the larger condyles from the older cats were mounted on specially prepared larger lower aluminium stubs (15 mm in diameter and 1 mm thick).
Maturation change in articular cartilage
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Fig. 3. Numerous closely packed humps (H) are seen on the surface of an air dried condyle from a 2 days old kitten. x 1150. RESULTS
Two days to two months old specimens Naked eye inspection, as well as examination with a x 7 magnifying glass, of fresh and processed specimens from these kittens showed that marked shrinkage and irregularity of the surface developed in air dried specimens, but this was not evident in critical point dried specimens. Such shrinkage was more marked in younger members of this group. The difference between air dried and critical point dried specimens could also be readily demonstrated at low magnification in the scanning electron microscope. Figure 1 shows a markedly shrunken air dried right medial femoral condyle with a distorted surface from a 3 weeks old kitten, while Figure 2 illustrates the critical point dried left medial femoral condyle from the same animal. It is evident that the critical point dried specimen is much larger (i.e. less shrunken) and the surface is smoother. Besides the gross irregularities engendered by shrinkage, the air dried specimens showed a surface so densely covered with humps that the articular cartilage had a cobble-stoned appearance (Fig. 3). Once again this change was somewhat more marked in the younger than in the older (1 month and 2 months old) kittens, so that in the latter the humps were less pronounced and not quite so closely packed as in the former. At higher magnifications the surface of the humps appeared quite rough and wrinkled (Fig. 4). The difference between air dried and critical point dried specimens (at higher magnifications than in Figures 1 and 2) is illustrated in Figures 5 and 6, which show the right and left medial femoral condyle respectively from the 3 weeks old kitten. It will be noted that closely packed humps are seen on the air dried specimen, but on
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F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
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Fig. 5. Air dried right medial femoral condyle from a 3 weeks old kitten showing numerous humps (H). For comparison with Fig. 6. x 1000.
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Fig. 7. Air dried femoral condyle from a 4 weeks old kitten showing humps (arrowheads) amongst parallel ridges (arrows). x 580. 23
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354 F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Fig. 8. Critical point dried condyle from a 4 weeks old kitten. Some of the humps appear to have ruptured and produced craters (arrowheads). x 650.
9! Fig. 9. Critical point dried condyle from a 2 weeks old kitten showing a crater presumably produced by the collapse of a hump. Note small shrouded mass (arrow) in the centre of the crater which probably represents a shrunken chondrocyte. The general surface of the specimen is beset by innumerable fine holes (arrowheads). x 2500
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355 the critical point dried specimen the humps appear fewer, less elevated, and separated by a relatively flat but finely wrinkled surface. In areas of the air dried specimens where considerable surface distortion and caving in or curling had occurred, the humps were seen to lie amongst a series of parallel ridges or undulations (Fig. 7). In some of the specimens dried by the critical point method a few of the humps seemed to have ruptured, producing a crater-like appearance (Fig. 8). In some instances a crater seemed to have been produced by a collapse of the superficial surface of a hump; in such instances a small shrouded mass, probably representing a shrunken chondrocyte, was seen in the centre of the crater (Fig. 9). A fairly constant and characteristic appearance of kitten cartilage dried by the critical point method was the presence of innumerable extremely fine holes which gave the surface (at high magnifications) a porous appearance. Six to eight months old specimens The air dried specimens from this age group showed humps, humps in pits, and pits. In some animals the humps predominated, in others there were more pits. A feature of interest detected in two of these animals was that, while the anterior part of the condyle showed humps and humps in pits, in the posterior part humps were absent and only quite shallow pits, difficult to record, were present (Figs 10, 11). Such features were even more difficult to discern in specimens dried by the critical point method, although at higher magnifications, and at high tilt angles, one could discern quite large shallow pits (Fig. 12).
