PF-127水凝胶联合骨髓间充质干细胞外泌体来源miR-132诱导成骨分化修复牙槽骨缺损

PF-127 Hydrogel Combined with Bone Marrow Mesenchymal Stem Cell-Derived Exosome-miR-132 to Induce Osteogenic Differentiation for Repair of Alveolar Bone Defects

目的:探究PF-127水凝胶联合骨髓间充质干细胞外泌体来源miR-132诱导成骨分化对牙槽骨缺损的修复作用。方法:培养骨髓间充质干细胞,(bone mesenchymal stem cells,BMSCs),并转染anti-miR-132、anti-miR-NC质粒。制备BMSCs来源外泌体(bone mesenchymal stem cells-exosomes,BMSCs-exo),蛋白质印迹法鉴定表达标志物。制备PF-127水凝胶和BMSCs-Exo复合物,PKH67标记法检测BMSCs的摄取;茜素红染色检测BMSCs的成骨能力;逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)检测细胞中miR-132表达和碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)mRNA表达。建立牙槽骨缺损大鼠模型,将大鼠随机分为PF-127水凝胶组、PF-127水凝胶+BMSCs-exo-anti-miR-NC组和PF-127水凝胶+BMSCs-exo-anti-miR-132组。微型计算机断层扫描(Micro-computed tomography,micro-CT)评估牙槽骨缺损情况;蛋白质印迹法检测牙槽骨组织中ALP、OCN、Runx2蛋白表达。结果:anti-miR-132组BMSCs中miR-132表达明显低于anti-miR-NC组(P<0.05),提示anti-miR-132成功转染至BMSCs中。BMSCs-exo-anti-miR-NC组和BMSCs-exo-anti-miR-132组中外泌体标志物CD9和CD63显著表达。和BMSCs-exo-anti-miR-NC组相比,BMSCs-exo-anti-miR-132组中miR-132表达明显降低(P<0.05)。和PF127水凝胶组相比,BMSC-exo-anti-miR-NC组、PF127水凝胶+BMSC-exo-anti-miR-NC组、PF127水凝胶+BMSC-exo-anti-miR-132组中miR-132表达均明显降低,茜素红染色相对活性、细胞中Runx2、ALP和OCN mRNA表达均明显增加,且PF127水凝胶+BMSC-exo-anti-miR-132组变化最显著(P<0.05)。和Control组相比,PF-127水凝胶+BMSCs-exo-anti-miR-NC组和PF-127水凝胶+BMSCs-exo-anti-miR-132组BV/TV比值、BMD、Tb.N和Tb.Th及细胞中Runx2、ALP、OCN蛋白表达均明显升高,Tb.Sp明显降低(P<0.05),且PF-127水凝胶+BMSCs-exo-anti-miR-132组变化最显著。结论:PF-127水凝胶联合骨髓间充质干细胞来源外泌体来源干扰miR-132表达可促进骨髓间充质干细胞的成骨分化,从而促进牙槽骨缺损大鼠的修复。

Objective:To investigate the effect of PF-127 hydrogel combined with bone marrow mesenchymal stem cell-derived exosome-miR-132 on the repair of alveolar bone defects by inducing osteogenic differentiation.Methods:Bone marrow mesenchymal stem cells(BMSCs)were cultured and transfected with anti-miR-132 and anti-miR-NC plasmids.Bone mesenchymal stem cell-derived exosomes(BMSCs-exo)were prepared and identified for expression of marker proteins by Western blot.PF-127 hydrogel and BMSCs-Exo complexes were prepared and BMSCs uptake was detected by PKH67 labeling;osteogenic ability of BMSCs was detected by alizarin red staining;reverse transcription-polymerase chain reaction(RT-PCR)was used to detect miR-132 expression and mRNA expression of alkaline phosphatase(ALP),osteocalcin(OCN),and Runt-related transcription factor 2(Runx2)in cells.An alveolar bone defect model was established in rats and the rats were randomly divided into three groups:PF-127 hydrogel group,PF-127 hydrogel+BMSCs-exo-anti-miR-NC group,and PF-127 hydrogel+BMSCs-exo-anti-miR-132 group.Micro-computed tomography(micro-CT)was used to evaluate the alveolar bone defect;Western blot was used to detect the expression of ALP,OCN,and Runx2 proteins in alveolar bone tissue.Results:miR-132 expression was significantly lower in the anti-miR-132 group BMSCs than in the anti-miR-NC group(P<0.05),indicating successful transfection of anti-miR-132 into BMSCs.Exosome markers CD9 and CD63 were significantly expressed in the BMSCs-exo-anti-miR-NC and BMSCs-exo-anti-miR-132 groups.Compared with the BMSCs-exo-anti-miR-NC group,miR-132 expression was significantly lower in the BMSCs-exo-anti-miR-132 group(P<0.05).Compared with the PF127 hydrogel group,miR-132 expression was significantly lower in the BMSC-exo-anti-miR-NC group,PF127 hydrogel+BMSC-exo-anti-miR-NC group,and PF127 hydrogel+BMSC-exo-anti-miR-132 groups,and the relative activity of alizarin red staining,Runx2,ALP,and OCN mRNA expression in cells were significantly increased,with the most significant changes in the PF127 hydrogel+BMSC-exo-anti-miR-132 group(P<0.05).Compared with the control group,BV/TV ratio,BMD,Tb.N,Tb.Th,and Runx2,ALP,and OCN protein expression in cells were significantly higher in the PF-127 hydrogel+BMSCs-exo-anti-miR-NC and PF127 hydrogel+BMSCs-exo-anti-miR-132 groups,and Tb.Sp was significantly lower(P<0.05),with the most significant changes in the PF127 hydrogel+BMSCs-exo-anti-miR-132 group.Conclusion:PF-127 hydrogel combined with bone marrow mesenchymal stem cell-derived exosome-miR-132 to interfere with miR-132 expression can promote osteogenic differentiation of bone marrow mesenchymal stem cells,thereby promoting the repair of alveolar bone defects in rats.