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The major thrust of this research proposal is to examine the role of energy metabolism in the initiation of mineralization of the epiphyseal growth cartilage. We will test the hypothesis that a change in energy metabolism results in phosphate and calcium fluxes leading to mineral deposition. Utilizing innovative high resolution image processing techniques we will generate metabolic maps of the growth cartilage and relate cell redox, adenine nucleotide levels and oxygen tension, at the cytochromes, to maturation and calcification of the cartilage. These maps will permit us to explore the influence of the vascular canals of the growth cartilage on chondrocyte metabolism and the development of mineralization. We will also relate energy status of the cells to their mineral content. We will use cell cultures to determine how calcium and phosphate ions and vitamin D control the redox status of chondrocytes. For this purpose, culture systems will be used in which we can monitor matrix vesicle biogenesis and chondrogenesis; these functions will be related to energy metabolism of the cells, vesicle formation and mineralization. Another goal of the proposal is to ascertain whether changes in redox and energy charge lead to changes in membrane lipids; we will measure membrane lipids and the generation of inositol phosphates by the growth cartilage can cells in culture and examine their influence on cell calcium transport. Finally, we will also determine how energy metabolism modulates membrane permeability and potential. This will be achieved using dyes to probe plasma and mitochondrial membranes. Further, we will measure cytosolic calcium concentrations and relate ion flux to membrane potential, cell redox, energy metabolism and mineralization.
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