Summary
Chondrocyte cultures grown in centrifuge tubes with intermittent centrifugation differentiate into hypertrophic chondrocytes and form calcification. We examined chondrocytes cultured in this system electron microscopically. Rat growth-plate chondrocytes were seeded in a plastic centrifuge tube and cultured in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 μg of ascorbic acid per ml. Specimens were examined by using electron microscopy and selected-area electron-diffraction techniques. In the early stage of culture, a few chondrocytes were scattered and extracellular matrices were not observed. In the middle stage of the cultures, the chondrocytes resembled proliferative cells. Matrix vesicles appeared to be budding from the cell surfaces of chondrocytes and were observed sparsely in the extracellular matrices, which were well formed around the chondrocytes. Matrix vesicles increased substantially during the following cultures. In the mature stage of the cultures, crystal formation related to matrix vesicles was observed. In the 33-day cultures, several masses of calcified matrix were formed and it was confirmed to be apatite by selected-area electron diffraction analysis. The chondrocytes appeared hypertrophic during this same stage. The 56-day culture was similar to the 33-day culture. It was concluded that this culture system provides an extracellular-matrix mineralization which is produced by chondrocytes per se.
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References
Brighton CT (1987) Longitudinal bone growth: the growth plate and its dysfunctions. AAOS Instruct Course Lee 36:3–36
Anderson HC (1967) Electron microscopic studies of induced cartilage development and calcification. J Cell Biol 35:81–101
Bonucci E (1967) Fine structure of early cartilage calcification. J Ultrastruct Res 20:33–50
Miki T, Yamamuro T (1987) The fate of hypertrophic chondrocytes in growth plates transplanted intramuscularly in the rabbit. Clin Orthop 218:276–282
Caplan A, Pechak DG (1987) The cellular and molecular embryology of bone formation. J Bone Miner Res 5:117–183
Hall BK (1987) Earliest evidence of cartilage and bone development in embryonic life. Clin Orthop 225:255–272
Binderman I, Greene RM, Pennypacker JP (1979) Calcification of differentiating skeletal mesenchyme in vitro. Science 206:222–225
Suzuki F, Takase T, Takigawa M, Uchida A, Shimonura Y (1981) Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells. Proc Natl Acad Sci USA 78:2368–2372
Gerstenfeld LC, Landis WJ (1991) Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system. J Cell Biol 112:501–513
Wuthier RE (1977) Electrolytes of isolated epiphyseal chondrocytes, matrix vesicles, and extracellular fluid. Calcif Tissue Res 23:125–133
Brucker P, Horler I, Mendler M, Houze Y, Winterhalter KH, Eich-Bender SG, Spycher M (1989) Induction and prevention of chondrocyte hypertrophy in culture. J Cell Biol 109:2537–2545
Castagnola P, Dozin B, Moro G, Cancedda R (1988) Changes in the expression of cellular genes show two steps in chondrocyte differentiation in vitro. J Cell Biol 106:461–467
Gibson GJ, Schor SL, Grant ME (1982) Effects of matrix macromolecules on chondrocyte gene expression: synthesis of a low molecular weight collagen species of cell cultures within collagen gels. J Cell Biol 93:767–774
Gibson GJ, Beaumont BW, Flint MH (1984) Synthesis of a low molecular weight collagen of chondrocytes from the presumptive calcification region of the embryonic chick sterna. The influence of culture with collagen gels. J Cell Biol 99:208–216
Solursh M, Jensen KL, Zanetti NC, Linsenmayer TF, Reiter RS (1984) Extracellular matrix mediates epithelial effects on chondrogenesis in vitro. Dev Biol 105:451–457
Solursh M, Jensen KL, Reiter RS, Schmid TM, Linsenmayer TF (1986) Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm and sternal chondrocytes. Dev Biol 117:90–101
