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Caveolar system of the articular chondroycte
JOURNAL OF ULTRASTRUCTURE RESEARCH
74, 1-10 (1981)
Caveolar System of the Articular Chondrocyte N O R M A N J, W I L S M A N , CORNELIA E . F A R N U M , AND D E B R A K . R E E D - A K S A M I T
Department of Veterinary Biology, University of Minnesota, St. Paul, Minnesota 55108 Received July 18, 1980 Tangential articular chondrocytes contain a complex system of membrane invaginations called caveolae. Caveolae are about 600 A in diameter, open to the extracellular space, and may attract and sequester calcium ions. On the average each chondrocyte has approximately 17 700 caveolae. This chondrocytie caveolar system appears remarkably similar to the caveolar system of smooth muscle ceils, and it is proposed that chondrocytic caveolae may contribute to an excitationcontraction coupling mechanism similar to the mechanism in smooth muscle cells.
Numerous circular- to flask-shaped invaginations, 60-90 nm in diameter, have been frequently observed in association with the chondrocytic plasma membrane. These invaginations, variously referred to as micropinocytotic vesicles, surface vesicles, intermediate vacuoles, or caveolae are clearly distinguished from coated vesicles by size, number, and membrane characteristics and have been assumed to have either an endocytotic or exocytotic role (3, 7, 11,
17, 20, 27, 37, 42, 43, 53). In smooth muscle cells an extensive system of similar invaginations of the plasma membrane has been studied in considerable detail. These invaginations, generally referred to as a caveolar system, have been shown to be permanent invaginations of the smooth muscle plasma membrane, restricted to the surface of the cell, and continuous with the extracellular space. Because of the surface position of this system and its abilities to accumulate calcium, this caveolar system is considered to be both homologous with and analagous to the T-axial tubular sYstem of skeletal muscle cells and thus an integral part of the contractile mechanism of smooth muscle cells (5, 12,
15, 18, 19, 52). While chondrocytes are known to be metabolically active ceils (29, 30), chondrocytes are generally not perceived of as actively contractile cells. However, in culture chondrocytes are in constant movement
and it has been proposed that in situ these cells may behave similarly (9, 21, 39, 43). While the mechanism of this contractile response in chondrocytes is unknown, chondrocytes do possess 60-A filaments which have some actin-like properties (24) and which could be similar to the contractile filaments of smooth muscle cells. It is our hypothesis that the plasma membrane invaginations of chondrocytes are similar to the caveolar system of smooth muscle cells and thus may be involved in an excitationcontractile coupling mechanism of the chondrocyte. The purpose of this paper is to describe the system of plasma membrane invaginations in normal articular chondrocytes and to compare its morphology, numerical density, and histochemical properties with the caveolar system of the smooth muscle cell. Chondrocytes from the tangential zone of articular cartilage were chosen as the model chondrocyte on which to investigate our hypothesis. MATERIALS AND METHODS Whole thickness slabs of articular cartilage were collected from the weight-bearing surface of the lateral femoral condyle of three anesthetized normal horses (2-4 years old) and four anesthetized dogs (2-3 years old). A portion of each slab was trimmed into l-ram3 blocks and fixed in 2.0% purified gluteraldehyde-2.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4; 726 mOSm) at room temperature for 2 hr. Blocks were subsequently rinsed and postfixed in aqueous 1% osmium tetroxide-/.5% potassium ferrocyanide at room 0022-5320/81/010001-10502.00/0 Copyright ~) 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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WILSMAN, FARNUM, AND REED-AKSAMIT
temperature for 1 hr. Potassium ferrocyanide was included in the postfixation solution to increase the preservation of glycogen and other complex carbohydrates (14).
Potassium Pyroantimonate Histochemistry Potassium pyroantimonate will precipitate in tissue in the presence of a number of cations; however, by using appropriate controls this technique can be made specific for calcium ions (38). A portion of each slab was trimmed into blocks less than 0.3 mm thick in aqueous 2.5% potassium pyroantimonate-2.0% osmium tetroxide (PAO) at 4°C for 2 hr. The PAO solution was made according to Simson and Spicer (38). Blocks were subsequently rinsed in water made slightly alkaline with potassium hydroxide. As a positive control, parotid salivary gland was collected, trimmed, and stained and fixed with PAO. As a negative control thinly sliced blocks were first placed in a 10 mM solution of ethylene glycol tris-N, N'-tetraacetic acid (EGTA) in a calcium magnesiumfree buffer at 4°C for 1 hr (6, 7, 38). Subsequently these blocks were quickly rinsed and then stained in PAO as described.
Lanthanum Hydroxide Histochemistry Lanthanum does not enter the intact cell and therefore is used as a marker for the extracellular space (22, 35). Blocks of cartilage were trimmed, fixed in 2% gluteraldehyde-2% paraformaldehyde, and rinsed as described. Blocks were then postfixed in aqueous 2.0% osmium tetroxide-l.5% potassium ferrocyan i d e - l % lanthanum hydroxide at room temperature for 1 hr. The lanthanum hydroxide was prepared from lanthanum nitrate as described by Revel and Karnovski (35).
they would be sectioned either parallel with or perpendicular to the articular surface. Ultrathin sections from the PAO and lanthanum experiments were examined both unstained and stained. Other sections were first stained with uranyl acetate and lead citrate and then examined in a Zeiss EM-10A electron microscope.
