This proposal has two objectives, both involved with the nature and role of the extracellular matrix components of bone and dentin. The first objective is to understand the molecular mechanisms involved in the construction of the mineralized extracellular matrix. The second objective is to examine the mechanisms by which the osteoinductive or morphogenic non-collagenous protein (NCP) components of bone and dentin exert their influence in directing cells to form bone. In bone and dentin the hydroxyapatite crystals are deposited within a preexisting collagenous fiber array and have a spatial orientation directed by that array. The means by which collagen directs this ordered mineralization is a primary target of Objective I. The basic hypothesis is that particular NCP interact with collagen fibers at specific loci and nucleate matrix directed mineralization. In dentin, the key NCP may be phosphophoryn (PP). The site of localization of PP on collagen will be determined and the effect of PP on apatite nucleation on collagen measured. In parallel work, the mRNA for PP will be isolated and used for cell free translation of PP and construction of a cDNA library. These can be used to probe gene organization in odontoblasts, and to determine the amount of PP mRNA in teeth with dentin mineralization disorders (e.g. DI-II, hereditary opalescent dentin). Antibodies to PP will be used to isolate the primary gene product, and this will be characterized. The secretory pathway for PP in rat incisor will be determined by immunocytochemical techniques. The lower molecular weight NCP fraction has components which function as osteogenic or osteoinductive agents. The identification and isolation of the specific component and the determination of its means of action is the focus of Objective II. A cell culture system will be used to determine the presence of a cell surface receptor and the extent of binding and internalization of the osteogenic component. Isolation will be carried out by HPLC and other chromatographic techniques. These studies should all have a significant impact on our understanding of both heritable and metabolic bone disease relating to growth and development and mineralization disorders, and on the ability to repair bone defects.
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