| The ultimate objective of periodontal regeneration therapy is to reconstitute the lost or injured tissues of periodontitis to restore their architecture and function, including cementum, periodontal ligament and alveolar bone on a diseased root surface. Because of the limited sources of periodontal ligament cells (PDLCs) in the periodontal defects, it is difficult to obtain the satisfactory regeneration. And then tissue engineering which is a new vista for periodontal regeneration is supposed to solve the problem. Tissue engineering is the product of co-development and combination of modern cell biology, biomaterial science, and engineering. The aim of tissue engineering is to investigate and construct tissue and organ substitutes. The purpose of this study is to estimate the potential effects of tissue engineering on the periodontal regeneration and provide the experiment evidences for the scaffolds selection. Human periodontal ligament fibroblasts(PDLFs) were cultured in vitro and the osteoblasts-like phenotypes of the cells were investigated in our experiment. By combing the human periodontal ligament fibroblasts with three absorbable guided tissue regeneration membranes in vitro, we evaluate their biocompatibility and construct membrane-like tissue with BME-10X. After implanting the Sprague-Dawley rat PDLFs and BME-10X complex in the rat calvaria intact bone surface, the ability for bone formation of this complex was observed. This study has included four parts:Part one: The culture of human periodontal ligament fibroblasts and study on their osteoblast-Iike phenotypes in vitroMethods: The human PDLFs were digested with collagenase from human teeth. Their alkaline phosphatase (ALP) levels and the ability to produce mineralized nodules in culture was examined compared with human fetus dermal fibroblasts. Results: Human PDLFs have significantly higher levels of ALP when compared with human fetus dermal fibroblasts (p<0.05) and can produce mineral-like nodules in vitro about 30 days. Conclusions: Human PDLFs cultured in vitro have phenotypes typical of osteoblasts, indicating that they are potential cells in periodontal tissue engineering.Part two: Biocompatibility studies on three guided tissue regeneration membranesMethods: Human PDLFs were cultured combined with three kinds of guided tissue regeneration membranes in vitro, and the morphological characters, cell attachment, and proliferation were detected. Three commercially available membranes examined were: BME-10X BioMesh and Bio-Gide. Results: Human PDLFs could be attached to and extended on three kinds of membranes, and normally grown, proliferated in a multi-layer fashion. No significant difference in long term attachment (24h) were observed between three kinds of membrane (P>0.05). The rate of cell proliferation with time differed among the membranes was examined, Bio-Gide and BME-10X could significantly promote cell proliferation (p<0.05) . Conclusions: The results show that Bio-Gide and BME-10X have good biocompatibility with human PDLFs and thus are potential scaffolds for periodontal tissue engineering.Part three: Tissue engineered periodontium formation by using guided tissue regeneration collagen membrane combined with cultured human periodontal ligament fibroblasts in vitroMethods: Human PDLFs were cultured combined with the BME-10X in vitro. Histological examination was performed with light microscopy H.E stained and transmission electron microscopy at 7 and 14 day. Results: Seeded human PDLFswere well attached to BME-10X and proliferated in a multi-layer fashion.membrane-like tissue was observed at 14 day. Conclusions: BME-10X has a goodbiocompatibility with human PDLFs, it can be used as scaffold for periodontal tissueengineering.Part four: A study of bone formation induced by subperiosteal implantation ofSprague-Dawley rat PDLFs and BME-10X complex on calvaria surface in ratsMethods: Sprague-Dawley rat PDLFs were cultured combined with the BME-10X in vitro. Histological examinati...
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