Stereology Numerical Density The ayerage number of caveolae per chondrocyte, N/o, was determined from (49)
Nc = Nv(¢, 0)/19. . . . . la/D(c~,
0)chond~ocyte
,
(1)
where A70 (~b,0) is the average number of caveolar profiles per chondrocytic profile on sections cut at the user-defined orientation (qS,0). In this study (6,0) is defined as perpendicular to the articular surface. Assuming caveolae are uniform spheres, /)/caveo~a is simply the diameter of the caveola, a n d / ~ (~b,0)/chonaro~yte is the mean projected diameter of the chondrocyte perpendicular to the defined plane of section (49). Using tissue prepared for standard electron microscopy, five blocks of equine articular cartilage were randomly selected and one ultrathin section of the tangential region was cut and collected on a Formvarcoated 2 x 1-mm slit grid. With the section in the electron microscope the corners of the section were digitalized using the stage traverse and 10 pairs of random coordinates were computer-generated for each section. The cell profile nearest to each pair of coordinates was photographed at x 10 000. The number of caveolar profiles in each of the 50 chondrocytic profiles was counted and the mean number of caveolar profiles per chondrocytic profile, Np (~b,0), was calculated.
Dcareola All Blocks Following fixation all blocks were rapidly dehydrated in ethanol, cleared in propylene oxide, and embedded in Epon-Araldite. Blocks were oriented such that
Twenty caveolar profiles in each chondrocytic profile were randomly selected and their diameters, d, were measured. D'-/eaveol a w a s then calculated from d by
FIGS. 1-15. Electron micrographs of articular chondrocytes from the tangential zone of equine articular cartilage (Figs. 1-9 and 14-15) and canine articular cartilage (Figs. 10-13). FIGS. I AND 2. Typical tangential articular chondrocytes in sections perpendicular to the articular surface (Fig. 1) and parallel with the articular surface (Fig. 2). Both cells include fields of perinuclear filaments (F). Figure 1 includes a single row of caveolar profiles (between arrows) and a cilium (C). Fig. 1, x 24 000; Fig. 2, x 9500. FIG. 3. Field of transversly sectioned filaments, adjacent to the nucleaus (N). Based on size (100 A) and position these are most likely intermediate filaments, x 200 000. FIGS. 4-7. Chondrocytic plasma membrane sectioned in a plane perpendicular to te cell surface. Caveolae appear as a single row of invaginations of the plasma membrane. Communication of the inner area of a caveola and the extracellular matrix is through a constricted neck. On occasion caveolae appear to contact mitochondria (Figs. 4 and 5, arrows). In other sections profiles of endoplasmic reticulum, similar to the peripheral elements of the sarcoplasmic reticulum of smooth muscle cells, can be seen immediately deep to and even indented by caveolae (Figs. 6 and 7, 0). x 121 000.
j~
c
W I L S M A N , F A R N U M , AND R E E D - A K S A M I T
Caveolar Morphology
dC - T + [(T - dC) z + 2dT(sin 1C Dear =
+ C(1
C2)~)1~
sin-lC + C(1
, (2)
C2)½
where C = (1 - sZ)1/2 and s equals the ratio o f the smallest diameter to the largest diameter of caveolar profiles and T = section thickness (16).
D ((~,O)chondrocyte From each of five blocks used in this study, 30 serial 1-/xm-thick sections were cut and stained. All chondrocytes that appeared in the 15th section were followed in preceding and succeeding sections and the number of sections, n, in which a chondrocyte appeared was counted. The projected diameter in micrometers for a chondrocyte was estimated at n - 1 to account for the fact that end sections would be incompletely filled. Then D((b ,0)/chondrocytewas estimated by the mean of all individual measurements. RESULTS
Both equine and canine articular cartilage are about 1 mm thick, divisible into tangential, intermediate, radiate, and calcified zones, and in general resemble previous descriptions of the structure of articular cartilage (17, 40, 43). In particular, tangential zone chondrocytes are oblate ellipsoids so that in sections cut perpendicular to the articular surface cell profiles are oval and about 12/xm long and 3/xm high. In sections cut parallel with the articular surface cell profiles are circular and about 12/xm in diameter (Figs. 1, 2). In all profiles large arrays of highly oriented filaments were observed. Filaments 60 A in diameter were located near the plasma membrane and extended into cell processes (Figs. 5, 12) while dense fields of 100-A-diameter filaments occupied a perinuclear position (Figs. 2, 3).
In sections cut perpendicular to the plasma membrane of the chondrocyte caveolae appeared as a single row of circular-to-oval invaginations of the plasma membrane. Each invagination was bounded by unit membrane coated with dense amorphous material on its concave side. Most caveolae opened to the extracellular matrix through a constricted neck (Figs. 4-8). While some caveolae did not appear to connect to the plasma membrane, the continuity of these caveolae and the extracellular space out of the plane of section was confirmed by finding lanthanum precipitates within each of these caveolae (Fig. 9). In grazing sections oriented tangentially to the plasma membrane, large fields of evenly spaced caveolar profiles were observed (Figs. 10, 11). Almost always caveolae appeared single although aggregates of two to five caveolae were seen. Occa-' sionally a connection between adjacent caveolae was present (Fig. 11). Caveolae occurred around the entire periphery of the cell including the periphery of cell processes (Fig. 12); however, in grazing sections through cell processes it appeared as if the entire process was filled with caveolae (Fig. 13). Immediately deep to the caveolae a sparse system of smooth endoplasmic reticulum was observed (Figs. 6-8). The terminal portions of this reticulum consisted of 1500-A dilations while the deeper aspects of these membranes had ribosomes. Occasionally mitochondria were found immediately deep to caveolae and in some cases
FIG. 8. Interrelationships of a caveolar chain (continuity out o f the plane of section), with a mitochondrion and peripheral elements of endoplasmic reticulum (Q). x 121 000. FIG. 9. Precipitate of the extracellular marker lanthanum is found lining the plasma membrane and caveolar profiles. While some caveolae do not appear to connect with the plasma membrane (arrows), continuity with the extracellular space out of the plane of section is demonstrated by finding lanthanum precipitates within these caveolar profiles, x 34 000. Fins. 10 AND 11. Fields of caveolar profiles viewed in grazing sections o f the chondrocytic plasma membrane. While most caveolae appear singular, a number of examples of fused caveolae can be seen. Figure 10 contains 560 caveolae. Fig. 10, × 14 000; Fig. 11, x 121 000.
