Objective: To explore the construction and biocompatibility in vitro evaluation of the electrospun-graphene (Gr)/silk fibroin (SF) nanofilms.
Methods: The electrostatic spinning solution was prepared by dissolving SF and different mass ratio (0, 5%, 10%, 15%, and 20%) of Gr in formic acid solution. The hydrophilia and hydrophobic was analyzed by testing the static contact angle of electrostatic spinning solution of different mass ratio of Gr. Gr-SF nanofilms with different mass ratio (0, 5%, 10%, 15%, and 20%, as groups A, B, C, D, and E, respectively) were constructed by electrospinning technology. The structure of nanofilms were observed by optical microscope and scanning electron microscope; electrochemical performance of nanofilms were detected by cyclic voltammetry at electrochemical workstation; the porosity of nanofilms were measured by n-hexane substitution method, and the permeability were observed; L929 cells were used to evaluate the cytotoxicity of nanofilms in vitro at 1, 4, and 7 days after culture. The primary Sprague Dawley rats' Schwann cells were co-cultured with different Gr-SF nanofilms of 5 groups for 3 days, the morphology and distribution of Schwann cells were identified by toluidine blue staining, the cell adhesion of Schwann cells were determined by cell counting kit 8 (CCK-8) method, the proliferation of Schwann cells were detected by EdU/Hoechst33342 staining.
Results: The static contact angle measurement confirmed that the hydrophilia of Gr-SF electrospinning solution was decreased by increasing the mass ratio of Gr. Light microscope and scanning electron microscopy showed that Gr-SF nanofilms had nanofiber structure, Gr particles could be dispersed uniformly in the membrane, and the increasing of mass ratio of Gr could lead to the aggregation of particles. The porosity measurement showed that the Gr-SF nanofilms had high porosity (>65%). With the increasing of mass ratio of Gr, the porosity and conductivity of Gr-SF nanofilm increased gradually, the value in the group A was significantly lower than those in groups C, D, and E ( P<0.05). In vitro L929 cells cytotoxicity test showed that all the Gr-SF nanofilms had good biocompatibility. Toluidine blue staining, CCK-8 assay, and EdU/Hoechst33342 staining showed that Gr-SF nanofilms with mass ratio of Gr less than 10% could support the survival and proliferation of co-cultured Schwann cells.
Conclusion: The Gr-SF nanofilm with mass ratio of Gr less than 10% have proper hydrophilia, conductivity, porosity, and other physical and chemical properties, and have good biocompatibility in vitro. They can be used in tissue engineered nerve preparation.
目的: 探索以石墨烯（graphene，Gr）和丝素蛋白（silk fibroin，SF）为原料，采用静电纺丝技术制备 Gr-SF 纳米膜方法，并评价Gr-SF 纳米膜体外生物相容性。.
方法: 取 Gr 和 SF 溶于甲酸溶液，按照不同 Gr 质量分数（0、5%、10%、15%、20%）配制静电纺丝溶液，测定溶液静态接触角。采用静电纺丝技术制备 0、5%、10%、15%、20% Gr-SF 纳米膜（分别为 A、B、C、D、E 组），光镜和扫描电镜观察其结构，电化学工作站循环伏安法测定电导率，正己烷溶液置换法测定孔隙率，采用 L929 细胞以细胞计数试剂盒 8（cell counting kit 8，CCK-8）法评价细胞毒性。取原代培养的 SD 大鼠雪旺细胞与各组Gr-SF 纳米膜共培养 3 d，甲苯胺蓝染色观察雪旺细胞生长形态和分布，CCK-8 法检测细胞黏附情况，EdU/Hoechst33342 染色检测细胞增殖情况。.
结果: 静态接触角检测显示随 Gr 质量分数增大，Gr-SF 静电纺丝溶液亲水性降低。光镜和扫描电镜观察示 Gr-SF 纳米膜具有纳米纤维结构，Gr 颗粒分散在纳米膜中，Gr 质量分数增加会促使颗粒聚集。孔隙率检测示 Gr-SF 纳米膜均具有高孔隙率（>65%），随 Gr 质量分数增加，Gr-SF 纳米膜的孔隙率和电导率均逐渐增加，C、D、E 组均高于 A 组（ P<0.05）。体外 L929 细胞毒性检测显示 Gr-SF 纳米膜具有良好生物相容性。甲苯胺蓝染色、CCK-8 检测及 EdU/Hoechst33342 染色示，Gr 质量分数在 10% 以内的 Gr-SF 纳米膜可支持共培养的雪旺细胞成活和增殖。.
结论: 采用静电纺丝技术制备的 10%Gr-SF 纳米膜具有合适的亲水性、导电性、孔隙率等理化特性，同时具有良好的体外生物相容性，可用于组织工程神经制备。.
Keywords: Electrospinning; Schwann cells; electrical conductivity; graphene; nanofilm; silk fibroin.