The major objective of the proposed studies is the elucidation of the mechanism of cell controlled biomineralization. The differentiating chick limb-bud mesenchymal cell micromass culture system will be studied. The Principal Investigator proposes to utilize this culture system because of the economy and reproducibility of the system, the availability of markers, and her previous experience with these cultures. The Principal Investigator has already demonstrated that conclusions derived from this avian model are generally applicable to mineralization mechanisms in other species. Prior results showed that phosphorylation of matrix proteins is a key step in the mineralization process and that mineralization is actively controlled by cells, even after the first mineral crystals are deposited. In the proposed studies, the Principal Investigator and her collaborators will focus on the initial events in the mineralization process and on those events that control the cell mediated proliferation of mineral crystals. The Principal Investigator proposes that an understanding of these events is crucial for the development of techniques for promoting bone ingrowth, treating osteoarthritis and its complications, and treating developmental abnormalities. The Principal Investigator will study the mechanisms of cell mediated calcification using physico-chemical and physical chemical techniques, supplemented with techniques from cell and molecular biology. Three specific hypotheses will be tested with evaluations based on Fourier Transform infrared microscopy, x-ray diffraction, electron microscopy, and chemical analyses. The first hypothesis to be tested in Aim 1 is that appropriate cell mediated phosphoprotein phosphorylation and dephosphorylation regulate the site of initial mineral deposition, the rate and extent of mineral deposition, and the properties of the mineral crystals. Studies will include inhibition of casein kinase-like enzymes and phosphoprotein phosphatases as well as overexpression of the phosphoprotein(s) identified as important for control of mineralization. The second hypothesis to be tested in Aim 2 is that both the cellular production and the extracellular post translational processing of collagens regulates the site of initial mineral deposition, the rate and extent of mineral deposition and the mineral crystals' properties. Experiments will be performed by antibody blocking of specific collagen epitopes (i.e. immunoblocking) and by altering collagen cross-links. The third hypothesis to be tested in Aim 3 is that both mature cells and mature matrices are needed for initiation of calcification and progression of crystal growth. The maturation process will be varied by exposing the cultures to load (i.e. stretching), and to specific growth factors (OP-l, vitamin A, dexamethasone) which are known to accelerate chondrocyte maturation. Emphasis will be placed on defining the characteristics of the mineral formed and on defining if mineralization is associated with alterations in the degree of phosphorylation of phosphoproteins or if it is collagen type or structure specific.
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