CHONDROCYTIC CAVEOLAE
®
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WILSMAN, FARNUM, AND REED-AKSAMIT
mitochondria and caveolae appeared to be attached to each other (Figs. 4, 5).
Potassium Pyroantimonate Histochemistry In sections from tissue that had been fixed and stained with 2% potassium antimonate (PAO), an electron-dense precipitate was observed along the entire plasma membrane including the cell processes. In places where cell processes were sectioned tangentially the entire process appeared to be filled with precipitate. H e a v y deposits o f precipitate were also observed in mitochondria and surrounding lipid droplets. T h e Golgi region and the euchromatic regions o f the nucleus showed light staining precipitate, while no precipitates were present in the dense fields o f filaments, lysosomes, or the heterochromatic portions of the nucleus (Fig. 14). T h o s e sections from tissue p r e t r e a t e d with E G T A prior to PAO showed essentially no precipitate anywhere in the cell (Fig. 15).
Stereology Numerical Density Np (4~,0). The mean number of caveolar profiles per chondrocytic profile averaged over all 50 chondrocytes observed was 88.6 (SD = 21.1). LSc~veo~. . The average diameter, d, of the 1000 caveolar profiles measured was 550 A (SD = 41A) and s, the ratio of smallest to largest, was 0.387. If section thickness is assumed to be 700 A then /)/caveota, calculated from Eq. (2), was estimated at 594 A. (q5,O) chondrocyte. Overall 82 tangential chondrocytes were available for study in the five series of serial sections. The
mean / ) (~b,0) chondrocyte was 11.9 ~ m (SD = 2.1/zm). Nc determination. With the n e c e s s a r y parameters determined the average n u m b e r o f caveolae per chondrocyt__e was calculated from Eq. (1). The mean, N/c, was estimated at 17 700 caveolae per chondrocyte. DISCUSSION There is evidence to suggest that articular chondrocytes migrate through their surrounding dense extracellular matrix. It is known that articular chondrocytes are born in a narrow zone subjacent to the tangential zone and presumably migrate from this region (27, 28, 34). Second, chondrocytes in cell culture are known to be in almost constant motion and presumably this reflects similar motion in vivo (9, 21, 25, 39, 43). Third, there is evidence that while most of the increase in cell density adjacent to cartilage lesions is due to mitosis, some of the increase in cell density may be due to a recruitment of chondrocytes from the surrounding matrix (23, 41, 43, 44, 48). The excitation-contraction coupling mechanism of smooth muscle cells consists of an extensive system of calcium-attracting caveolae functioning analogously to the T-axial tubular system of skeletal muscle, a sarcoplasmic reticulum, and dense fields of thick and thin filaments (5, 15). What we propose is that the contractile mechanism of chondrocytes may resemble (although is probably less developed than) the contractile mechanism of smooth muscle cells, and our initial evaluation of this proposal has been to compare the extensive system of invaginations of the chondrocytic plasma
FIGS. 12 AND 13. Caveolae as they appear in a cell process that has been sectioned transversly (Fig. 12) and horizontally (Fig. 13). × 121000. FIG. 14. Chondrocyte fixed and stained with potassium antimonate-osmium tetroxide (PAO) and examined without additional staining. PAO deposits are found along the entire length of plasma membrane including cell processes. × 15 000. FIG. 15. Chondrocyte pretreated with EGTA prior to reaction with PAO. Since pretreatment with EGTA essentially eliminates the reaction product, we concluded the reaction product in Fig. 14 was caused by calcium ions. × 24 000.
•
~l~
~,
x
8
WILSMAN, FARNUM, AND REED-AKSAMIT
membrane with the caveolar system of the smooth muscle cell. This study has demonstrated that the caveolar systems of smooth muscle cells and chondrocytes are remarkably similar in shape, size, position, numerical density, calcium affinity, and membrane characteristics. The caveolae of chondrocytes are flask- or circular-shaped structures about 600 A in diameter and similar in both size and shape to individual caveolae in smooth muscle cells (18, 19, 38). Chondrocytic caveolae, like smooth muscle caveolae, open to the extracellular space through a narrow neck. In some micrographs this opening was out of the plane of section; however, in micrographs prepared with the extracellar marker lanthanum hydroxide those caveolae with no visible connection with the extracellular matrix contained lanthanum precipitate indicating a communication with the matrix out of the plane of section. Thus caveolae of chondrocytes like caveolae of smooth muscle cells appear to be permanent fixtures of the plasma membrane in communication with the extracellular space. Potassium antimonate will precipitate in tissue sections in the presence of a number of cations (38); however, the lack of precipitate seen in this study following pretreatment with EGTA indicates that the precipitate seen in this study is specific for calcium ions (6, 7). Dense PAO precipitate was deposited inside the plasma membrane in a narrow zone containing the caveolae. The specificity of the localization of precipitate suggests a structural basis for its deposition. This indirectly suggests that chondrocytic caveolae like smooth muscle caveolae attract and perhaps concentrate and sequester calcium ions (7, 22, 32, 33). Other authors have also suggested a direct relationship between positive PAO staining and chondrocytic surface vesicles (6, 7, 26). However, direct visualization of PAO precipitate within caveolar profiles is not possible because membranes even with
