Summary
We studied the response to culture conditions and the differentiative ability in suspension culture in alginate gels of resting chondrocytes from the preosseous cartilage of adult pig scapula. It was found that the maximum rate of chondrocyte duplication is reached at the fourth day in culture whereas the rate of proteoglycan synthesis and alkaline phosphatase expression do not gain a maximum value before the seventh day. During the culture time, the chondrocytes undergo differentiation as it is demonstrated by the alkaline phosphatase specific activity increase and by morphological criteria (hypertrophy, increase of the number of mitochondria per cell, increased endoplasmic reticulum, matrix vesicle production). The alginate gels can be easily dissolved to obtain cell populations in which the variation of cytosolic calcium concentration following a proliferative stimulus can be conveniently observed using the conventional procedure of Fura 2.
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Hayashi M, Minomiya Y, Parsons S, Hayashi K, Olsen BR, Trelstad RL (1986) Differential localization of mRNA of collagen types I and II in chick fibroblast, chondrocytes and corneal cells by in situ hybridization using cDNA probes. J Cell Biol 102:2302–2309
Kosher RA (1983) The chondroblast and the chondrocyte. In: Hall BK (ed) Cartilage, vol 1. Academic Press, New York, p 59
von der Mark H, von der Mark K, Gay S (1976) Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II antibodies and their application to early stages of the chick embryo. Dev Biol 48:237–249
Heinegárd D, Paulsson M (1987) Structural and contractile proteins, part D; extracellular matrix. In: Cunningham LW (ed) Methods of enzymology. Academic Press, Orlando, 145, p 336–363
Pollesello P, D'Andrea P, Martina M, de Bernard B, F. Vittur (1990) Modification of plasma membrane of differentiating preosseous chondrocytes: evidence of a degradative process in the mechanism of matrix vesicle formation. Exp Cell Res 188: 214–218
Schmid TM, Conrad HE (1982) Unique low molecular weight collagen secreted by cultured chick embryo chondrocytes. J Biol Chem 257: 12444–12450
Capasso O, Tajana G, Cancedda R (1984) Location of 64K collagen producer chondrocytes in developing chicken embryo tibiae. Mol Cell Biol 4:1163–1168
Kielty CM, Kwan APL, Holmes DF, Schor SL, Grant ME (1985) Type X collagen a product of hypertrophic chondrocytes. Biochem J 227:545–554
Vittur F, Zanetti M, Stagni N, de Bernard B (1977) Are newly synthesized proteoglycans responsible for calcification in cartilage? Bull Mol Biol Med 2:189–198
Bonucci E (1967) Fine structure of early cartilage calcification. J Ultrastruct Res 20:33–50
Anderson HC (1969) Vesicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol 41:59–72
Boyan BD, Schwartz Z, Carnes DL, Ramirez V (1988) The effects of vitamin D metabolites on the plasma and matrix vesicle membranes of growth and resting cartilage cells in vitro. Endocrinology. 122:2851–2860
Boyan BD, Schwartz Z, Swain LD, Carnes DL, Zislis T (1988) Differential expression of phenotype by resting zone and growth region costochondral chondrocytes in vitro. Bone 9:185–194
Wuthier RE, Chin JE, Hale JE, Register TC, Hale LV, Ishikawa Y (1985) Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture. J Biol Chem 260: 15972–15979
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. J Biol Chem 264:21346–21355
Benya PD, Shaffer JD (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30:215–224
Quarto R, Dozin B, Tacchetti C, Campanile G, Malfatto C, Cancedda R (1990) In vitro development of hypertrophic chondrocytes from selected clones of dedifferentiated cells. J Cell Biol 110:1379–1386
D'Andrea P, Grandolfo M, de Bernard B, Vittur F (1990) Serum induced cytosolic calcium movements and mitogenesis in cultured preosseous chondrocytes. Exp Cell Res 191:22–26
Tacchetti C, Quarto R, Campanile G, Cancedda R (1989) Calcification of in vitro developed hypertrophic cartilage. Dev Biol 132:442–447
Guo J, Jourdian GW, Mac Callum DK (1989) Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect Tissue Res 19:277–297
Vittur F, Pugliarello MC, de Bernard B (1971) Chemical modifications of cartilage matrix during endochondral calcification. Experientia 27:126–127
Brukner P, Hörler I, Mendler M, Houze Y, Winterhalter KH, Eich-Bender SG, Spicher MA (1989) Induction and prevention of chondrocyte hypertrophy in culture. J Cell Biol 109:2537–2545
Meldolesi J, Huttner WB, Tsien RY, Pozzan T (1984) Free cytoplasmic Ca2+ and neurotransmitter release: studies on PC 12 cells and synaptosomes exposed to α-latrotoxin. Proc Natl Acad Sci USA 81:620–624
Labarca C, Paigen K (1980) A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344–352
Stagni N, Furlan G, Vittur F, Zanetti M, de Bernard B (1979) Enzymatic properties of the Ca2+-binding glycoprotein isolated from preosseous cartilage. Cacif Tissue Int 29:27–32
Takagi M (1990) Ultrastructural cytochemistry of cartilage proteoglycans and their relation to the calcification process. In: Bonucci E, Motta PM (eds) Ultrastructure of skeletal tissues. Kluver Academic Publishers, Boston, p 111
Ali SY, Sajdera SW, Anderson HC (1970) Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage. Proc Natl Acad Sci USA 67:1513–1520
Gunter TE, Zuscik MJ, Puzas JE, Gunter KK, Rosier RN (1990) Cytosolic free calcium concentrations in avian growth plate chondrocytes. Cell Calcium 11:445–457
Iannotti JP, Brighton CT (1989) Cytosolic ionized calcium concentration in isolated chondrocytes from each zone of the growth plate. J Orthop Res 7:511–518
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Grandolfo, M., D'Andrea, P., Paoletti, S. et al. Culture and differentiation of chondrocytes entrapped in alginate gels. Calcif Tissue Int 52, 42–48 (1993). https://doi.org/10.1007/BF00675625
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DOI: https://doi.org/10.1007/BF00675625