FORMATION OF NODULAR STRUCTURES RESEMBLING MATURE ARTICULAR-CARTILAGE IN LONG-TERM PRIMARY CULTURES OF HUMAN FETAL EPIPHYSEAL CHONDROCYTES ON A HYDROGEL SUBSTRATE

Objective. To establish long-term cultures of human fetal epiphyseal chondrocytes under conditions that allow the preservation of a cartilage-specific phenotype. Methods. Chondrocytes isolated from 20-24-week human fetal epiphyseal cartilage were cultured for up to 180 days on plastic dishes previously coated with the hydrogel, poly-(2-hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characteristics of the cultures were examined at various intervals, and the expression of genes encoding types I, II, and IX collagen and aggrecan core protein was determined by Northern hybridizations of total cellular RNA with human-specific complementary DNAs. Results. Human fetal epiphyseal chondrocytes cultured for 180 days under conditions that prevented their attachment to the underlying substratum formed nodular structures with morphologic and structural characteristics resembling mature articular cartilage. The cells in the center of the nodules remained spherical and were surrounded by an abundant cartilaginous extracellular matrix, as evidenced by histochemical and ultrastructural examinations. The cells in the periphery of the nodules acquired a discoid morphology and were surrounded by a sparse extracellular matrix. Biosynthetic studies demonstrated the maintenance of a cartilage-specific phenotype throughout the 180 days of culture, with the production of aggrecan and types II, IX, and XI collagens but not type I collagen. Northern hybridizations showed high levels of messenger RNAs (mRNAs) for aggrecan core protein, type II procollagen, and type M collagen, but type I procollagen mRNA was not detectable even at 180 days of culture. Conclusion. The human chondrocyte culture system described here allows the maintenance of a chondrocyte-specific phenotype for prolonged periods (up to 180 days). The long-term chondrocyte cultures formed nodular structures that resemble mature articular cartilage morphologically, ultrastructurally, biosynthetically, and in the pattern of cartilage-specific gene expression.