Understanding of the regulation of growth, differentiation and mineralization in skeletal tissues is key to developing effective therapies for calcific diseases. A key system critical to bone growth and fracture repair is growth plate (GP) cartilage. Expression of local growth factors, and modulation by endocrine agents, plays a critical role in normal development and calcification of GP and fracture-callous cartilage. The ability now to grow cultures of GP chondrocytes in which normal matrix synthesis and mineralization occur, provides and opportunity to explore the effects of these factors in isolation from unknown systemic factors. The long-term objective of this proposal is to utilize this culture system to elucidate the mechanism of biomineralization in GP cartilage and to clarify the role that growth factors and hormones play in regulating GP development. Humoral factors have been selected for study based on their known effects on skeletal cells and GP chondrocytes, and on our recent findings. These include the lipophilic hormones, the calcific hormones, local growth factors, mineralization, when added alone or in combination to primary cultures of avian GP chondrocytes at both pre-and post-confluent stages of culture, will be analyzed using several biochemical and morphological parameters. The three major goals in this proposal are: 1) to study the effect of these factors on GP chondrocytes evaluating their influence on: a) cell growth (total cellular protein, RNA and DNA levels), b) extracellular matrix production (rate of synthesis of collagen and proteoglycans), c) differentiation (expression of alkaline phosphatase activity, type X collagen, the acidic phospholipid-dependent Ca2+ binding proteins (annexins), and their co-localization in the cells and extracellular matrix), and d) mineralization of the cultures (Ca2+ and Pi content and matrix vesicle production). 2) to develop a rapidly mineralizing GP cultures system using a programmed sequence using a combination fracture repair, but also be a paradigm for development of """"""""tissue engineered"""""""" cartilage for replacement in diseased joints. 3) to establish spatial and temporal relationships between key cellular proteins, enzymes, lipids, and mineral ions calcification of this complex tissue. State-of-the-art fluorescent probes will be used to monitor the location of these key entities using powerful new laser confocal microscopes to study their localization in fixed tissues and in living cells with a degree of precision heretofore impossible. These studies should enable major strides toward the goal of understanding the processes occurring during growth plate development, and should uncover critical parameters required for the rational design of treatments to promote cartilage repair and rapid bone fracture healing.
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