The overall project goals are development of Raman imaging with application to changes in bone mineral crystallite lattice and matrix collagen secondary structure in tissue subjected to tensile and compressive loading. Line-focused 532 nm laser light will be used to excite spectra of tissue pretreated by chemical and photochemical bleaching of fluorophores. An imaging spectrograph and low-light level CCD will provide spatially resolved spectra and ability to measure 0.1-0.2 cm(-1) changes peak in position with integration times as low as 0.05 sec. The project is the first phase of a study of the failure mechanism of osteoporotic tissue at the level of atomic and molecular structure, with the goal of new intervention strategies for minimization of fracture risk. The project will also serve as a model for study of biomechanics and other metabolic diseases and genetic defects of musculoskeletal tissue. Techniques will be developed using bovine bone and then ported to archived human tissue specimens. Our hypotheses are: Ion spacing in bone mineral change under mechanical stress during elastic deformation, while plastic deformation (cracking, compression or fracture) results in permanent changes 2) The matrix responds to mechanical stress in the elastic regime by perturbation of cross-links and changes in helix pitch of the strands of the collagen fibrils, resulting in changes in helix and coil conformations and hydrogen bonding. In plastic deformation cross-links between collagen fibrils are ruptured. These hypotheses can be tested by spectroscopic imaging of tissue because vibratioal frequencies and intensities respond to changes in local environment. Static Raman imaging will explore chemistry of tissue that has been subjected to fatigue loading. Dynamic imaging will follow changes in bone tissue chemical parameters with applied loads. Both univariate and multivariate methods will be used for data reduction. Systematic studies of age effects will be performed on murine tissue with exploratory work on human tissue at age extremes.
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