精氨酸–谷氨酸–天冬氨酸–缬氨酸改性丝素导管的制备及性能研究

Study on Preparation and Performance of Arg-Glu-Asp-Val Modified Silk Fibroin Tube

摘要: 目的：制备由REDV多肽修饰且具沟槽结构的丝素蛋白支架。方法：在含有30微米沟槽结构的PDMS膜上制备丝素薄膜，卷成管。通过多巴胺改性在丝素移植物表面共价连接REDV多肽进行修饰。扫描电镜观察其内表面微观形貌，电子万能试验机(TFW-58)检测REDV多肽修饰的丝素蛋白支架力学性能的改变。将RSC96细胞种植在丝素蛋白支架内表面1-3 d，观察丝素蛋白质支架内表面的沟槽结构对细胞趋向生长的影响。使用CCK-8 (cell counting Kit-8)测定法检测RSC96细胞在丝素移植物表面的增殖情况。采用SD大鼠制备坐骨神经损伤模型，分别用丝素导管(SF)、具沟槽丝素导管(TOPOLOGY)、REDV多肽修饰丝素导管(REDV)、REDV修饰且具沟槽结构丝素导管(TOPOLOGY/REDV)进行桥接修复。术后12 w测量靶肌湿重比、移植段坐骨神经纵切免疫荧光染色。结果：扫描电镜观察，TOPOLOGY组及TOPOLOGY/REDV组均具有均一的沟槽结构；力学测试结果表明REDV多肽修饰的丝素蛋白支架力学性能有所改善；RSC96细胞与丝素移植物共培养发现移植物表面沟槽结构对于细胞的趋向生长具有引导作用；CCK-8细胞活力检测表明REDV多肽修饰的丝素蛋白支架较单纯的丝素蛋白支架而言生物相容性有所改善；术后12 w靶肌恢复湿重比及坐骨神经纵切免疫荧光染色结果表明REDV多肽修饰且具沟槽结构的丝素蛋白支架对于细胞的吸附生长、损伤神经的修复具有促进作用。结论：REDV多肽修饰且具沟槽结构的丝素蛋白支架具有良好的生物相容性，对于细胞的粘附及趋向生长具有促进作用，为丝素蛋白在神经损伤再生领域提供了新的应用。

Abstract: Purpose: To prepare a silk fibroin scaffold with grooved structure modified by REDV polypeptide. Method: A silk fibroin film was prepared on a PDMS film containing a 30-micron groove structure and rolled into a tube. The REDV polypeptide is covalently attached to the surface of the silk fibroin graft for modification by dopamine modification. Scanning electron microscope was used to observe the micro morphology of the inner surface, and the electronic universal test (TFW-58) machine was used to detect the changes in the mechanical properties of the silk fibroin scaffold modified with REDV polypeptide. RSC96 cells were planted on the inner surface of the silk fibroin scaffold for 1 - 3 days, and the influence of the groove structure on the inner surface of the silk fibroin scaffold on cell growth was observed. CCK-8 (cell counting Kit-8) assay was used to detect the proliferation of RSC96 cells on the surface of silk fibroin grafts. SD rats were used to prepare sciatic nerve injury models. Silk fibroin catheters (SF), grooved silk fibroin catheters (TOPOLOGY), REDV polypeptide modified silk fibroin catheters (REDV), REDV modified silk fibroin catheters with grooved structure (TOPOLOGY/REDV) were used to perform bridge repair. 12 weeks after the operation, the target muscle wet weight ratio and the longitudinal section of the transplanted sciatic nerve were measured by immunofluorescence staining. Results: Scanning electron microscopy showed that both the TOPOLOGY group and the TOPOLOGY/REDV group had a uniform groove structure; the mechanical test results showed that the mechanical properties of the silk fibroin scaffold modified with REDV polypeptides had been improved; RSC96 cells and silk fibroin grafts were co-cultured, and it was found that the groove structure on the surface of the grafts had a guiding effect on the growth of cells; CCK-8 cell viability test showed that the silk fibroin scaffold modified by REDV polypeptide is more biological than simple silk fibroin scaffold. The CCK-8 cell viability test showed that the silk fibroin scaffold modified by REDV polypeptide has improved biocompatibility compared with the pure silk fibroin scaffold; the target muscle restored wet weight ratio and sciatic nerve longitudinal section immunofluorescence staining results 12 weeks after operation, which shows that the silk fibroin scaffold modifies with REDV polypeptide and has a grooved structure that can promote the adhesion and growth of cells and the repair of damaged nerves. Conclusion: REDV polypeptide modified silk fibroin scaffold with groove structure has good biocompatibility, promotes cell adhesion and growth tendency, and provides a new application for silk fibroin in the field of nerve injury and regeneration.