This proposal intends to obtain new information concerning vertebrate skeletal calcification in bone, cartilage, and tendon. Normal chicks, mice, rats, and turkeys; bone and cartilage cultures; and an abnormal osteogenesis imperfecta mouse mutant will be used as models. Studies in these examples will examine certain structural and chemical interactions in their respective extracellular matrices, intracellular matrices, and intra-and extracellular interfaces. Work will be done systematically, defining protein secretory pathways, cell matrix adhesion relations, and mineral-matrix associations. Ultrastructural, biochemical, immunochemical, and biophysical methods will be applied to extend the applicants' previous result describing the presence, nature, location, and interaction of organic matrix and mineral components in vertebrate calcification. The specific hypothesis to be examined is: the organic matrix of vertebrate calcifying tissues critically influences the deposition of mineral in terms of crystal size, shape, location, orientation, and distribution.
Specific Aims, to address structure- function relations at atomic, molecular, and macromolecular levels of hierarchy, are to: (1) identify the presence and location of collagenous and non-collagenous proteins involved in mineralization, as well as selected integrins and cytoskeletal elements at the cell-matrix interface, utilizing conventional and high voltage electron microscopy, three-dimensional (3D) image reconstruction and video techniques, and 2D and 3D immunochemistry; (2) determine the sites of mineral deposition by similar means and also electron diffraction, x-ray probe microanalysis, and novel Fourier transform infrared spectroscopy; (3) characterize interaction between extracellular matrix proteins and mineral through correlation of the methods above and by atomic force microscopy and computer modeling (molecular dynamics); and (4) assess the effects in bone cell culture on matrix-mineral interaction following modulation of protein transport with the inhibitor, monensin. It is suggested that outcome data will contribute to knowledge of structure and function of cells, the cell-matrix interface, and extracellular matrices and events fundamental to vertebrate calcification.
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