The proposed study will identify the microchemical composition of the initial and subsequent mineral phases in sections of normal and vitamin Ddeficient osteomalacic bone tissues. in addition, to determine the temporal relationship, this study will use high voltage electron stereomicroscopy together with computer graphic reconstructions to obtain threedimensional locations of the minerals in respect to the tissue ultrastructure. While the presence of hydroxyapatite has been well established, the same is not true for secondary calcium phosphate mineral phases with lower calcium/phosphate ratios, such as brushite, octacalcium phosphatem, and tricalcium phosphate (which exist in some pathologicallyrelated calcification in teeth). No investigation has yet been reported on diseased bone tissues involving inadequate mineralizaiton resulting from deficient calcium and/or phosphate levels. These secondary calcium phosphate phases inherit higher solubility and dissolution rate than the hydroxypatite phase. Therefore, determining the presence of these phases in metabolic bone diseases, such as osteomalacia, or other diseases involving insufficient amount of minerals in osteoid, is critical in not only understanding the physicochemistry of mineral formation in bone but also to developing concepts and potential solutions to bone tissue pathologies. In order to eliminate chemical fixation and staining, we will employ the ultracryomicrotomy method for specimen preparation. To maximize instrumental sensitivity for the detection of small amounts of inorganic constitutents, high voltage electron microdiffraction techniques will be adapted. The high voltage electron microscope is equipped with an environmental chamber that allows the examination of the tissue in their natural state, in their unfrozen hydrated condition. In addition, high voltage stereomicroscopy and threedimensional computer reconstruction will be used to obtain the structural information, and thus, spatial and temporal sequence will be determined. These are the most uptodate methods for obtaining the most complete and intimate knowledge of the inorganic constituents of bone tissue heretofore obtained.
