| Periodontal regeneration is expected to reconstitute the injured tissues of the periodontal attachment apparatus physiologically and functionally, including cementum, periodontal ligament and alveolar bone. Traditional periodontal therapy and guided tissue regeneration failed to fullfil the goal. Tissue engineering has been introduced into the field of periodontology, which contribute a new vista to periodontal regeneration. But there are some difficulties in finding proper seeded cells. Owing to its heterogeneity, periodontal ligament (PDL) cell is reagrded as the main source of cell to regenerate periodontal tissues and expected to be the seeded cells in previous studies,, because of their potential of differentiation to fibroblasts, osteoblasts, or cementoblasts. However, there are also some disadvantages for PDL cell to be seeded cells, for example , extraction must be carried out to obtain PDL cell, which is often refused by patients in clinic.Gingival fibroblast is also the constituent of periodontal tissues, and easier to obtain clinically than PDL cell, gingival fibroblast have strong proliferation potential and even show some characters of osteoblasts when they are induced by some bone-inducing factors such as human bone morphogenetic protein-2(hBMP2) . hBMP2 is a kind of strong bone-inducing factor and plays a crucial role in periodontal regeneration. So, combining GINGIVAL FIBROBLAST with hBMP2 may be used in periodontal tissue engineering to fulfill the periodontal therapy goal.This study tries to reform gingival fibroblast to be the seeded cells in periodontal tissue engineering by transfecting hBMP2 gene into gingival fibroblast. Several experiments as below are carried on in this dissertation:1. The phagemid expression vector for hBMP2was amplified in E. coli., then, the plasmid was extracted, purified, and identified. All of these were the preparation for following experiments.2. hBMP2 gene was transfected into gingival fibroblast by using LipofectAMINE. Positive clones were selected with G-418. The expression of hBMP2 protein in gingival fibroblast transfected were determined by in situ hybridization, immunohistochemistry and sandwich-in ELISA. We also proved the hBMP2 protein has biological activity by investigating ALP activity and osteocalcin (OC) quantity in MC3T3-E1 cells treated with the conditioned media from the gene-transfected gingival fibroblast cultures. The results showed that hBMP2 protein was successfully expressed in gingival fibroblast transfected with hBMP2 gene.3 Observed the biological behaviours of gingival fibroblast transfected with hBMP2 gene stably, it was found that the shape of parts of gingival fibroblast changed from fusiform to polygonal; compared with parental cells, there were no obvious difference in doubling time and proliferative ability ; but enhanced both ALP activity and OC quantity of the hBMP2 gene transfected cells (P < 0.01); the ALP synthesis was upregulated in the hBMP2 gene transfected cells byhistochemistry staining, furthermore, mineral nodules developed in mineralization culture medium after gene transfection. Our results showed that the hBMP2 gene transfected cells expressed osteoblastic phenotype.4 We investigated the biological behaviors of hBMP2-transfected gingival fibroblast cultured in calcium alginate gel. The results showed that the cells spread very well in calcium alginate gel, secreted extracellular matrix, and expressed hBMP2 protein.5 To evaluate their effects in periodontal tissue regeneration, we compounded the hBMP2-transfected gingival fibroblast to calcium alginate gel and implanted them to artificial furcation defects of dogs. The periodontal apparatus attachments regenerated within 8 weeks. Root resorption and ankylosis were not found in experiment group. In experimental group, the quantities of new formed bone, cementum and connective tissue were more significantly than them in negative control group which implanted gingival fibroblast (P<0.01), but had no obvious differen...
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