One of the major goals of periodontics has been the regeneration of a new connective tissue attachment following disease. The interactions of the periodontal ligament (PDL) fibroblasts with their extracellular matrix (ECM) and the changes associated with progression of periodontal disease and wound healing are complex processes which are likely to be particularly critical in understanding periodontal regeneration. The major goals of this study are to obtain a greater understanding of the role of fibronectin (FN) in the behavior of normal and diseased PDL fibroblasts and its role in tissue remodeling. We have chosen FN because of (1) its known importance in establishment of a stable pericellular matrix in many connective tissue cell types, (2) its known importance to the process of wound healing in other tissues such as skin, (3) its ability to promote migration of connective tissue cells, and (4) the possibility that degeneration of FN initiated by wounding or by the onset of periodontal disease, is likely to play a role in perpetuating the cycle of periodontal tissue destruction. We propose to characterize the production and stability of endogenous FN synthesized by normal PDL fibroblasts and to determine how PDL fibroblasts interact with exogenously added FN, FN isoforms, and different functional recombinant FN fragments. We will also test the hypotheses that the stability of FN produced by fibroblasts from diseased PDL is altered, and that the responses of diseased PDL fibroblasts to exogenous FNs differ from those of fibroblasts from normal PDL. Specifically, we will culture human PDL fibroblasts from normal and diseased tissues and analyze their endogenous FN at the protein and RNA level, using immunofluorescence and northern blotting techniques. The stability of endogenous FN produced by these cells will be analyzed by zymographic detection on substrate gels. To further characterize the interaction of FN with PDL fibroblasts, we will survey the FN receptors expressed on the surface on the PDL cells, using immunohistochemical and immunoprecipitation techniques. To determine how PDL fibroblasts interact with various forms of exogenously added FNs, we will use specific attachment and migration assays. Finally, to test our hypothesis that PDL fibroblasts from healthy and diseased tissue interact differently with FN, we will compare these cells by examining levels of expression of FN protein and mRNA, stability of FN protein and histology of the cells. From this study, we will gain a better understanding of the unique interactions that take place in the periodontium between PDL fibroblasts and the ECM, and of the effects of FN on the migration of PDL fibroblasts and their ability to synthesize a stable matrix. These studies relate directly to periodontal regeneration therapy and to wound healing in general.
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