poststaining are not resolvable using PAO procedures. Perhaps the only structural difference between caveolae of chondrocytes and those of smooth muscle cells is that the caveolae of smooth muscle cells, especially those of coronary artery smooth muscle, are frequently seen in the form of fused caveolae or branching chains of caveolae (15). While some complex fused caveolae were observed in chondrocytes these most frequently occurred only as doublets and the elongate chains and multiple configurations of caveolae described in smooth muscle were lacking. This structural difference is perhaps refleeted in a comparison of the volume densities of caveolae in chondrocytes and smooth muscle cells. Using an estimate of 17700 caveolae per chondrocyte and assuming that caveolae are spheres of 600 A in diameter and that chondrocytes are oblate ellipsoids with a major axis of 11.9/,m and a minor axis of 3 txm, it is possible to estimate the volume density, Vv, of caveolae in chondrocytes at 0.01. This estimate compares with an estimate of 0.020.03 for the volume density of caveolae in smooth muscle cells (32). Forbes has recently suggested that a universal aspect of excitation-contraction coupling in muscle cells is sarcoplasmic invaginations ranging in complexity from the T-axial tubular system of striated muscle to caveolar chains of varying lengths to single caveolae (15). The presence of caveolae in chondrocytes suggests that these cells also possess this surface modification and thus may be capable of contraction and movement in vivo. All of these invaginated structures potentially extend the extracellular fluid space and its ions to deeper regions within the cell. Other components of an excitation-contractile coupling mechanism for chondrocytes such as an analog to the sarcoplasmic reticulum and contractile filaments remain unstudied. In many chondrocytic profiles dilated smooth endoplas-
CHONDROCYTIC CAVEOLAE
mic reticulum was observed immediately deep to the surface caveolae and extending to close approximation with mitochondria. These structures are suggestions of the previously described peripheral or junctional sarcoplasmic reticulum of smooth muscle cells (15, 52). Obviously any excitation-contraction coupling mechanism requires the presence of contractile filaments. Chondrocytes are known to possess dense fields of 100-A filaments; however, these 100-A filaments have the morphological characteristics of intermediate filaments and their function is unknown. They are considered by some to be a sign of degeneration and impending cell death (4, 31, 42). However, we have found that although the density of 100-A filaments varies in different profiles of any particular cell, the presence of dense fields of filaments is clearly visible in all chondrocytes followed in serial section. It has been estimated that these filaments comprise approximately 30-40% of the volume fraction of normal equine articular chondrocytes (2) and we have not been able to correlate the p r e s e n c e of 100-A filaments with any changes in other cell components which would indicate cell degeneration. The 60-A filaments subjacent to the plasma membrane appear similar in size and position to chondrocytic filaments previously shown to have the actin-like property of forming arrowhead complexes with heavy meromyosin (24). We would propose that the 60-A filaments are most likely contractile filaments. Articular cartilage is, of course, aneural. Therefore excitation depolarization of the plasma membrane is not achieved by the peripheral nervous system. However, each articular chondrocyte is known to possess one cilium (50, 51) presumed to be nonmotile because of the lack of dynein arms and regularly arranged radial spokes (1, 13, 36, 46). There are a number of known examples where deflection of a nonmotile cilium results in depolarization of the cell
membrane (8, 10, 45, 47). Therefore it is possible to hypothesize that perhaps chondrocytes possess a similar mechanism to initiate the event of excitation contraction. The presence of caveolae in articular chondrocytes is clearly not a new observation as caveolae have been described by numerous authors. We believe the significance of this present study, in light of the extensive studies of the caveolar system in smooth muscle cells, lies in defining the extent to which a caveolar system exists in articular chondrocytes and redefining the function that these structures may play in the biology of the articular chondrocyte.
REFERENCES 1. AFZELIUS, B. A. (1976) Science 193, 317. 2. ALTON, E. (1980) M.S. thesis, University of Minnesota. 3. BARKER, W. D., AND MARTINEK, J. (1976) Clin. Orthop. Rel. Res. 115, 286. 4. BARNETT, C. H., COCHRANE, W., AND PALFREY, A. J. (1963) Ann. Rheum. Dis. 22, 389. 5. BoIs, R. M. (1973) Anat. Rec. 177, 61. 6. BRIGHTON, C. T., AND HUNT, R. M. (1974) Clin. Orthop. Rel. Res. 100, 406. 7. CARSON,F. L., DAVIS, W. L., MATTHEWS, J. L., AND MARTIN, J. H. (1978) Anat. Rec. 190, 23. 8. CHAPMAN, K. M., DUCKROW, R. B., AND MORAY, D. T. (1973) Nature (London) 244, 453. 9. CHESTERMAN, P. J., AND SMITH, A. U. (1968) J. Bone Joint Surg. 50-B, 184. 10. COGGESHALL,R. E. (1969) J. Morphol. 127, 113. 11. DAVIS, D. V., BARNETT, C. H., COCHRANE, W., AND PALFREY, A. J. (1962) Ann. Rheum. Dis. 21, 11. 12. DEVINE, C. E., SIMPSON, F. O., AND BERTAUD, W. S. (1971) J. Cell Sci. 8, 427. 13. FAWCETT, D. W. (1977) N. Engl. J. Med. 297, 46. 14. FAWCETT, D. W., AND DYM, M. (1974) J. Reprod. Fert. 38, 401. 15. FORBES, M. S., RENNELS, M. J., AND NELSON, E. 0979) J. Ultrastruct. Res. 67, 325. 16. FREEDMAN, L. S. (1973) J. Microsc. 100, 219. 17. FREEMAN, M. A. R. (1973) Adult Articular Cartilage, Pitman, London. 18. GABELLA,G. (1973) Phil. Trans. Roy. Soc. 265, 7. 19. GABELLA, G. (1971) J. Cell Sci. 8, 601. 20. GHADIALLY, F. N., MEHTA, P. N., AND KIRKALDY-WILLIS, H. (1970) J. Bone Joint Surg. 52-A, 1147.
