DIFFERENTIATION AND MINERALIZATION IN CHICK CHONDROCYTES MAINTAINED IN A HIGH CELL-DENSITY CULTURE - A MODEL FOR ENDOCHONDRIAL OSSIFICATION

Chondrocytes isolated from the proliferative and differentiating zones of 3-wk-old chick growth plates were cultured in the presence of 10% fetal bovine serum (FBS) and ascorbic acid for up to 21 d in a high ce ll density culture within Eppendorf tubes. Tile proliferative, differe ntiating, and calcification properties of the chondrocytes were examin ed by immunolocalization and by enzyme histochemical and biochemical m ethods. The cells maintained a chondrocyte phenotype throughout cultur e: they were round in shape and synthesized both collagen type II and proteoglycans. The expression of a hypertrophic phenotype was evident by Day 3 of culture and fi om this time onwards characteristics of ter minal differentiation were observed. The cells were positive for both alkaline phosphatase (ALP) activity and c-myc protein and the surround ing matrix stained strongly for collagen type X. Small foci of mineral ization associated with individual chondrocytes were first evident by Day 6 and more widespread areas of mineralization occupying large area s of matrix were present by Day 15. Mineralization occurred without th e addition of exogenous phosphate to the medium. This culture system d isplays characteristics that are similar in both morphological and dev elopmental terms to that of chick chondrocyte differentiation and calc ification in vivo and therefore offers an excellent in vitro model for endochondral ossification.