The Experimental Study Of Fabricating Cell Sheet By Using The Periodontal Ligament Derived Stem Cells For The Periodontal Regeneration.

The cell-based therapy for the periodontal diseases has showed the predominant advantages over the traditional protocols. While the cell therapy was still confronted with many difficulties such as: uneasiness to localize on the host bed, uncertain relationship between the cell amount and the regenerative efficiency as well as inevitable host response to exogenous carriers. The engineered cell sheet contained the rich extracellular matrix which favors the cellular activities. However the factors which mediate the fabrication of the cell sheet are complicated. The cell-cell interaction, cell density, cell state, the changes of the induction environment all act on the synthesis and reconstruction of the extracellular matrix and influence the configuration of the cell-cell, cell-ECM as well as their biological activity. Specialized oral environment and function restrict the maneuverability of the cell sheet application and cell activities. So it is necessary to improve the biological and mechanical properties of the cell sheet by meliorating the fabrication mode and supplying the special inducing environment, and improve the biocompatility of the lesioned root and periodontal socket in aims to facilitate the successful regeneration of periodontal tissue. Three parts and six experiments were designed and operated. The main results were listed as follows:The first part was designed to isolate and characterize the SD rat PDLSC. The biological properties of the stem cells were observed and showed heterogenicity of stem cells. When the cell sheet was fabricated using the rPDLSC, the rich extracellular matrix was detected and showed time-lapse response. The transplanted cell sheet could regenerate the periodontium-like and cementum-like tissue and showed obvious osseogenesis compared with the single cell suspension. Then the experiement was designed to evaluate potential influence of the different osseous environment on the maturity and biological properties of the cell sheet. The effect of the direct and indirect induction of the CBB particles with different density was compared and the results indicated that the direct induction alter the cell-cell sense and may boost the differentiation and aging of the stem cells during the fabrication of cell sheet.The second part was designed to optimize the fabrication of the three-dimensional culture of the cell sheet. The multilayered cell sheet was used to compound the cell pellets. The results showed the rich ECM molecules in the multilayer cell sheet benefit the interactive adhesion among the cell sheets and the cellular activities amid the cell pellets also demonstrated that the massive increase in ECM secretion improved the adhesion capability and scarcely inhibited the cellular activity. The histological analysis of in vitro co-culture of the pellets with the blocks of ceramic bovine bone (CBB) and dentin slice for 2 weeks indicated the formation of the fiber attachment to the matrix. The SEM observation also indicated the favorable integration of the fibers between the cell pellets and surface of the dentin and CBB particles.The third part was designed to obaserve the potential regenerative capability of differently-cultivated cell sheet and the influence of the different host environment. The cell pellets, cell sheet with or without the support of dentin slice were transplanted into the omentum sac, periodontal defect respectively to evaluate the regeneration potential. All the transplants in the omentum sac and periodontal defects showed the formation of regular aligned cementum/PDL-like complex, however the mineral deposit and fiber alignment were more obvious in the modified cell pellet group than that in the monolayered cell sheet group. Meanwhile, the uneven reconstruction of the cementum/PDL-like complex was observed around the recipient sites in the latter group. In sum, modifiedly prepared PDLSC pellet may be a promising alternative to the periodontal defect repair for future clinical application.