The overall objective of these studies is to elucidate the mechanism of cell-controlled biomineralization using the differentiating chick limb-bud mesenchymal cell micromass culture system. In this system, mesenchymal cells proliferate and differentiate into chondrocytes and under well-defined conditions, recapitulate the cascade of events in the epiphyseal growth plate, concluding with cartilage calcification. Results obtained to date reveal that conclusions derived from this avian model are generally applicable to mineralization mechanisms in other species. The theme of the current proposal is the importance of both mature cells and mature matrices for biomineralization. The primary outcome of the studies is the deposition of a """"""""physiologic"""""""" calcified matrix, assessed using Fourier Transform Infrared imaging (FTIRI) at approximately 7 um spatial resolution and transmission electron microscopy (TEM). Four hypotheses will be tested, specifically that: (1) Both mature cells and mature matrices are required for initiation of calcification and progression of crystal growth. (2) Chondrocyte apoptosis is not essential for cartilage calcification. (3) Time-dependent modification of phosphoproteins alters cartilage calcification. (4) Degradation of cartilage proteoglycans by matrix metalloproteinases and cathepsins facilitates mineralization of the mature matrix.
Four aims will test these specific hypotheses.
Aim 1 will compare the rates of mineralization and the properties of the mineral formed by immature cells on mature and immature matrices and mature cells on immature and mature matrices. BMP-6 will be used to enhance maturation; PTHrP to block it. 45Ca uptake and FTIRI will be the primary outcomes; TEM and x-ray diffraction will provide confirmation of results.
In Aim 2, rates of mineralization and properties of the mineral will be compared in cultures in which apoptosis has been decreased using caspase inhibitors, and those in which apoptosis has been stimulated. The amount of mineral formed and the crystallinity of the mineral around chondrocyte nodules will be correlated with the increase or decrease in the number of apoptotic cells relative to baseline. Effects on cell proliferation will also be documented.
In Aim 3, the effect of phosphoprotein (PP) modulation on the extent and rates of mineralization, and mineral and matrix properties will be determined, and the proteins facilitating matrix mineralization identified. Modulation during specific stages of culture development will include: inhibiting enzymes that phosphorylate/de-phosphorylate PPs, overexpressing PPs using the recombinant competent avian retrovirus system (RCAS), addition of synthetic PP peptidomimetics, and/or antibody blocking of specific PPs. In Am 4, mineralization rates, amounts and characteristics of the mineral and the proteins (especially proteoglycans) in the matrix following addition of specific cathepsin and metalloproteinase inhibitors and the products of these enzymes will be monitored. Results of these proposed should provide new insights in calcification mechanisms in general, and new information on how to treat conditions in which cartilage calcification is aberrant.
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