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21. GREEN, W. T. (1971) Clin. Orthop. Rel. Res. 75, 248. 22. HAN, S. S., KIM, S. K., AND CHO, M. I. (1976) J. Anat. 122, 559. 23. HULTH, A., LINDBERG, L., AND TELHAG, H. (1972) Clin. Orthop. Rel. Res. 84, 197. 24. ISHIKAWA,H., BISCHOFF, R., AND HOLTZER, H. (1969) J. Cell Biol. 43, 312. 25. KAWlAK, J., MOSKALEWSKI, S., AND DARZYNKIEWICZ, A. (1965) Exp, Cell Res. 39, 59. 26. LEWINSON, D,, AND SILBERMANN, M. (1978) Metab. Bone Dis. Rel. Res. 1, 243. 27, MANKIN, H. J. (1964) Clin. Orthop. Rel. Res. 34, 170. 28. MANKIN, H. J. (1962) J. Bone Joint Surg. 44-A, 682. 29. MANKIN, H. J., AND LIPPIELLO, L. (1969) J. Bone Joint Surg. 51-A, 862. 30. MANKIN, H. J., AND LIPPIELLO,L. (1969) J. Bone Joint Surg. 51-A, 1591. 31. MEACHIM, G., AND RoY, S. (1967) Ann. Rheum. Dis. 26, 50. 32. MCGUFEEE, L. J., AND BAGLEY, R. M. (1976) Amer. J. Physiol. 230, 1217. 33. POPESCU, L. M. (1974) Stud. Biophys. 44, 141. 34. PRIBYLOVA,E., AND HERT, J. (1971) Folia Morphol. 19, 233. 35. REVEL, J. P., AND KARNOVSKY, M. J. (1967) J. Cell Biol. 33, C7-C12. 36. SATIR, P. (1965) J. Cell Biol. 39, 77. 37. SILBERMANN,M., AND LEWINSON, D. (1978) J. Anat. 125, 55.
38. SIMSON, J. A. V., AND SPICER, S. S. (1975) J. Histochem. Cytochem. 23, 575. 39. SMITH, A. U. (1965) Nature London 205, 782. 40. SOKOLOFF, L. (1978) The Joints and Synovial Fluid, Academic Press, New York. 41. SOKOLOEF,L. (1974) J. Rheum. 1, 9. 42. SPRINZ, R., AND STOCKWELL, R. A. (1976) J. Anat. 122, 91. 43. STOCKWEEE, R. A. (1979) Biology of Cartilage Cells, Cambridge Univ. Press, Cambridge. 44. TELHAG, H. (1972) Clin. Orthop. Rel. Res. 86, 224. 45. THURM, U. (1968) Syrup. Zool. Soc. London 23, 199. 46. WARNER,F. D., AND SATIR, 1°. (1974) J. CellBiol. 63, 35. 47. WEIDERHOLD,M. L. (1976) Annu. Rev. Biophys. Bioeng. 5, 39. 48. WEISS, C., AND MIROW, S. (1972) J. Bone Joint Surg. 54-A, 954. 49. WILSMAN,N. J. (1979) J. Histochem. Cytochem. 27, 1551. 50. W1LSMAN,N. J., FARNUM, C. E., AND REED-AKSAMIT, D. K. (1980) Anat. Rec., 197, 355. 51. WILSMAN, N. J. (1978) J. Ultrastruct. Res. 64, 270. 52. WOOTTON, G. S., AND GOOt~FbRD, P. J. (1975) Cell Tissue Res. 161, 119. 53. ZIMNY, M. L., AND REDLER, I. (1969) J. Bone Joint Surg. 51-A, 1179.
74, 1-10 (1981)
Caveolar System of the Articular Chondrocyte N O R M A N J, W I L S M A N , CORNELIA E . F A R N U M , AND D E B R A K . R E E D - A K S A M I T
Department of Veterinary Biology, University of Minnesota, St. Paul, Minnesota 55108 Received July 18, 1980 Tangential articular chondrocytes contain a complex system of membrane invaginations called caveolae. Caveolae are about 600 A in diameter, open to the extracellular space, and may attract and sequester calcium ions. On the average each chondrocyte has approximately 17 700 caveolae. This chondrocytie caveolar system appears remarkably similar to the caveolar system of smooth muscle ceils, and it is proposed that chondrocytic caveolae may contribute to an excitationcontraction coupling mechanism similar to the mechanism in smooth muscle cells.
Numerous circular- to flask-shaped invaginations, 60-90 nm in diameter, have been frequently observed in association with the chondrocytic plasma membrane. These invaginations, variously referred to as micropinocytotic vesicles, surface vesicles, intermediate vacuoles, or caveolae are clearly distinguished from coated vesicles by size, number, and membrane characteristics and have been assumed to have either an endocytotic or exocytotic role (3, 7, 11,
17, 20, 27, 37, 42, 43, 53). In smooth muscle cells an extensive system of similar invaginations of the plasma membrane has been studied in considerable detail. These invaginations, generally referred to as a caveolar system, have been shown to be permanent invaginations of the smooth muscle plasma membrane, restricted to the surface of the cell, and continuous with the extracellular space. Because of the surface position of this system and its abilities to accumulate calcium, this caveolar system is considered to be both homologous with and analagous to the T-axial tubular sYstem of skeletal muscle cells and thus an integral part of the contractile mechanism of smooth muscle cells (5, 12,
15, 18, 19, 52). While chondrocytes are known to be metabolically active ceils (29, 30), chondrocytes are generally not perceived of as actively contractile cells. However, in culture chondrocytes are in constant movement
and it has been proposed that in situ these cells may behave similarly (9, 21, 39, 43). While the mechanism of this contractile response in chondrocytes is unknown, chondrocytes do possess 60-A filaments which have some actin-like properties (24) and which could be similar to the contractile filaments of smooth muscle cells. It is our hypothesis that the plasma membrane invaginations of chondrocytes are similar to the caveolar system of smooth muscle cells and thus may be involved in an excitationcontractile coupling mechanism of the chondrocyte. The purpose of this paper is to describe the system of plasma membrane invaginations in normal articular chondrocytes and to compare its morphology, numerical density, and histochemical properties with the caveolar system of the smooth muscle cell. Chondrocytes from the tangential zone of articular cartilage were chosen as the model chondrocyte on which to investigate our hypothesis. MATERIALS AND METHODS Whole thickness slabs of articular cartilage were collected from the weight-bearing surface of the lateral femoral condyle of three anesthetized normal horses (2-4 years old) and four anesthetized dogs (2-3 years old). A portion of each slab was trimmed into l-ram3 blocks and fixed in 2.0% purified gluteraldehyde-2.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4; 726 mOSm) at room temperature for 2 hr. Blocks were subsequently rinsed and postfixed in aqueous 1% osmium tetroxide-/.5% potassium ferrocyanide at room 0022-5320/81/010001-10502.00/0 Copyright ~) 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
WILSMAN, FARNUM, AND REED-AKSAMIT
temperature for 1 hr. Potassium ferrocyanide was included in the postfixation solution to increase the preservation of glycogen and other complex carbohydrates (14).
