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

Objective. To establish long-term cultures of human fetal epiphyseal c hondrocytes under conditions that allow the preservation of a cartilag e-specific phenotype. Methods. Chondrocytes isolated from 20-24-week h uman fetal epiphyseal cartilage were cultured for up to 180 days on pl astic dishes previously coated with the hydrogel, poly-(2-hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characte ristics of the cultures were examined at various intervals, and the ex pression of genes encoding types I, II, and IX collagen and aggrecan c ore protein was determined by Northern hybridizations of total cellula r RNA with human-specific complementary DNAs. Results. Human fetal epi physeal chondrocytes cultured for 180 days under conditions that preve nted their attachment to the underlying substratum formed nodular stru ctures with morphologic and structural characteristics resembling matu re articular cartilage. The cells in the center of the nodules remaine d spherical and were surrounded by an abundant cartilaginous extracell ular matrix, as evidenced by histochemical and ultrastructural examina tions. The cells in the periphery of the nodules acquired a discoid mo rphology and were surrounded by a sparse extracellular matrix. Biosynt hetic studies demonstrated the maintenance of a cartilage-specific phe notype throughout the 180 days of culture, with the production of aggr ecan and types II, IX, and XI collagens but not type I collagen. North ern hybridizations showed high levels of messenger RNAs (mRNAs) for ag grecan core protein, type II procollagen, and type M collagen, but typ e I procollagen mRNA was not detectable even at 180 days of culture. C onclusion. The human chondrocyte culture system described here allows the maintenance of a chondrocyte-specific phenotype for prolonged peri ods (up to 180 days). The long-term chondrocyte cultures formed nodula r structures that resemble mature articular cartilage morphologically, ultrastructurally, biosynthetically, and in the pattern of cartilage- specific gene expression.