【摘要】 目的 制备纳米硅酸镁锂功能化聚己内酯(nanosilicate functionalized polycaprolactone,PCL/LAP)电纺膜，评价其对骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)介导骨再生修复的作用。方法 通过静电纺丝技术制备PCL/LAP电纺膜，并与大鼠BMSCs细胞共培养，通过细胞骨架染色、活/死细胞染色和CCK-8法检测PCL/LAP电纺膜的细胞相容性。利用划痕实验、Transwell迁移实验和qRT-PCR检测细胞迁移相关基因Pdgf、Tgfβ和Mmp2表达，评估PCL/LAP电纺膜对BMSCs迁移能力的影响。采用碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色和qRT-PCR检测成骨分化基因(Alp、Col1a1、Runx2、Bglap和Bmp2)与成血管相关基因(Angpt1、Fgf2和Vegfa)的表达水平，Western blot检测RUNX2蛋白表达，评估PCL/LAP电纺膜对BMSCs成骨分化与成血管的调控作用。PCL和PCL/LAP电纺膜条件培养液分别作用血管内皮细胞EAhy926，通过qRT-PCR检测成血管相关基因(KDR、ENOS和HIF1A)的表达。结果 BMSCs在PCL/LAP电纺膜表面黏附良好，细胞活力无明显影响(P>0.05)。PCL/LAP不仅促进BMSCs增殖(P<0.05)、迁移，还能增强ALP活性和促进矿化结节形成(P<0.05)，上调成骨分化相关基因与蛋白的表达(P<0.05)。PCL/LAP电纺膜可促进BMSCs高表达成血管相关因子基因(P<0.05)，间接调控血管内皮细胞成血管分化基因的表达(P<0.05)。结论 PCL/LAP电纺膜具有优异的生物相容性，可促进BMSCs的增殖、迁移、成骨分化及其介导的成血管分化，有望作为屏障膜应用于骨缺损的再生修复治疗。更多还原

【Abstract】 Objective To fabricate nanosilicate functionalized polycaprolactone( PCL/LAP) electrospun membrane and evaluate its role in bone marrow mesenchymal stem cells( BMSCs)-induced bone repairing. Methods The PCL/LAP electrospun membranes were fabricated via electrospinning technology and co-cultured with rat BMSCs. The cytocompatibility of the membranes was evaluated through cytoskeleton staining,live/dead cell staining and CCK-8 assay. The migration capacity of BMSCs was assessed using scratch assay,Transwell migration experiments and expression of migration-related genes( Pdgf and Tgfβ) was evaluated by qRT-PCR. The osteogenic differentiation and pro-angiogenesis potential were determined by alkaline phosphatase( ALP) staining,alizarin red staining,expression levels of osteogenesis-related genes( Alp,Col1a1,Runx2,Bglap and Bmp2) and angiogenesis-related genes( Angpt1,Fgf2 and Vegfa) along with RUNX2 protein expression. PCL and PCL/LAP electrospun membranes conditioned medium was subsequently used to stimulate vascular endothelial cells( EAhy926). The expression of angiogenesis-associated genes( KDR,ENOS and HIF1A)was quantified by qRT-PCR. Results BMSCs adhered well to the surface of the PCL/LAP membranes,with no significant impact on cell viability( P>0.05). PCL/LAP membranes not only promoted the proliferation( P<0.05),migration( P<0.05),but also enhanced ALP activity and mineralized nodule formation( P<0.05),increased osteogenic differentiation gene and protein expression( P<0.05) of BMSCs. Moreover,PCL/LAP promoted the expression of angiogenic genes of BMSCs( P<0.05),to indirectly regulate angiogenesis-related gene expression in vascular endothelial cells( P <0.05). Conclusion PCL/LAP electrospun membranes exhibit excellent biocompatibility and can promote proliferation,migration,osteogenic differentiation and BMSC-mediated angiogenic differentiation,showing great potential for bone defect repairing as barrier membrane.更多还原