REGULATION OF MATRIX GENE EXPRESSION IN CHONDROCYTES

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Defective repair of cartilage matrix is a major feature accounting for loss of function of articular cartilage in osteoarthritis (OA) and other arthritides. In the past, definition of the mechanisms that induce and maintain anabolic activities in human chondrocytes have been hampered by the lack of suitable chondrocyte culture systems. Recently, we reported the development and characterization of immortalized human chondrocyte lines that express cartilage-specific matrix proteins under defined culture conditions. In this proposal, a newly established temperature-sensitive articular chondrocyte line (tsT/AC62) will be used to define at the cellular and molecular level the mechanisms involved in the induction and maintenance of "appropriate" patterns of synthesis of cartilage-specific collagen, and other matrix proteins. It is our hypothesis that down-regulation of proliferation and maintenance in a 3-dimensional environment in the presence of critical anabolic factors will permit expression of the chondrocyte phenotype and matrix deposition in vitro and in vivo. Our approach will be to develop 3- dimensional culture models in which the chondrocyte phenotype is enhanced and maintained in vitro and identify critical factors controlling cartilage-specific gene expression and matrix synthesis both in vitro and in vivo by the following specific aims: (1) Determine factors that maintain and enhance chondrocyte phenotype in immortalized human chondrocyte culture models in vitro. (2) Identify molecular mechanisms involved in induction and maintenance of differentiated chondrocyte phenotype by direct analysis of regulatory sequences of the cartilage-specific type II collagen gene (COL2A1). (3) Examine factors influencing cartilage formation in vivo. Implantation of genetically engineered human chondrocytes in a three-dimensional collagen matrix in SCID mice will validate the results obtained in vitro and test the capacity of these cells to participate in authentic cartilage repair. The availability of human chondrocyte culture models that can be manipulated reproducibly to undergo defined programs of differentiation will allow us to examine in greater detail the molecular mechanisms regulating COL2A1 expression both in vitro and in vivo. An understanding of critical signals that induce and maintain chondrocyte phenotype, but that may not be present in the microenvironment of the adult OA chondrocyte, will eventually provide rational strategies for ex vivo gene therapy for improving cartilage repair with chondrocyte transplantation approaches.