负载葛根素的聚己内酯/明胶纳米纤维膜诱导成骨和抗菌的研究

Study on induced osteogenesis and antibacterial activity of polycaprolactone/gelatin nanofiber membrane loaded with puerarin

目的:用静电纺丝技术将聚己内酯(PCL)、明胶(GEL)、葛根素(PUE)制备成PCL/GEL@PUE纳米纤维膜,验证其生物相容性、机械性能、成骨及抗菌性能。方法:在电纺前溶液中分别加入0%(w/v)、0.1%(w/v)、0.2%(w/v)的PUE,制成PCL/GEL、PCL/GEL@0.1%PUE、PCL/GEL@0.2%PUE 3组纳米纤维膜。利用扫描电子显微镜(SEM)观察纳米纤维膜的结构;傅里叶红外光谱分析仪(FTIR)确认制备成功;力学性能测试纳米纤维膜机械强度。将骨髓间充质干细胞接种于各纳米纤维膜上,通过细胞计数试剂(CCK-8)法、钙黄绿素/碘化丙啶(Calcein-AM/PI)染色检测细胞相容性;茜素红染色检测成骨性能。将耐甲氧西林金黄色葡萄球菌与各组纳米纤维膜共培养,并涂琼脂板,计数菌落,检测抑菌率。结果:SEM结果显示,加入PUE后纤维变细,孔径变小,表面变粗糙;FTIR结果显示PCL/GEL@PUE纳米纤维膜构建成功;力学测试结果表明,加入PUE后,纳米纤维膜机械性能增强。CCK-8及Calcein-AM/PI染色结果显示,各组纳米纤维膜在第7天时能明显促进骨髓间充质干细胞增殖,其中PCL/GEL@0.2%PUE组细胞相容性最好。茜素红染色结果表明,PCL/GEL纳米纤维膜无明显成骨作用,而PCL/GEL@0.1%PUE、PCL/GEL@0.2%PUE纳米纤维膜成骨作用递增。抗菌实验结果表明,PCL/GEL纳米纤维膜无抗菌作用,而PCL/GEL@0.1%PUE、PCL/GEL@0.2%PUE纳米纤维膜分别有48%、86%的抑菌率。结论:负载PUE[0.1%(w/v)、0.2%(w/v)]的PCL/GEL纳米纤维膜具有良好的成骨性能和抗菌性能,其中以PCL/GEL@0.2%PUE组效果最好。

Objective:To prepare PCL/GEL@PUE nanofiber membrane with polycaprolactone(PCL),gelatin(GEL),and puerarin(PUE)by electrospinning technology,and to verify its biocompatibility,mechanical properties,osteogenesis and antibacterial properties.Methods:PCL/GEL,PCL/GEL@0.1%PUE and PCL/GEL@0.2%PUE of three groups of nanofiber membranes were prepared by adding 0%(W/V),0.1%(W/V),and 0.2%(W/V)of PUE into the pre-electrospinning solution.The structures of nanofiber membranes were observed by scanning electron microscope(SEM);the preparation was confirmed by Fourier infrared spectroscopy(FTIR);mechanical properties tested the mechanical strength of nanofiber membranes.The bone marrow mesenchymal stem cells were inoculated on the nanofiber membranes and the cell compatibility was tested by CCK-8 test and Calcein-AM/PI staining;alizarin red S staining was used to detect the osteogenic properties.Methicillin-resistant Staphylococcus aureus was co-cultured with nanofiber membranes of each group and coated with agar plates to count the colonies and test the bacteriostasis rate.Results:The results of SEM showed that the fibers became thinner,the pore diameter became smaller,and the surface became rougher after adding PUE;FTIR results showed that PCL/GEL@PUE nanofiber membrane was successfully constructed;the mechanical test results showed that the mechanical properties of nanofiber membranes were enhanced after adding PUE.The results of CCK-8 and Calcein-AM/PI staining showed that the nanofiber membranes in each group could significantly promote the proliferation of bone marrow mesenchymal stem cells on the 7th day,and the PCL/GEL@0.2%PUE group had the best cytocompatibility.Alizarin red S staining showed that polycaprolactone/gelatin nanofiber membrane had no obvious osteogenic effect,while PCL/GEL@0.1%PUE and PCL/GEL@0.2%PUE nanofiber membranes had increasing osteogenic effect.The results of the antibacterial experiment showed that PCL/GEL nanofiber membrane had no antibacterial effect,while PCL/GEL@0.1%PUE and PCL/GEL@0.2%PUE nanofiber membranes had 48% and 86% antibacterial rates,respectively.Conclusion:PCL/GEL nanofiber membranes loaded with PUE[0.1%(w/v),0.2%(w/v)]show good osteogenic and antibacterial properties,and PCL/GEL@0.2%PUE group shows the best effect.