ESTABLISHMENT OF BONE MORPHOGENETIC PROTEIN-2 RESPONSIVE CHONDROGENICCELL-LINE

A clonal cell line named RMD-1 was established from the skeletal muscl e of a 20-day fetal rat. RMD-1 represents a morphologically homogeneou s population of undifferentiated mesenchymal cells, expressing alpha-s mooth muscle actin and type I collagen, but no cartilage-associated ge nes. When cultured in agarose gel containing 100 ng/ml of recombinant human bone morphogenetic protein 2 (rhBMP-2; BMP-2), RMD-1 cells forme d colonies and showed chondrocyte-like features as assessed by their u ltrastructure, metachromatic staining with toluidine blue, and the pro duction of large hydrodynamic-size proteoglycans. RMD-1 cells also dif ferentiated into chondrocytes when the cells were plated at high densi ty (over 2.5 x 10(5) cells/cm(2)) on type I collagen and incubated in medium containing 0.5% fetal bovine serum and 100 ng/ml of BMP-2, This chondrogenic differentiation was evidenced by a distinct morphologica l change into spherical cells, an increase in the levels of sulfated g lycosaminoglycans, a decrease in type I collagen mRNA and the expressi on of cartilage-associated genes, including type II collagen, type IX collagen, aggrecan and alkaline phosphatase. In the presence of ascorb ic acid and 10% serum, RMD-1 cells increased in size and expressed typ e X collagen as well as high alkaline phosphatase activity, then induc ed matrix mineralization. Thus, RMD-1 is a unique cell line that can d ifferentiate from undifferentiated mesenchymal cells into hypertrophic chondrocytes.