| ObjectiveDental implantation is an effective and prevalent treatment modality for replacingmissing teeth and restoring maxillofacial defects. With the development of denal implant, itbegans to widely used in patients with diabetes mellitus. However, the lower success ratesof dental implants in diabetic patients than in normal patients has been a problem todentists. The studies have proved that poor osseointegration around dental implants wasthe mian reason that caused implants failure in diabetic patients.Diabetes is a kind of metabolic disease characterized by high blood glucose. Thestudies have proved that diabetes can lead to bone metabolism disorders, which may causereduced bone mass, delayed bone union, and osteoporosis in humans and animal models.Moreover, diabetes is one of the risk factors that causes periodontal disease and alveolarbone loss. The quality of jaw bones is the foundation to dental implantation restoration.There’s no doubt that the adverse effects of diabetes on bone will affect the success rate ofimplant.Bone mesenchymal stem cells (BMSCs) are capable of self-renewing anddifferentiating into multiple cell lineages, which make them an excellent cell source inbone regeneration. Bone formation is a complicated process, and the key point of thisprocess is bone marrow stromall cells (BMSCs) differentiated into osteoblast cells (OBs),and osteoblast synthesis extracellular matrix and mineralized. This process is preciselyregulated by a network which is composed of many signaling pathways and transcription factors. In particular, the modulation of the molecular differentiation into bone in MSCs bythe BMP signaling pathway is well known and BMP-2is one of the most effective inducersin this pathway. Moreover, more and more studys indicated that microRNAs (miRNAs),20–25nucleotides noncoding RNA molecules in the cells of animal and plant, also playedan important role in this regulating networks.Several studies indicated that high glucose inhibits osteogenic differentiation ofBMSCs and the proliferation and mineralization of osteoblasts, which were considered thereason of diabetes causing bone loss. However, the exact mechanism still remain unknown.Therefore, our purpose is to study the biological function of BMSCs, explore the role ofBMP signaling pathway and sceen differential expression of miRNAs in osteogenicdifferentiation of BMSCs in high glucose microenvironment, which is helpful to explainhow high glucose microenvironment affects the osteogenic ablity of BMSCs.MethodsPart I:We isolated bone marrow stromall cells from madibulars of C57male mice andcultured in normal DMEM. Passage4BMSCs were used in our study. We tested theexpression of mesenchymal stem-cell markers of the cells by FCM and investigated thedifferentiation potential of BMSCs to undergo osteoblastic/adipogenic differentiation test.Part II:We exposed mice BMSCs to medium containing four different concentrations ofglucose respectively. We tested cell activity by MTT and used FCM to tested theproliferation index and the number of apoptotic cells. The Alizarin red S staining, ALPactivity test and Ca/P content secreted by cells were performed to observe the osteogenicability of BMSCs in different concentrations of glucose.Part III:We explore the role of the BMP signaling pathway in the progression of boneregeneration in high glucose condition. Real time-PCR were used for determining the gene expression (RUNX2，ALP and OCN) to further verify the inhibition of high gluose onosteogenic differentiation of BMSCs. Western blot analysis was used to test the proteincontent of BMP-2. The cells treated with or without (as control)100ng/mL BMP-2addition and then the gene expression of RUNX2，ALP and OCN were mesured again.Part Ⅳ:We screened the differential expression of microRNA in BMSCs cultuerd in normalor high glucose medium by microRNA microarrays (version11.0). The realtime-PCRwas used to validate the differential expression of miRNA. Those that were overexpression and could target BMP-2were chosed.PartⅤ:We detected the expression of miRNAs chosed above during the osteogenicdifferentiation of BMSCs by real time PCR and miR-467f was finally chosed for furtherstudy. BMSCs were transfected by miR-467f mimics, then cultured in the in osteogenicinduction medium. The marker genes of osteogenic differentiation were detectd by the realtime PCR. The ability of mineralization was detected by alizarin red. miRNA targetprediction software and dual luciferase reporter assay were used to validate the target genesof miR-467f. Cells were transfected by miR-467f and siBMP-2mimics respectively, thencultured in the in osteogenic induction medium, finally detceted the phenotype ofosteogenic differentiation.Results:Part I:The isolated cells expressed positive for CD9but negative for CD45and CD14.moderately positive for CD146,which conformed to stem cell markers. And these cellscould be induced to osteogenesis and Adipogenesis in special medium.Part II:We found that a high glucose concentration (<25mM) stimulated BMSC proliferationbut inhibited osteogenic differentiation in a dose-dependent manner. However, high glucose concentration (>35mM) deteriorated the BMSC proliferation deteriorated andcaused cell apoptosis. The osteogenic ability of BMSC decreased as the glucoseconcentration increased.Part III:The expresion of RUNX2, ALP, and OCN,three key osteoblast marker genes, and thelevel of BMP-2protein were all decresed in25mM high glucose condition. After treatmentwith ectogenic BMP-2. the intracellular BMP-2in both osteogenic groups (5.5mM and25mM glucose) was significantly elevated. Moreover, The expression of RUNX2, ALP, andOCN was also increased, which suggested the osteogenic ability of BMSCs in high glucosemicroenvironment was improved by ectogenic BMP-2addition.Part Ⅳ:Compared with the control group, the expression of9kinds of miRNAs increased,while the expression of16miRNAs decreased in BMSCs cultured in high glucosecondition. The results were further confirmed by realtime-PCR. miR-467f、miR-2183、miR-3472'miR-345-5p were chosed by high expression and being involved inosteogenesis probably.PartⅤ:The results of real time PCR showed that the level of miR-467f decreasedtime-dependently during the the osteogenic differentiation of BMSCs. Transfected bymiR-467f mimics, BMSCs showed that the mRNA expression of RUNX2,ALP and OCNand the ability of mineralization were attenuated. miRNA target prediction softwareTargetscan showed that BMP-2was a candidate target gene of miR-467f. Dual luciferasereporter assay demonstrated that miR-467f could interact with the3′UTR of BMP-2directly. Moreover, overexpression of miR-467f in BMSCs inhibited the endogenousexpression of BMP-2mRNA and protein. And BMSCs transfected by miR-567f andsiBMP-2mimics showed the similar inhibition of the osteogenic differetiation.Conclusions: 1. High glucose in the microenvironment markedly affects biological function of BMSCsobtained from the mandibulars of mice. Cell proliferation were improved but osteogenicdifferentiation were inhibited in high glucose condition.2. There were differential expression miRNAs in BMSCs cultured in high glucose. ThemiR-467f, one of high expression miRNA, decreased during the osteogenic differetiationof BMSCs. Meanwihle overexpression miR-467f could attenuate the osteogenicdifferetiation of BMSCs.3. BMP-2, a key protein of the BMP signaling pathway, was one of the target genes ofmiR-467f.It plausible that miR-467f attenuates the osteogenic differetiation of BMSCsthrough the inhibtion of BMP-2.4.In high glucose microenvironment, the level of miR-467f is increased and it can inhibitthe expression of BMP-2, which wouid be one of the reasons that high glucose can inhibitthe osteogenic ability of BMSCs. |
PDF Full Text Request