丝素蛋白/生物活性玻璃复合纤维膜的制备及性能表征

Preparation and characterization of silk fibroin/bioactive glass composite fiber membrane

背景:在构建具有生物功能的引导骨再生膜时,单一材料因功能不足而无法满足临床上的需要,因此将多种材料复合已成为目前组织修复工程的一种趋势。目的:通过静电纺丝技术制备丝素蛋白/生物活性玻璃复合纤维膜,表征其理化性能和体外生物相容性。方法:将0.8 g丝素蛋白溶解于10 mL六氟异丙醇中制备静电纺丝溶液,利用静电纺丝技术制备丝素蛋白纳米纤维膜(记为SF纤维膜);将0.1,0.3,0.5,0.8 g的生物活性玻璃分别加入静电纺丝溶液中,利用静电纺丝技术制备丝素蛋白/生物活性玻璃复合纤维膜(依次记为SF/1BG、SF/3BG、SF/5BG、SF/8BG纤维膜)。表征5组纤维膜的理化性能及生物相容性。结果与结论:①扫描电镜下可见5组纤维表面光滑、连续且均匀,无串珠样结构,丝纤维之间无明显粘连,均表现出随机排布的无序多孔结构,添加生物活性玻璃后纤维膜的纤维直径减小。傅里叶红外光谱与X射线衍射检测显示,纤维膜中丝素蛋白与生物活性玻璃的化学结构稳定。SF、SF/1BG、SF/3BG、SF/5BG、SF/8BG纤维膜表面的水接触角分别为105.02°,72.58°,78.13°,79.35°,72.50°。②将骨髓间充质干细胞分别接种于5组纤维膜上,CCK-8检测显示相较于SF、SF/8BG纤维膜,SF/1BG、SF/3BG、SF/5BG纤维膜可促进骨髓间充质干细胞的增殖;活/死细胞染色显示5组纤维膜表面的细胞活力较好,其中SF/5BG纤维膜表面的细胞数量更多、分布更均匀;罗丹明-鬼笔环肽染色与扫描电镜观察显示相较于SF纤维膜,SF/5BG纤维膜更有利于骨髓间充质干细胞的黏附。将骨髓间充质干细胞分别接种于5组纤维膜上进行成骨诱导分化,SF/3BG、SF/5BG组碱性磷酸酶活性高于其他3组(P<0.05,P<0.01,P<0.001);茜素红染色显示,添加生物活性玻璃后纤维膜的钙结节形成增加,并且以SF/5BG组钙结节形成最多。③结果表明,丝素蛋白/生物活性玻璃复合纤维膜具有良好的生物安全性和生物相容性。

BACKGROUND:In the construction of guided bone regeneration membrane with biological function,a single material cannot meet the clinical needs due to its insufficient function,so the composite of multiple materials has become a trend of tissue repair engineering.OBJECTIVE:To prepare silk fibroin/bioactive glass composite fiber membranes by electrospinning technology,and to characterize the physicochemical properties and biocompatibility in vitro.METHODS:The solution of electrospinning was prepared by dissolving 0.8 g silk fibroin protein in 10 mL hexafluoro-isopropanol alcohol,and the nanofiber membrane of silk fibroin protein was prepared by electrospinning technology(denoted as SF fiber membrane).0.1,0.3,0.5,and 0.8 g of bioactive glass were added to the electrospinning solution,and the silk fibroin/bioactive glass composite fiber membrane was prepared by electrospinning technology(recorded as SF/1BG,SF/3BG,SF/5BG,and SF/8BG fiber membrane in turn).The physicochemical properties and biocompatibility of five groups of fiber membranes were characterized.RESULTS AND CONCLUSION:(1)The scanning electron microscopy results showed that nanofibers of the prepared composite membrane were smooth,continuous and uniform and had no beaded structure.There was no obvious adhesion between the silk fibers,and they all showed random arrangement of disordered porous structures.The fiber diameter of the fiber membrane decreased after the addition of bioactive glass.Fourier infrared spectroscopy and X-ray diffraction detection results showed that the chemical structure of silk fibroin protein and bioactive glass in fiber membrane was stable.The water contact angles of SF,SF/1BG,SF/3BG,SF/5BG,and SF/8BG were 105.02°,72.58°,78.13°,79.35°,and 72.50°,respectively.(2)Bone marrow mesenchymal stem cells were inoculated on five groups of fiber membranes.CCK-8 assay results showed that SF/1BG,SF/3BG,and SF/5BG fiber membranes could promote the proliferation of bone marrow mesenchymal stem cells compared with SF and SF/8BG.Live cell/dead cell staining showed that the cell vitality on the surface of the five groups of fiber membranes was better,and the number and distribution of cells on the surface of SF/5BG fiber membrane were more uniform.Rhodamine phalloidin staining and scanning electron microscopy exhibited that compared with SF fiber membrane,the SF/5BG fiber membrane was more favorable to the adhesion of bone marrow mesenchymal stem cells.Bone marrow mesenchymal stem cells were inoculated on the fiber membrane of the five groups for osteogenic induction differentiation,and the alkaline phosphatase activity of the SF/3BG and SF/5BG groups was higher than that of the other three groups(P<0.05,P<0.01,P<0.001).Alizarin red staining showed that the formation of calcium nodules in fiber membrane increased after the addition of bioactive glass,and the formation of calcium nodules in the SF/5BG group was the most.(3)The results show that silk fibroin/bioactive glass composite fiber membrane has good biosafety and biocompatibility.