The long term objective of this application is to obtain new information of the organization and chemistry of vertebrate skeletal tissues: bone, cartilage, and tendon. Investigation of calcification in normal and abnormal samples will examine interaction between mineral crystals and extracellular matrix and the precise role of collagen, vesicles, and certain phosphorylated proteins in mineral nucleation, growth and development. Mineralization will be studied initially in normal chicks, rats, mice, and turkeys as vertebrate model systems and later in examples of abnormal calcification including rickets, osteoporosis, calcergy, calciphylaxis, and osteopetrosis. Ultrastructural, biochemical, immunocytochemical, and biophysical methods will be applied to determine the presence, nature, location, and relation of mineral and organic matrix components in the respective tissues. Specifically, the hypothesis to be tested is: Certain phosphoproteins located in extracellular organic matrices of vertebrate tissues are required for the deposition of mineral in calcification. These phosphoproteins are associated with collagen and/or extracellular vesicles to provide critical nucleation sites for mineral formation. Growth and development of mineral subsequent to nucleation is under strict control of stereochemical and physicochemical constraints. The precise aims for examining this hypothesis are to 1) characterize in the calcifying systems the macromolecular stereochemistry of collagen fibrils and their spatial and structural relationship with phosphoproteins or extracellular vesicles using conventional and high voltage electron microscopy, three-dimensional computer image reconstruction, microscopic tomography, and simulated video techniques; 2) identify microscopic sites of mineral deposition through electron diffraction-dark field imaging, high resolution electron probe microanalysis, and topographic imaging in conjunction with conventional and anhydrous sample preparation and 3) determine the spatial and temporal interaction between mineral and phosphoproteins, collagen, and other organic matrix constituents through correlation of the above imaging methods. The data will contribute to knowledge of normal structure, organization, and physiological functions of cells and extracellular matrices of mineralizing tissues and fundamental events of vertebrate calcification.
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