The overall aim of this project is a complete understanding of the chemistry of mineralization of bone tissue through use of real-time correlative Raman microspectroscopy/fluorescence imaging methods. Our hypothesis is that mineralization follows a three-step sequence (disordered calcium phosphate to octacalcium phosphate (OCP)-like mineral to carbonated apatite) that is mediated by non-collagenous proteins, including bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), osteocalcin (OC) and osteopontin (OPN) and that this process can be followed in real time. We further hypothesize that the development of first-deposited mineral into carbonated apatite proceeds through the OCP-like intermediate and that the transformation of this mineral occurs through a layer-by-layer mechanism over a conversion time of approximately 3 hours. Our test system will be fetal murine calvarial tissue sections. For each of the four proteins, mineralization will be perturbed by adenovirus-based overexpression the of protein to calvarial tissue sections under culture. To visualize the infected osteoblasts we will use adenoviruses that code for both the selected non-collagenous protein and green fluorescent protein (GFP). The GFP will serve as a fluorescent marker for infected osteoblasts. The tissue sections will be continuously monitored for 12-24 hours by near-infrared Raman microspectroscopy, using fluorescent markers to identify the locations of osteoblasts with overexpressed protein. After the incubation the tissue section will be visualized by histochemistry to confirm the loecation of overexpressed protein. Correlative Raman/GFP fluorescence imaging will establish co-localization of new mineral and OPN. Point mutations will be used to evaluate effects of the deletion of specific domains in the overexpressed proteins. These experiments will be complemented by model compound studies on synthetic OCP and carbonated apatites, including measurement of powder diffraction patterns and computation of vibrational spectroscopic band positions by density function theory. Overall, the project will allow us to establish the mechanism and kinetics of the bone mineral transformation from first deposited disorded calcium phosphate to final stable apatitic carbonate. The information so gained will be invaluable in guiding the development of new anabolic agents for treatment of osteoporosis and other skeletal diseases. Our success in this grant cycle will lead to an extension to closely related methods for real-time measurement of the more complicated processes of matrix and cross-link formation.
The proposal uses optical measurements to follow development of bone mineral through several stages. It is known that this process malfunctions in metabolic bone diseases, including osteoporosis and in genetic defects of bone. The information gained in this project will help in the design of new therapeutic agents for some of these diseases.
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