Potassium Pyroantimonate Histochemistry Potassium pyroantimonate will precipitate in tissue in the presence of a number of cations; however, by using appropriate controls this technique can be made specific for calcium ions (38). A portion of each slab was trimmed into blocks less than 0.3 mm thick in aqueous 2.5% potassium pyroantimonate-2.0% osmium tetroxide (PAO) at 4°C for 2 hr. The PAO solution was made according to Simson and Spicer (38). Blocks were subsequently rinsed in water made slightly alkaline with potassium hydroxide. As a positive control, parotid salivary gland was collected, trimmed, and stained and fixed with PAO. As a negative control thinly sliced blocks were first placed in a 10 mM solution of ethylene glycol tris-N, N'-tetraacetic acid (EGTA) in a calcium magnesiumfree buffer at 4°C for 1 hr (6, 7, 38). Subsequently these blocks were quickly rinsed and then stained in PAO as described.
Lanthanum Hydroxide Histochemistry Lanthanum does not enter the intact cell and therefore is used as a marker for the extracellular space (22, 35). Blocks of cartilage were trimmed, fixed in 2% gluteraldehyde-2% paraformaldehyde, and rinsed as described. Blocks were then postfixed in aqueous 2.0% osmium tetroxide-l.5% potassium ferrocyan i d e - l % lanthanum hydroxide at room temperature for 1 hr. The lanthanum hydroxide was prepared from lanthanum nitrate as described by Revel and Karnovski (35).
they would be sectioned either parallel with or perpendicular to the articular surface. Ultrathin sections from the PAO and lanthanum experiments were examined both unstained and stained. Other sections were first stained with uranyl acetate and lead citrate and then examined in a Zeiss EM-10A electron microscope.
Stereology Numerical Density The ayerage number of caveolae per chondrocyte, N/o, was determined from (49)
Nc = Nv(¢, 0)/19. . . . . la/D(c~,
0)chond~ocyte
,
(1)
where A70 (~b,0) is the average number of caveolar profiles per chondrocytic profile on sections cut at the user-defined orientation (qS,0). In this study (6,0) is defined as perpendicular to the articular surface. Assuming caveolae are uniform spheres, /)/caveo~a is simply the diameter of the caveola, a n d / ~ (~b,0)/chonaro~yte is the mean projected diameter of the chondrocyte perpendicular to the defined plane of section (49). Using tissue prepared for standard electron microscopy, five blocks of equine articular cartilage were randomly selected and one ultrathin section of the tangential region was cut and collected on a Formvarcoated 2 x 1-mm slit grid. With the section in the electron microscope the corners of the section were digitalized using the stage traverse and 10 pairs of random coordinates were computer-generated for each section. The cell profile nearest to each pair of coordinates was photographed at x 10 000. The number of caveolar profiles in each of the 50 chondrocytic profiles was counted and the mean number of caveolar profiles per chondrocytic profile, Np (~b,0), was calculated.
Dcareola All Blocks Following fixation all blocks were rapidly dehydrated in ethanol, cleared in propylene oxide, and embedded in Epon-Araldite. Blocks were oriented such that
Twenty caveolar profiles in each chondrocytic profile were randomly selected and their diameters, d, were measured. D'-/eaveol a w a s then calculated from d by
FIGS. 1-15. Electron micrographs of articular chondrocytes from the tangential zone of equine articular cartilage (Figs. 1-9 and 14-15) and canine articular cartilage (Figs. 10-13). FIGS. I AND 2. Typical tangential articular chondrocytes in sections perpendicular to the articular surface (Fig. 1) and parallel with the articular surface (Fig. 2). Both cells include fields of perinuclear filaments (F). Figure 1 includes a single row of caveolar profiles (between arrows) and a cilium (C). Fig. 1, x 24 000; Fig. 2, x 9500. FIG. 3. Field of transversly sectioned filaments, adjacent to the nucleaus (N). Based on size (100 A) and position these are most likely intermediate filaments, x 200 000. FIGS. 4-7. Chondrocytic plasma membrane sectioned in a plane perpendicular to te cell surface. Caveolae appear as a single row of invaginations of the plasma membrane. Communication of the inner area of a caveola and the extracellular matrix is through a constricted neck. On occasion caveolae appear to contact mitochondria (Figs. 4 and 5, arrows). In other sections profiles of endoplasmic reticulum, similar to the peripheral elements of the sarcoplasmic reticulum of smooth muscle cells, can be seen immediately deep to and even indented by caveolae (Figs. 6 and 7, 0). x 121 000.
j~
c
W I L S M A N , F A R N U M , AND R E E D - A K S A M I T
Caveolar Morphology
dC - T + [(T - dC) z + 2dT(sin 1C Dear =
+ C(1
C2)~)1~
sin-lC + C(1
, (2)
C2)½
where C = (1 - sZ)1/2 and s equals the ratio o f the smallest diameter to the largest diameter of caveolar profiles and T = section thickness (16).