Twelve and twenty months old specimens In the air dried specimens from these animals innumerable pits were easily detected. This gave the surface a beaten-copper, or golf ball-like, appearance, characteristic of adult cartilage viewed with the scanning electron microscope. Only a rare small flat hump in a pit was seen in the 12 months old cat, but we could not find this feature in the 20 months old animal. However, in this animal occasional long straight fibres were seen traversing the surface in some areas of the specimen (Fig. 13). DISCUSSION
Undulations and ridges The present study provides further evidence in support of the idea that ridges and undulations are not a constant feature of the normal articular surface as has been suggested by some authors (McCall, 1968; Gardner & Woodward, 1969; Walker et al. 1969; Redler & Zimny, 1970; Gardner, 1972; Mow et al. 1974; Redler, 1974), for such features were only seen in some areas of air dried kitten cartilage, where marked shrinkage and distortion of the specimen has occurred, but not in specimens dried by the critical point method, where such changes were absent or minimal. This observation is in keeping with our past study, where we found ridges on the surface of air dried young rabbit cartilage, but not on specimens dried by the critical point method (Ghadially et al. 1977b). That shrinkage and distortion can produce ridges and undulations is also evidenced by the fact that such features are not present on mature rabbit or dog cartilage attached to bone, but they are readily produced when cartilage is detached from bone or injured in various ways (Ghadially et al. 1976, 1977 a). The observations of 23-2
356 F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Fig. 10. Surface of anterior portion of an air dried femoral condyle from a 6-8 months old cat. Note humps (arrows) and pits (arrowheads). x 580.
iI
-.
Fig. 11. Posterior end of condyle shown in Fig. 10. Note that only pits (P) are present. x 580.
Maturation change in articular cartilage
Fig. 12. Critical point dried condyle from a 6-8 months old cat. Note quite large but shallow pits (P) on the surface. x 1100.
'3
Fig. 13. Pits (P) and fibres (arrowhead) seen on the surface of an air dried femoral condyle from a 20 months old cat. Also seen are altered erythrocytes or cellular debris (arrow) acquired during specimen collection and preparation. x 1000.
357
358
F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
Clarke (1971, 1973 a) on adult human cartilage are essentially similar, for he too failed to find ridges on cartilage attached to bone, but found some adjacent to the fractured edges of the specimen. It seems to us that the artefactual nature of ridges and undulations has now been unequivocally established, and that this phenomenon when seen on normal cartilage is largely the result of tissue shrinkage and distortion.
Humps and pits These features are not quite as distinct and different as their names suggest, for small humps can at times be seen in the floor of pits, and large humps at times have a narrow moat around them suggesting that they too have originated from the floor of the pit. Both pits and humps are thought to reflect the presence of underlying chondrocytes and lacunae. The former feature is thought to be due to a caving in of the surface over a shrunken chondrocyte and lacuna (Clarke, 1973 a; Ghadially et al. 1976) while it has been suggested that the latter feature indicates a chondrocyte elevated above the general articular surface (Ghadially et al. 1977b). Support for this idea comes from our present study which shows that ruptured humps (Fig. 8) have a hollow interior, which one could interpret as a lacuna from which the cell had been lost. Even more intriguing is the appearance seen when the surface of a hump collapses (Fig. 9), for then a small central elevated mass is seen which can be interpreted as a shrunken chondrocyte lying under a shroud-like veil derived from the collapsed surface of the hump. The question now arises as to whether such craters occur in vivo, reflecting the way chondrocytes are lost from the surface as cartilage matures, or whether this is a preparative artefact. It is well known that the cellularity of the superficial zone of articular cartilage diminishes markedly as animals mature, but there is probably no further significant decrease in cellularity of cartilage as the animal becomes older (Stockwell & Meachim, 1972). It is thought that such reduction in cellularity is achieved by in situ necrosis of chondrocytes, and not by a shedding of cells from the surface (for references and review see Ghadially & Roy, 1969). Such contentions, however, are based on a study of articular cartilage from more mature animals, and not from very young animals such as were used in this study. Thus no firm answer can be given to the question posed by the craters found in this study. Whether pits and humps occur in vivo is debatable, but the results of our present study clearly show that tissue shrinkage is at least a factor in their production, for these features