The broad, long-term objective of this proposal is the development of therapeutic agents that enhance repair to aged, damaged or surgically altered cartilage. In vitro, perlecan promotes the chondrogenic differentiation of C3H10T1/2 cells, and the phenotypic maintenance of normal human and exostoses chondrocytes. However, it is unknown if perlecan alone can promote chondrogenesis in vitro.
The Specific Aim of this proposal, is to determine the requirement for perlecan function in chondrogenesis in vitro. To investigate the role of perlecan in chondrogenesis in vitro, approaches that enhance or inhibit perlecan expression will be employed. The first approach will utilize an inducible expression system to over-express domain I of perlecan in multipotential, C3H10T1/2, cells. Induced overexpression of perlecan domain I is hypothesized to promote the aggregation and chondrogenic differentiation of C3H10T1/2 cells. Employing a ribozyme construct, the second approach will determine if inhibition of endogenous perlecan expression prevents the chondrogenic conversion of C3H10T1/2 cells. Inhibition of perlecan expression is hypothesized to prevent the chondrogenic conversion of C3H10T1/2 cells. These experiments are designed to determine if C3H10T1/2 cells must produce endogenous perlecan to sustain the differentiative process, or may progress through this program without expression of perlecan the gene. This information has therapeutic importance since situations may exist in which individuals cannot produce cartilage due to defects in perlecan expression or constituent HS chain assembly, eg., exostoses. In this event, perlecan administration with or without cell therapy may be effective in stimulating chondrogenesis in aging or damaged cartilage.
