The long-term objective of this research is to characterize the mechanisms of hormonal regulation of adult cartilage matrix synthesis and assembly by articular chondrocytes. The experimental goal is to use synthesis of keratan sulfate proteoglycans and Type II collagen as markers of the adult cartilage phenotype and to determine under in vitro serum-free conditions the unique combination of connective tissue specific hormones necessary for reconstruction of the cartilage matrix. Results obtained during years 01-03 of this grant show that IGF- I/GH in combination selectively stimulate proteoglycan sysnthesis and enhance levels of keratan sulfate in the culture. In addition, the combination of insulin, IGF-I and fibroblast growth factor are sufficient in a defined serum-free medium to stimulate the proliferation of adult bovine articular cartilage cells. The continuation of this research in years 04-06 will focus on the process of synthesis and assembly of the extracellular matrix by articular chondrocytes in a defined culture environment. The Specific Aims are all predicated on the hypothesis of hormonal control of cartilage matrix synthesis and are directed to the following topics: (1) Analysis of the extracellular matrix synthesized by adult articular cartilage cells grown to confluency in a serum-free medium containing insulin, IGF-I and FGF. (2) Investigate the ability of connective tissue specific hormones, parathyroid hormone, growth hormone, thyroxine, thyrocalcitonin, estrogen and testosterone, to stimulate matrix synthesis of a mature phenotype as adjuncts to insulin, IGF-I and FGF. (3) Investigate the influence of hormones, attachment factors, protease inhibitors and cellular geometry on the assembly of proteoglycans into matrix. (4) Extend the conditions for serum- free culture to human costal and articular chondrocytes and quantitate the characteristics of the medium and cell-associated proteoglycans. Techniques include serum-free chondrocyte culture, quantification and fractionation of proteoglycan, analysis of collagen synthesis, microanalysis of glycosaminoglycan and quantitation of DNA and RNA. The studies are significant to clinical orthopedics and reconstructive surgery; the information regarding hormonal control of cartilage synthesis is directly applicable to inhibition of cartilage destruction, stimulation of cartilage repair and preparation of transplantable cartilage.