CELL-PROLIFERATION, EXTRACELLULAR-MATRIX MINERALIZATION, AND OVOTRANSFERRIN TRANSIENT EXPRESSION DURING IN-VITRO DIFFERENTIATION OF CHICK HYPERTROPHIC CHONDROCYTES INTO OSTEOBLAST-LIKE CELLS

Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage-dep endent culture conditions in the presence of ascorbic acid (Descalzi C ancedda, E, C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Bio l. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondr ocytes undergoing this differentiation process to the growth of hypert rophic chondrocytes maintained in suspension culture as such. The prol iferation rate is significantly higher in the adherent hypertrophic ch ondrocytes differentiating to osteoblast-like cells. In cultures suppl emented with retinoic acid the proliferation rate is further increased . In both cases cells stop proliferating when mineralization of the ex tracellular matrix begins. We also report on the ultrastructural organ ization of the osteoblast-like cell cultures and we show virtual ident ity with cultures of osteoblasts grown from bone chips. Cells are embe dded in a dense meshwork of type I collagen fibers and mineral is obse rved in the extracellular matrix associated with collagen fibrils. Dif ferentiating hypertrophic chondrocytes secrete large amounts of an 82- kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteobl ast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after ret inoic acid addition, when the cartilage-bone-specific collagen shift o ccurs, and decays between the 5th and the 10th day, when cells have fu lly acquired the osteoblast-like phenotype. Similar results were obtai ned when retinoic acid was added to the culture at the 50 nM ''physiol ogical'' concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.