| Inflammation leads to absorption of bone tissue around natural teeth and implants,inhibits the differentiation of mesenchymal stem cells (MSCs) from the periodontal ligament or bone marrow, and affects the reconstruction of the bone. It is the key of the tooth or implants loosen or even lost. How to improve the biological functions of BMSCs in inflammatory microenvironment is a hot issue in oral science .The implant-bone integration process is similar to the healing process of fractures, in which BMSCs plays important roles. Former studies explored various ways to enhance the implant integration, including the chemical or bio-chemical ways, the design and surface treatment of the dental implant, as well as the use of physical influence. The promotion of various forms of exogenous electric field on bone formation and bone remodeling has been confirmed by a few studies. Previous studies confirmed that DCEF ,as a more secure and reliable physical factor, could improve abilities of the migration and proliferation of BMSCs. Yet studies about the effects of DCEF on the osteogenic differentiation of BMSCs in inflammatory microenvironment was rarely found. In this study, exogenous inflammatory factors were used to induce inflammatory microenvironment, and rBMSCs responses were studied. Our study will contribute to developing a new study a new strategy and a new basis for the treatment of periodontitis and peri-implantitis.Experiment 1 : rBMSCs extraction, culture and identificationObjective: To isolate and extract rBMSCs and transmit them to the third generation for subsequent testing.Methods: Whole bone marrow adherent culture method, osteogenic induction alizarin red staining, adipogenic induced oil red O staining and flow cytometry to identify cell surface markers.Results: The P3 generation rBMSCs showed strong proliferative ability. Their morphologies were in long spindle shape and more stable than the original cells.Osteogenic and adipogenic cells showed osteogenic and adipogenic ability respectively.The cell surface markers CD 105 and CD90 were positive, CD45 and CD 14 were negative,which accorded with the phenotypic characteristics of mesenchymal stem cells.Conclusion: The extracted target cells were of high purity, and had the potential of osteogenic and adipogenic differentiation. They showed the biological characteristics of mesenchymal stem cells, which met the requirements of subsequent experiments.Experiment 2 : Comparison of the effects of different concentrations of TNF-a on rBMSCs in inflammatory response.Objective: By comparing the proliferation inhibition rate and the expression of autoinflammatory cytokines in each group, determined the effects of different concentrations of TNF-a on rBMSCs in inflammatory response, thus to screen out the optimal concentration and to provide a stable and reliable method of inflammatory response for subsequent experiments.Methods: Inflammatory induced by different concentrations of TNF-α, cell growth curves were determined by MTT assay, the levels of TNF-α and IL-1β in the cells were measured by ELISA kit, and their genes expression levels were detected by real-time quantitative PCR.Results: When the concentration of TNF-a was less than 10ng / mL, the cell proliferation ability was not significantly different. When the concentration of TNF-a was 12ng / mL,the the proliferation ability was inhibited. When TNF-a concentration was 10ng / mL, the cell inflammatory factor secretion was the highest than other groups, showing significant inflammatory characteristics.Conclusion: lOng / mL TNF-α could be used to induce rBMSCs to produce inflammatory response.Experiment 3: Effects of inflammatory microenvironment on osteogenic differentiation of rBMSCs.Objective: To compare the differences of osteogenic differentiation of rBMSCs under inflammatory environment.Methods: Inflammation induction, osteogenesis induced and alizarin red staining,Calcium ion deposition detection, expression levels of genes of BSP, OCN and Runx2 were measured by real - time quantitative PCR, Western blot was used to detect the expression of osteogenesis - associated protein ALP and Runx2.Results: Alizarin red staining after osteogenesis induction: The number of mineralized nodules of rBMSCs in inflammatory microenvironment was lower than that in normal rBMSCs. The expression of ALP and Runx2 in rBMSCs in inflammatory microenvironment was significantly lower than that in normal rBMSCs, and similar trends were founded in genes expression of BSP, OCN and Runx2.Conclusion: The inflammatory microenvironment established in vitro could inhibit osteogenic differentiation of rBMSCs.Experiment 4:Effects of DCEF on osteogenic differentiation of rBMSCs in inflammatory microenvironmentObjective: To investigate the effects of DCEF on the expressions of osteogenesis -associated proteins and the expression osteogenesis - associated genes of rBMSCs in inflammatory microenvironment.Methods: Under DCEF, MTT assay cell growth curve, osteogenesis induced and alizarin red staining, calcium ion deposition detection, the expression levels of genes of BSP,OCN and Runx2 were measured by real - time quantitative PCR, and Western blot was used to detect the expression of osteopontin ALP and Runx2.Results: The proliferation capacities of experimental cells under DCEF were improved.Results of alizarin red staining showed that the color in H+cytokines+ DCEF group was darker than that of the rBMSCs group,which was also lighter than the normal rBMSCs+ DCEF group. The number of mineralized nodules in DCEF groups were significantly higher than that with no DCEF(P <0.05). Real-time quantitative PCR results showed that:Genes expression levels of BSP, OCN and Runx2 in H+cytokines+DCEF group were significantly higher than those in the control group (P <0.05). Western blot results showed that: the expression levels of ALP and Runx2 protein in H+cytokines+ DCEF group were significantly higher than those in control group.Conclusion: DCEF could improve the proliferation ability and osteogenic differentiation of rBMSCs in inflammatory microenvironment. |
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