D ((~,O)chondrocyte From each of five blocks used in this study, 30 serial 1-/xm-thick sections were cut and stained. All chondrocytes that appeared in the 15th section were followed in preceding and succeeding sections and the number of sections, n, in which a chondrocyte appeared was counted. The projected diameter in micrometers for a chondrocyte was estimated at n - 1 to account for the fact that end sections would be incompletely filled. Then D((b ,0)/chondrocytewas estimated by the mean of all individual measurements. RESULTS
Both equine and canine articular cartilage are about 1 mm thick, divisible into tangential, intermediate, radiate, and calcified zones, and in general resemble previous descriptions of the structure of articular cartilage (17, 40, 43). In particular, tangential zone chondrocytes are oblate ellipsoids so that in sections cut perpendicular to the articular surface cell profiles are oval and about 12/xm long and 3/xm high. In sections cut parallel with the articular surface cell profiles are circular and about 12/xm in diameter (Figs. 1, 2). In all profiles large arrays of highly oriented filaments were observed. Filaments 60 A in diameter were located near the plasma membrane and extended into cell processes (Figs. 5, 12) while dense fields of 100-A-diameter filaments occupied a perinuclear position (Figs. 2, 3).
In sections cut perpendicular to the plasma membrane of the chondrocyte caveolae appeared as a single row of circular-to-oval invaginations of the plasma membrane. Each invagination was bounded by unit membrane coated with dense amorphous material on its concave side. Most caveolae opened to the extracellular matrix through a constricted neck (Figs. 4-8). While some caveolae did not appear to connect to the plasma membrane, the continuity of these caveolae and the extracellular space out of the plane of section was confirmed by finding lanthanum precipitates within each of these caveolae (Fig. 9). In grazing sections oriented tangentially to the plasma membrane, large fields of evenly spaced caveolar profiles were observed (Figs. 10, 11). Almost always caveolae appeared single although aggregates of two to five caveolae were seen. Occa-' sionally a connection between adjacent caveolae was present (Fig. 11). Caveolae occurred around the entire periphery of the cell including the periphery of cell processes (Fig. 12); however, in grazing sections through cell processes it appeared as if the entire process was filled with caveolae (Fig. 13). Immediately deep to the caveolae a sparse system of smooth endoplasmic reticulum was observed (Figs. 6-8). The terminal portions of this reticulum consisted of 1500-A dilations while the deeper aspects of these membranes had ribosomes. Occasionally mitochondria were found immediately deep to caveolae and in some cases
FIG. 8. Interrelationships of a caveolar chain (continuity out o f the plane of section), with a mitochondrion and peripheral elements of endoplasmic reticulum (Q). x 121 000. FIG. 9. Precipitate of the extracellular marker lanthanum is found lining the plasma membrane and caveolar profiles. While some caveolae do not appear to connect with the plasma membrane (arrows), continuity with the extracellular space out of the plane of section is demonstrated by finding lanthanum precipitates within these caveolar profiles, x 34 000. Fins. 10 AND 11. Fields of caveolar profiles viewed in grazing sections o f the chondrocytic plasma membrane. While most caveolae appear singular, a number of examples of fused caveolae can be seen. Figure 10 contains 560 caveolae. Fig. 10, × 14 000; Fig. 11, x 121 000.
CHONDROCYTIC CAVEOLAE
®
6
WILSMAN, FARNUM, AND REED-AKSAMIT
mitochondria and caveolae appeared to be attached to each other (Figs. 4, 5).
Potassium Pyroantimonate Histochemistry In sections from tissue that had been fixed and stained with 2% potassium antimonate (PAO), an electron-dense precipitate was observed along the entire plasma membrane including the cell processes. In places where cell processes were sectioned tangentially the entire process appeared to be filled with precipitate. H e a v y deposits o f precipitate were also observed in mitochondria and surrounding lipid droplets. T h e Golgi region and the euchromatic regions o f the nucleus showed light staining precipitate, while no precipitates were present in the dense fields o f filaments, lysosomes, or the heterochromatic portions of the nucleus (Fig. 14). T h o s e sections from tissue p r e t r e a t e d with E G T A prior to PAO showed essentially no precipitate anywhere in the cell (Fig. 15).
Stereology Numerical Density Np (4~,0). The mean number of caveolar profiles per chondrocytic profile averaged over all 50 chondrocytes observed was 88.6 (SD = 21.1). LSc~veo~. . The average diameter, d, of the 1000 caveolar profiles measured was 550 A (SD = 41A) and s, the ratio of smallest to largest, was 0.387. If section thickness is assumed to be 700 A then /)/caveota, calculated from Eq. (2), was estimated at 594 A. (q5,O) chondrocyte. Overall 82 tangential chondrocytes were available for study in the five series of serial sections. The
mean / ) (~b,0) chondrocyte was 11.9 ~ m (SD = 2.1/zm). Nc determination. With the n e c e s s a r y parameters determined the average n u m b e r o f caveolae per chondrocyt__e was calculated from Eq. (1). The mean, N/c, was estimated at 17 700 caveolae per chondrocyte. DISCUSSION There is evidence to suggest that articular chondrocytes migrate through their surrounding dense extracellular matrix. It is known that articular chondrocytes are born in a narrow zone subjacent to the tangential zone and presumably migrate from this region (27, 28, 34). Second, chondrocytes in cell culture are known to be in almost constant motion and presumably this reflects similar motion in vivo (9, 21, 25, 39, 43). Third, there is evidence that while most of the increase in cell density adjacent to cartilage lesions is due to mitosis, some of the increase in cell density may be due to a recruitment of chondrocytes from the surrounding matrix (23, 41, 43, 44, 48). The excitation-contraction coupling mechanism of smooth muscle cells consists of an extensive system of calcium-attracting caveolae functioning analogously to the T-axial tubular system of skeletal muscle, a sarcoplasmic reticulum, and dense fields of thick and thin filaments (5, 15). What we propose is that the contractile mechanism of chondrocytes may resemble (although is probably less developed than) the contractile mechanism of smooth muscle cells, and our initial evaluation of this proposal has been to compare the extensive system of invaginations of the chondrocytic plasma
FIGS. 12 AND 13. Caveolae as they appear in a cell process that has been sectioned transversly (Fig. 12) and horizontally (Fig. 13). × 121000. FIG. 14. Chondrocyte fixed and stained with potassium antimonate-osmium tetroxide (PAO) and examined without additional staining. PAO deposits are found along the entire length of plasma membrane including cell processes. × 15 000. FIG. 15. Chondrocyte pretreated with EGTA prior to reaction with PAO. Since pretreatment with EGTA essentially eliminates the reaction product, we concluded the reaction product in Fig. 14 was caused by calcium ions. × 24 000.