Kato Y, Iwamoto M, Koike T, Suzuki F, Takano Y (1988) Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor β and serum factors. Proc Natl Acad Sci USA 85:9552–9556
Iwamoto M, Sato K, Nakashima K, Shimazu A, Kato Y (1989) Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor. Dev Biol 136:500–507
Shimomura Y, Yoneda T, Suzuki F (1975) Osteogenesis by chondrocytes from growth cartilage of rat rib. Calcif Tissue Res 19:179–187
Landis WJ, Glimcher MJ (1982) Electron optical and analytical observations of rat growth plate cartilage prepared by ultracryomicrotomy: the failure to detect a mineral phase in matrix vesicles and the identification of heterodispersed particles as the initial solid phase of calcium phosphate deposited in the extracellular matrix. J Ultrastruc Res 78:227–268
Arsenault AL, Hunziker EB (1988) Electron microscopic analysis of mineral deposits in the calcifying epiphyseal growth plate. Calcif Tissue Int 42:119–126
Wu LNY, Sauer GR, Genge BR, Wuthier RE (1989) Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media: evidence of an association between matrix vesicles and collagen. J Biol Chem 264:21345–21355
Ali SY, Gray JC, Wisby A, Phillips M (1977) Preparation of thin cryosections for electron probe analysis of calcifying cartilage. J Microsc 111:65–76
Akisaka T, Kawaguchi H, Subita GP, Shigenaga Y, Gay CV (1988) Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution. Calcif Tissue Int 42:383–393
John EH, Wuthier RE (1987) The mechanism of matrix vesicle formation: studies on the composition of chondrocyte microvilli and on the effects of microfilament-perturbing agents on cellular vesiculation. J Biol Chem 262:1916–1925
Genge BR, Wu LNY, Wuthier RE (1990) Differential fractionation of matrix vesicle proteins: further characterization of the acidic phospholipid-dependent Ca2+-binding proteins. J Biol Chem 265:4703–4710
Wu LNY, Genge BR, Lloid GC, Wuthier RE (1991) Collagen-binding proteins in collagenase-related matrix vesicles from cartilage: interaction between matrix vesicle proteins and different types of collagen. J Biol Chem 266:1195–1203
Genge BR, Sauer GR, Wu LNY, McLean FM, Wuthier RE (1988) Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization. J Biol Chem 263:18513–18519
Tacchetti C, Quarto R, Campanile G, Cancedda R (1989) Calcification of in vitro developed hypertrophic cartilage. Dev Biol 132:442–447
Tacchetti C, Quarto R, Nitsch L, Hartmann DJ, Cancedda R (1987) In vitro morphogenesis of chick embryo hypertrophic cartilage. J Cell Biol 105:999–1006
Akisaka T, Gay CV (1985) The plasma membrane and matrix vesicles of mouse growth plate chondrocytes during differentiation as revealed in freeze-fracture replicas. Am J Anat 173:269–286
Orney A, Atkin I, Levy J (1980) Ultrastrucual studies on the origin and structure of matrix vesicles in bone of young rats. Acta Anat 106:450–461
Reinholt FP, Engfeldt B, Hjerpe A, Jansson K (1982) Stereological studies on the epiphyseal growth plate with special reference to the distribution of matrix vesicles. J Ultrastruc Res 80:270–279
Brighton CT, Sugioka Y, Hunt RM (1973) Cytoplasmic structures of epiphyseal plate chondrocytes: quantitative evaluation using electron micrographs of rat costochondral junctions with special reference to the fate of hypertrophic cells. J Bone Joint Surg 55-A:771–784
Hunziker EB, Scheck RK, Cruz-Orive LM (1987) Quantification of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J Bone Joint Surg 69-A:162–173
Kato Y, Iwamoto M (1990) Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation. J Biol Chem 265:5903–5909
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Nakagawa, Y., Shimizu, K., Hamamoto, T. et al. Electron microscopy of calcification during high-density suspension culture of chondrocytes. Calcif Tissue Int 53, 127–134 (1993). https://doi.org/10.1007/BF01321891
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DOI: https://doi.org/10.1007/BF01321891