The primary goals are to determine the composition and functional features of the supramolecular complex of proteins that calcifies, and how cells regulate this process. Towards these aims, cell cultures that form mineralized tissues were established for biochemical analysis, and for studies at the genomic level in collaboration with Drs. Marian F. Young and Larry W. Fisher. Intrinsic factors were found to influence the biosynthetic output of these cells such as the animal species, position within the cell cycle, and importantly, the developmental age of the donor. Bone cells and their products were compared to those from other tissues, and from patients with different diseases. By histochemistry at the EM level, BSP was found in packets of matrix that, upon secretion, were the first sites of mineral deposition. Many of the bone matrix proteins, including BSP, were found to support cell attachment in vitro. By immunohistochemistry, the RGD-containing proteins and their receptors were found to be expressed at particular stages of maturation in vivo. Interestingly, one of the cell surface recept subunits that binds to fibronectin (alpha-4) was highly expressed in the osteoblastic layer. In cells from osteogenesis imperfecta patients, there were changes in post-translational modifications of not only collagen, but also proteoglycans, and in the absolute and relative amounts of the various components. These changes may cause the altered crystal structure found in OI bone, which ultimately results in fragility. In Turner's syndrome (karyotype 45, X0 and characterized by short stature and early onset osteoporosis), biglycan (whose gene is on the X chromosome) was found to be reduced by 50%. In various forms of osteosarcoma in rats and humans, both qualitative and quantitative differences in bone matrix proteins were detected, which may contribute to derangements in growth and ultrastructure observed in these tumors. Continued characterization of the interrelationship between cells and their extracellular environment will provide a clearer understanding of bone metabolism in health and in disease.
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