GENE AND PROTEIN EXPRESSION DURING DIFFERENTIATION AND MATRIX MINERALIZATION IN A CHONDROCYTE CELL-CULTURE SYSTEM

Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. N asal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10(5) cell/cm(2) and cultured in Dul becco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and beta-glycerophosphate. Firs t, cells lost their phenotype but in this condition they rapidly reexp ressed the chondrocyte phenotype and were able to form calcified carti laginous nodules with the morphological appearance of cartilage minera lization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, prot eoglycan core protein, characteristic markers of chondrocyte different iation, as well as alkaline phosphatase and osteocalcin associated wit h the mineralization process. Analysis of labeled collagen and immunob lotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our c ulture conditions promoted extracellular matrix mineralization and cel l differentiation towards the hypertrophic phenotype. This differentia tion process was characterized by the induction of type X collagen mRN A, alkaline phosphatase, and diminished expression of type TI collagen and core protein of large proteoglycan after an increase in their mRN A levels before the mineralizing process. These results revealed disti nct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stage s of the differentiation program in vitro.