were more prominent in air dried specimens where tissue shrinkage is quite marked, than in critical point dried specimens where such changes are minimal. The results of light microscopic studies aimed at resolving this dilemma are singularly difficult to interpret. The most extensive studies on this point are those by Gardner and his colleagues who at first (see review by Gardner, 1972) described systems of undulations of various dimensions on the surface of human articular cartilage, but later (Longmore & Gardner, 1975), talked about 'quaternary ridges' and 'tertiary hollows' (presumably the same as our pits). Nevertheless one remains unconvinced that such features occur in vivo. In their recent report Longmore & Gardner (1975) deal with human articular cartilage collected at autopsy from persons 0-47 years old. Pieces of cartilage detached from bone were examined by scanning electron microscopy, and cartilage attached to condyles was studied by reflected light interference microscopy. It seems remarkable that no mention of humps is made in the text, even though humps are
359 Maturation change in articular cartilage quite clearly seen in Figure 14 in their paper which depicts the scanning electron microscopic appearance of the articular surface from the femoral condyle of a 10 months old infant. Pits were detected by interference microscopy, but the significance of this observation is limited by the use of autopsy material in which shrinkage of chondrocytes and other post-mortem changes would be expected to occur. It may be argued that whether pits or humps develop depends upon variations in preparative procedures. However, such a contention appears to be untenable, for in the present study we found that in 6 to 8 months old cat cartilage, humps, humps in pits, and pits occurred in one and the same specimen. This clearly absolves the preparative procedures from responsibility and indicates that one must seek for an explanation of these differences (humps and pits) in the tissue itself. But this must not be construed to mean that pits and humps occur in vivo, or that they are not engendered by preparative procedures, only that artefacts though they may be, they are meaningful artefacts which show a difference between immature young and mature adult cartilage. Speculations regarding the nature of such differences have been discussed in a previous paper (Ghadially et al. 1977b) and will not be repeated here. In a previous study (Ghadially et al. 1977b) we showed that the surface of young rabbit and juvenile human cartilage shows humps but published reports on adult cartilage from these species indicate that it is covered by pits. The present study clearly establishes that the change of surface topography from humps to pits is a maturation phenomenon. In the cartilage of kittens up to 2 months old, innumerable humps are seen but no pits are found. At the age of 6-8 months humps begin to be transformed into or replaced by pits. This change seems to commence on the posterior part of the condyle, for here pits are found while humps and humps in pits are still present on the anterior part of the same condyle. One may well envisage that progression of such changes ultimately (12-20 months old specimens) produces the adult-type articular surface where the surface is covered with innumerable pits and only a rare hump in a pit, or no humps at all, are seen. Another probably significant change revealed by our study is the occurrence of fibres on the surface of the articular cartilage of the 20 months old cat. Since this feature was witnessed in only one animal no categorical statement can be made, but it seems likely that this is not a maturation change but a regressive age change involving loss of surface matrix and laying bare of superficial fibres. SUMMARY
The articular surface of the femoral condyle of kittens (2 days to 2 months old) and cats (6 months to 20 months old) was studied by examining air dried and critical point dried specimens in the scanning electron microscope. The surface of kitten cartilage was found to be populated by innumerable humps which were more prominent in air dried than in critical point dried specimens. Undulations and ridges were seen on air dried kitten cartilage, which was markedly shrunken and distorted, but undulations and ridges were absent from critical point dried specimens where shrinkage was more modest or even undetectable. The surface of the articular cartilage of 12 months and 20 months old cats was populated by innumerable pits. A rare hump in a pit was seen in specimens from the 12 months old cat, but not from the 20 months old animal. In 6-8 months old cats an intermediate situation prevails, for in some specimens pits were present on the
360 F. N. GHADIALLY, E. M. MOSHURCHAK AND J. A. GHADIALLY
posterior part of the condyle but humps and humps in pits were present on the anterior aspect. This study shows that the surface of young articular cartilage is populated by humps, but as the cartilage matures these formations are either transformed into pits or replaced by pits.
