(verbatim) Cells of the intervertebral disc (IVD) exhibit little capacity for functional matrix repair in situ, which may contribute to the progressive nature of disc degeneration. IVD cells respond to mechanical stimuli with altered biosynthesis in manner that depends on zone of cell origin (anulus fibrosus, transition zone or nucleus pulposus), although the mechanisms governing these responses are poorly understood. The central hypothesis of this proposal is that zonal variations in the local mechanical environment of IVD cells are dominant in regulating zonal variations in disc cell metabolism. Our preliminary analytical and finite element analyses suggest that the micromechanical environment of disc cells depends on four parameters: (1) the anisotropic, nonlinear and multiphasic properties of the extracellular matrix; (2) applied loading conditions; (3) cell mechanical properties; and (4) three-dimensional cell geometry. In this study, we propose a set of experiments to quantify zonal variations in these four parameters and to incorporate them in a computational model of IVD cell micromechanics. Independent tests will be performed to quantify material properties of the cell and extracellular matrix using materials testing and micropipette aspiration techniques. Three-dimensional cell geometry and the local boundary conditions in the IVD under compression will be obtained using confocal laser scanning microscopy. A finite element model of the micromechanical environment of IVD cells will be developed, which incorporates nonlinear, anisotropic and biphasic material behaviors and the measured material and geometric data. Finite element model predictions of the local mechanical environment of IVD cells under compression in the intact IVD (in situ), and isolated cells cultured in a three-dimensional alginate matrix (ex situ), will be obtained to precisely determine important local mechanical stimuli such as stress, strain, fluid pressure and fluid flow. To construct new and precise relationships between these stimuli and IVD cell metabolism, corresponding experiments to measure zonal variations in gene expression and biosynthesis of collagens and agrecan will be performed on intact IVD and cell-alginate constructs under compression. Comparison of data for in situ and ex situ experiments is expected to reveal the relative contributions of cell morphology, matrix properties and loading conditions to the micromechanical environment and metabolic response of IVD cells. Furthermore, the experimental and computational data to be obtained in this project will define new relationships between precisely determined mechanical stimuli and IVD cell metabolism that are prerequisite to understanding the mechanisms that govern cellular response to mechanical stimuli in vivo.
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