改良静电纺丝立体纳米纤维支架用于骨修复的研究

Modified Electrospinning Three-dimensional Nanofiber Scaffold for Bone Repair

目的:针对静电纺丝纳米纤维膜缺乏立体结构的问题,结合静电纺丝与静电喷雾技术制备立体纳米纤维支架,并评估其骨修复能力。方法:利用交替静电纺丝/静电喷雾制备聚己内酯(polycaprolactone, PCL)立体纳米纤维支架(PCL NFs+PCL MSs),采用扫描电镜观察支架形貌,并检测机械性能;将大鼠骨髓间充质干细胞接种在支架上,利用活死细胞染色和细胞骨架染色评估支架体外生物相容性;通过荧光定量聚合酶链反应(real-time fluorescence quantitative PCR,RT-qPCR)、碱性磷酸酶(alkaline phosphatase, ALP)染色和茜素红染色(alizarin red staining, ARS)和免疫荧光染色评估支架体外成骨性能,最后通过大鼠颅骨缺损模型评估支架体内成骨性能;全部实验采用单纯电纺支架(PCL NFs)作为对照。结果:PCL NFs+PCL MSs具有明显的纳米纤维和微球结构,相比PCL NFs, PCL NFs+PCL MSs具有更好的立体结构(厚度增加近3倍)和显著提高的机械性能及生物相容性,且细胞更易向PCL NFs+PCL MSs内部浸润。体外成骨诱导实验表明,PCL NFs+PCL MSs组rBMSCs成骨相关基因[骨形态发生蛋白-2(bone morphogenetic protein-2,BMP2)、ALP、Ⅰ型胶原蛋白(collagen typeⅠ, COL-1)、骨特异性转录因子2(Runt-related transcription factor 2,Runx2)、骨桥蛋白(osteopontin, OPN)]和蛋白(COL1和BMP2)表达显著上调,ALP和ARS染色也显示PCL NFs+PCL MSs具有更好的促rBMSCs成骨分化能力。动物实验结果同样提示相比PCL NFs, PCL NFs+PCL MSs展示出显著提高的骨修复性能。结论:交替静电纺丝与静电喷雾可成功制备立体多孔纳米纤维支架,相较于单纯静电纺丝纳米纤维膜,其更有利于细胞浸润,且骨修复性能显著提高,具有良好的临床应用前景。

Objective:To prepare three-dimensional nanofibrous scaffold by electrospinning and electrospraying technology,and to investigate its bone repair ability.Methods:The three-dimensional polycaprolactone scaffold(PCL NFs+PCL MSs)was prepared by alternating electrospinning/electrospraying.The morphology of the scaffold was observed by scanning electron microscope,and the mechanical properties were tested.Live/dead staining and cytoskeleton staining were performed to evaluate the biocompatibility of scaffold.The in vitro osteogenic performance of the scaffold was investigated using real-time quantitative PCR,alkaline phosphatase(ALP)staining,alizarin red staining(ARS),and immunofluorescence staining.Finally,the in vivo bone repair ability of the scaffold on rat skull defect model was evaluated.All experiments used pure electrospun nanofibrous scaffolds(PCL NFs)as controls.Results:PCL NFs+PCL MSs exhibited obvious nanofiber and microsphere structure.Compared to PCL NFs,PCL NFs+PCL MSs had better three-dimensional structure(nearly 3-fold increase in thickness),significantly improved mechanical properties,and biocompatibility.The cells were more easily ingrained into the interior of PCL NFs+PCL MSs.In vitro studies shown that the expression of osteogenic related genes[bone morphogenetic protein-2(BMP2),alkaline phosphatase(ALP),collagen typeⅠ(COL1),Runt-related transcription factor 2(RUNX2),and osteopontin(OPN)]and proteins(COL1,BMP2)in bone marrow mesenchymal stem cells of PCL NFs+PCL MSs were significantly upregulated.ALP and ARS staining also suggested that PCL NFs+PCL MSs had better osteogenic ability.In vivo study indicated that PCL NFs+PCL MSs had significantly improved bone repair ability.Conclusion:The three-dimensional nanofiber scaffold was successfully prepared by alternating electrospinning and electrospraying.This scaffold has significantly improved bone repair performance and good clinical application prospects.