| Periodontal regeneration is expected to reconstitute the lost or injured tissues to restore the architecture and function of the periodontal tissues, including cementum. periodontal ligament and alveolar bone. And the basis of periodontal regeneration is periodontal ligament cells(PDLCs), which are the main responsive cells to form the new attachment. But the sources of PDLCs are very limited in the periodontal defects, so that it is difficult to obtain the satisfactory regeneration. And then tissue engineering is supposed to be able to solve the problem. Tissue engineering is the emerging field of science aimed at developing techniques for the fabrication of new tissues to replace damaged tissues. To engineer living tissues, cultured cells are coaxed to grow on bioactive degradable scaffolds that provide the physical and chemical cues to guide their differentiation and assembly into three-dimensional tissues. Nowadays the form styles of tissue engineering have included cells/scaffolds, growth factors/scaffolds and cells/growth factors/scaffolds. In order to estimate the potential effects of tissue engineering on the periodontal regeneration, following the study on the cellular mechanism of the enhanced periodontal regeneration by rhBMP-2 and rh-bFGF, this study focuses on the research of periodontal regeneration by tissue engineering for the first time, so as to seek for the methods of obtaining ideal periodontal regeneration by tissue engineering, and to provide a new way for the therapy of the periodontal diseases. This study has included the three parts:1. The study on the cellular mechanism of the enhanced periodontal regeneration by rhBMP-2 and rh-bFGF1) The influences on the proliferation , DNA synthesis and cell cycle of human PDLCs by rhBMP-2 or/and rh-bFGF were evaluated by MTT colorimetric assay and flow cytometry(FCM), in order to analyze the possible mechanism of promoting the proliferation of PDLCs by rhBMP-2 or/and rh-bFGF. The results have indicated that rhBMP-2 and rh-bFGF were both able to enhance the proliferation and DNA synthesis of PDLCs, and rhBMP-2 and rh-bFGF had synergistic action. rhBMP-2 and rh-bFGF enhanced the proliferative responses of PDLCs in a dose-dependent manner,and the effect of rh-bFGF was more obvious than rhBMP-2 . Employing the FCM technique, we observed the increased S %, the decreased GI % and the enhanced (S+G2M)% when the cells were stimulated by rhBMP-2 and rh-bFGF,which indicated that the effects of enhancing the proliferation and DNA synthesis of PDLCs by rhBMP-2 and rh-bFGF may be accomplished by accelerating GI transition.2) In order to evaluate the effects of rhBMP-2 and rh-bFGF on protein synthesis of PDLCs, the influences of rhBMP-2 and rh-bFGF on the total protein content and the ultrastructure of human PDLCs were observed by using Coomassie brilliant blue staining and transmission electron microscope(TEM). The results has showed that rhBMP-2 significantly increased the total protein content of PDLCs at the concentrations from 25 to lOOug/L in a dose-dependent manner,in the contrary, rh-bFGF significantly decreased the total protein content of PDLCs,but the effect diminished from the 7th day. When combining rhBMP-2 with rh-bFGF, the increased total protein content was observed from the 5th day. At the same time, the enhanced DNA and protein synthesis activities of PDLCs stimulating by rhBMP-2 were observed by TEM, and the enhanced DNA synthesis and the unchanged protein synthesis activities of PDLCs stimulating by rh-bFGF were observed.3) The expression of osteocalcin(OCN) mRNA, osteopontin(OPN) mRNA and bone sialoprotein(BSP) mRNA of human PDLCs stimulating by rhBMP-2 andrh-bFGF were investigated by Western dot blot to further evaluate the influence of rhBMP-2 and rh-bFGF on the PDLCs differentiatation. The results indicated rhBMP-2 could increase the mRNA expression of OCN,OPN and BSP of PDLCs, and rh-bFGF had no influences on them, when combined with rhBMP-2, rh-bFGF didn't influence rhBMP-2's effects on the PDLCs d... |
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