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The objective of this proposal is to examine the linkage between phosphate metabolism, the redox status of hard tissue cells and the initiation of mineralization. We propose to investigate this relationship by performing experiments on the growth plate of the chick in situ and by studying isolated chondrocytes and osteoblasts. The in situ experiments will utilize "state-of-the-art" techniques to provide new insights into hard tissue metabolism. Spatial and temporal changes in phosphate pools will be mapped and related to changes to the redox status of the cells and the development of mineralization. We will disrupt these linkages by the administration of fluoride and the induction of metabolic bone diseases and determine how modulation of energy metabolism, phosphate pool size and mineral deposition affects the redox state; in other experiments, we will change the cell redox status and assess how this perturbation modulates phosphate metabolism. In vitro experiments will be performed to explore the mechanisms that link the three parameters described above. These studies will utilize cultured chondrocytes and osteoblasts and a novel technique that was developed in our laboratory to study vesicle biogenesis. We will use this in vitro technique to examine the role of vesicles in initiating mineralization. In these studies we will investigate mechanisms for ion accumulation by vesicles and relate those processes to cell redox and phosphate metabolism. It is hoped that the in vivo and in vitro experiments will provide new information concerning the role of cellular metabolism in controlling phosphate utilization and the initiation of mineralization.
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