•
~l~
~,
x
8
WILSMAN, FARNUM, AND REED-AKSAMIT
membrane with the caveolar system of the smooth muscle cell. This study has demonstrated that the caveolar systems of smooth muscle cells and chondrocytes are remarkably similar in shape, size, position, numerical density, calcium affinity, and membrane characteristics. The caveolae of chondrocytes are flask- or circular-shaped structures about 600 A in diameter and similar in both size and shape to individual caveolae in smooth muscle cells (18, 19, 38). Chondrocytic caveolae, like smooth muscle caveolae, open to the extracellular space through a narrow neck. In some micrographs this opening was out of the plane of section; however, in micrographs prepared with the extracellar marker lanthanum hydroxide those caveolae with no visible connection with the extracellular matrix contained lanthanum precipitate indicating a communication with the matrix out of the plane of section. Thus caveolae of chondrocytes like caveolae of smooth muscle cells appear to be permanent fixtures of the plasma membrane in communication with the extracellular space. Potassium antimonate will precipitate in tissue sections in the presence of a number of cations (38); however, the lack of precipitate seen in this study following pretreatment with EGTA indicates that the precipitate seen in this study is specific for calcium ions (6, 7). Dense PAO precipitate was deposited inside the plasma membrane in a narrow zone containing the caveolae. The specificity of the localization of precipitate suggests a structural basis for its deposition. This indirectly suggests that chondrocytic caveolae like smooth muscle caveolae attract and perhaps concentrate and sequester calcium ions (7, 22, 32, 33). Other authors have also suggested a direct relationship between positive PAO staining and chondrocytic surface vesicles (6, 7, 26). However, direct visualization of PAO precipitate within caveolar profiles is not possible because membranes even with
poststaining are not resolvable using PAO procedures. Perhaps the only structural difference between caveolae of chondrocytes and those of smooth muscle cells is that the caveolae of smooth muscle cells, especially those of coronary artery smooth muscle, are frequently seen in the form of fused caveolae or branching chains of caveolae (15). While some complex fused caveolae were observed in chondrocytes these most frequently occurred only as doublets and the elongate chains and multiple configurations of caveolae described in smooth muscle were lacking. This structural difference is perhaps refleeted in a comparison of the volume densities of caveolae in chondrocytes and smooth muscle cells. Using an estimate of 17700 caveolae per chondrocyte and assuming that caveolae are spheres of 600 A in diameter and that chondrocytes are oblate ellipsoids with a major axis of 11.9/,m and a minor axis of 3 txm, it is possible to estimate the volume density, Vv, of caveolae in chondrocytes at 0.01. This estimate compares with an estimate of 0.020.03 for the volume density of caveolae in smooth muscle cells (32). Forbes has recently suggested that a universal aspect of excitation-contraction coupling in muscle cells is sarcoplasmic invaginations ranging in complexity from the T-axial tubular system of striated muscle to caveolar chains of varying lengths to single caveolae (15). The presence of caveolae in chondrocytes suggests that these cells also possess this surface modification and thus may be capable of contraction and movement in vivo. All of these invaginated structures potentially extend the extracellular fluid space and its ions to deeper regions within the cell. Other components of an excitation-contractile coupling mechanism for chondrocytes such as an analog to the sarcoplasmic reticulum and contractile filaments remain unstudied. In many chondrocytic profiles dilated smooth endoplas-
CHONDROCYTIC CAVEOLAE
mic reticulum was observed immediately deep to the surface caveolae and extending to close approximation with mitochondria. These structures are suggestions of the previously described peripheral or junctional sarcoplasmic reticulum of smooth muscle cells (15, 52). Obviously any excitation-contraction coupling mechanism requires the presence of contractile filaments. Chondrocytes are known to possess dense fields of 100-A filaments; however, these 100-A filaments have the morphological characteristics of intermediate filaments and their function is unknown. They are considered by some to be a sign of degeneration and impending cell death (4, 31, 42). However, we have found that although the density of 100-A filaments varies in different profiles of any particular cell, the presence of dense fields of filaments is clearly visible in all chondrocytes followed in serial section. It has been estimated that these filaments comprise approximately 30-40% of the volume fraction of normal equine articular chondrocytes (2) and we have not been able to correlate the p r e s e n c e of 100-A filaments with any changes in other cell components which would indicate cell degeneration. The 60-A filaments subjacent to the plasma membrane appear similar in size and position to chondrocytic filaments previously shown to have the actin-like property of forming arrowhead complexes with heavy meromyosin (24). We would propose that the 60-A filaments are most likely contractile filaments. Articular cartilage is, of course, aneural. Therefore excitation depolarization of the plasma membrane is not achieved by the peripheral nervous system. However, each articular chondrocyte is known to possess one cilium (50, 51) presumed to be nonmotile because of the lack of dynein arms and regularly arranged radial spokes (1, 13, 36, 46). There are a number of known examples where deflection of a nonmotile cilium results in depolarization of the cell
membrane (8, 10, 45, 47). Therefore it is possible to hypothesize that perhaps chondrocytes possess a similar mechanism to initiate the event of excitation contraction. The presence of caveolae in articular chondrocytes is clearly not a new observation as caveolae have been described by numerous authors. We believe the significance of this present study, in light of the extensive studies of the caveolar system in smooth muscle cells, lies in defining the extent to which a caveolar system exists in articular chondrocytes and redefining the function that these structures may play in the biology of the articular chondrocyte.
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WILSMAN, FARNUM, AND REED-AKSAMIT
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