Analysis of cartilage maturation using micromass cultures of primary chondrocytes

A micromass culture (MM-C) system of primary immature chondrocytes for func tional analysis of soluble factors involved in the maturation step of carti lage was previously developed. Ectopically expressed BMP-2 was shown to ind uce the expression of the Ihh and Noggin genes. Here it is demonstrated tha t, upon longer culture, secreted bone morphogenetic protein-2 (BMP-2) furth er promotes the maturation step as judged by the induction of type X collag en and BMP-6 expression, which are known to be detectable in the later phas e of cartilage maturation. Induction of all of these genes by secreted BMP- 2 was not inhibited by ectopic expression of parathyroid hormone-related pe ptide (PTHrP) induced by retrovirus vector infection, although the same vir us vector showed strong inhibitory effects on the expression of type X coll agen gene or alkaline phosphatase activity in mature chondrocytes. These re sults suggest that the maturation-promoting activity exhibited by BMP-2 is dominant over the suppressive effect of PTHrP in immature chondrocytes. Whe n the BMP-6 gene was introduced into the same virus vector as that used for BMP-2, it induced the same sets of genes (Ihh, Noggin, type X collagen and endogenous BMP-6) as BMP-2 did. These results also suggest that BMP-6 woul d autonomously maintain and/or promote a later stage of chondrocytic matura tion.