This work was supported by grants from the Medical Research Council of Canada and the Canadian Arthritis and Rheumatism Society. REFERENCE S CLARKE, I. C. (1971a). Human articular surface contours and related surface depression frequency studies. Annals of the Rheumatic Diseases 30, 15-23. CLARKE, f. C. (1971 b). A method for the replication of articular cartilage surfaces suitable for the scanning electron microscope. Journal of Microscopy 93, 67-71. CLARKE, I. C. (1971 c). Surface characteristics of human articular cartilage - a scanning electron microscope study. Journal of Anatomy 108, 23-30. CLARKE, I. C. (1973a). Correlation of S.E.M. replication and light microscopy studies of the bearing surfaces in human joints. Scanning Electron Microscopy (Part 3). Proceedings of the Workshop on Scanning Electron Microscopy in Pathology (ed. 0. Johari & I. Corvin), pp. 659-666. Chicago: IH Research Institute. CLARKE, I. C. (1973 b). Quantitative measurement of human articular surface topography 'in vitro' by profile recorder and stereomicroscopy techniques. Journal of Microscopy 97, 309-314. GARDNER, D. L. (1972). The influence of microscope technology on knowledge of cartilage surface structure. Annals of the Rheumatic Diseases 31, 235-258. GARDNER, D. L. & WOODWARD, D. (1969). Scanning electron microscopy and replica studies of articular surface of guinea-pig synovial joints. Annals of the Rheumatic Diseases 28, 379-391. GHADIALLY, F. N., AILSBY, R. L. & ORYSCHAK, A. F. (1974). Scanning electron microscopy of superficial defects in articular cartilage. Annals of the Rheumatic Diseases 33, 327-332. GHADIALLY, F. N., GHADIALLY, J. A., ORYSCHAK, A. F. & YONG, N. K. (1976). Experimental production of ridges on rabbit articular cartilage: a scanning electron microscope study. Journal of Anatomy 121, 119-132. GHADIALLY, F. N., GHADIALLY, J. A., ORYSCHAK, A. F. & YONG, N. K. (1977a). The surface of dog articular cartilage: A scanning electron microscopic study. Journal of Anatomy 123, 527-526. GHADIALLY, F. N., MOSHURCHAK, E. M. & THOMAS, I. (1977b). Humps on young human and rabbit articular cartilage. Journal of Anatomy 124, 425-435. GHADIALLY, F. N. & Roy, S. (1969). Ultrastructure of Synovial Joints in Health and Disease. London: Butterworths. LONGMORE, R. B. & GARDNER, D. L. (1975). Development with age of human articular cartilage surface structure. A survey by interference microscopy of the lateral femoral condyle. Annals of the Rheumatic Diseases 34, 26-37. MCCALL, J. G. (1968). Scanning electron microscopy of articular surfaces. Lancet ii, 1194. MOW, VAN C., LAI, W. M. & REDLER, I. (1974). Some surface characteristics of articular cartilage. 1. A scanning electron microscopy study and a theoretical model for the dynamic interaction of synovial fluid and articular cartilage. Journal of Biomechanics 7, 449-456. REDLER, I. (1974). A scanning electron microscopic study of human normal and osteoarthritic articular cartilage. Clinical Orthopaedics and Related Research 103, 262-268. REDLER, I. & ZIMNY, M. L. (1970). Scanning electron microscopy of normal and abnormal articular cartilage and synovium. Journal of Bone and Joint Surgery 52-A, 1395-1404. STOCKWELL, R. A. & MEACHIM, G. (1972). The chondrocyte. In Adult Articular Cartilage (ed. M. A. R. Freeman). New York: Grune & Stratton. WALKER, P. S., SIKORSKI, J., DOWSON, D., LONGFIELD, M. D., WRIGHT, V. & BUCKLEY, T. (1969). Behaviour of synovial fluid on surfaces of articular cartilage. A scanning electron microscope study. Annals of the Rheumatic Diseases 28, 1-14.
