基于丝素定向微纳米结构与功能的脊髓重构及性能的研究

Repairing spinal cord injury (SCI) is a major challenge in the filed of nerve regeneration. A novel approach to overcome this challenge is to take advantage of biodegradable natural biomaterials with oriented micro and nano structures, since such structures can mimic the morphology of native spinal cord tissue. Our research team has previously found that silk fibroin (SF) scaffolds with oriented micro ridges could guide hippocampal neurons axonal elongation and migration to encourage SCI recovery. However, the extent of SCI recovery is still limited, which has motivated us to further improve the micro-environment of the scaffolds. Based on our previous research, we will use green and feasible approach of slightly dissolve the SF fibers by calcium salt to obtain the micro SF fibers with nano structures and nerve growth factor (NGF). We will improve the pore interconnection and the micro-environment of the silk fibroin (SF) based scaffolds by incorporating hyaluronic acid (HA), laminin (LN). To insight the methods of controlling micro and nano structures of SF, the influences of preparation conditions on the performance of the scaffolds will be investigated. Also, to evaluate the effects of scaffolds' morphology and structure on the behavior of nerve stem cells, the adhesion, proliferation, alignment and protein expression of the cells will be examined. In addition, we will investigate the SCI repair bioactivity of the scaffolds and how such scaffolds affect axon regeneration, with an animal model. This study will provide essential data for the development of SCI tissue regeneration.

脊髓缺损的修复是神经再生医学领域的难题，定向微纳米结构的生物可降解材料能够模拟脊髓组织结构，为脊髓修复提供了新方法。本项目组前期研究发现定向微纳米图案的丝素支架对神经细胞具有引导作用，有促脊髓缺损修复的生物活性，但损伤脊髓的功能恢复还不够理想，需深入研究。本研究在前期工作的基础上，利用绿色、简便的钙盐微溶技术，制备微纳米结构的丝素纤维，并负载神经生长因子、整合透明质酸（HA）和层粘连蛋白（LN），构建定向微纳米结构的多功能丝素基脊髓修复支架。通过研究制备条件对支架的理化性能的影响，找到控制支架的微纳结构和性能的方法。通过观察神经干细胞的发育、排列及其相关蛋白表达，明确材料的组成和微结构对细胞行为的影响；通过评估不同支架对大鼠脊髓缺损的修复效果，分析材料的组成和结构在体内不同时期对脊髓再生的影响，揭示支架的多功能信号协同促进脊髓修复的机制，为组织工程丝素支架修复脊髓缺损提供重要实验数据。

脊髓损伤的重建是再生医学领域研究的热点，自体神经移植仍是其治疗脊髓缺损的“金标准”，但也受限于供体不足、结构差异和二次手术等。具有促脊髓修复活性的丝素生物材料支架为脊髓的再生提供了替代方案。本研究采用定向温度场冷冻和钙盐微溶等技术，构建了具有定向微纳米结构和活性成分的丝素基三维支架，优化了支架的成型条件和活性成分，探明了支架形貌的调控规律；研究了支架对神经细胞生长的支撑作用，发现定向通道及微纳米结构对神经轴突的延伸具有引导作用；研究了支架对SD大鼠缺损脊髓重建的促进作用，结果表明定向通道的丝素/层粘连蛋白三维支架能有效地引导神经轴突的延伸，完成缺损区域的桥接，并在植入70天后完全降解，具有促脊髓再生的活性。本研究为基于丝素蛋白质人工脊髓的开发，提供了重要的理论依据和支持。项目资助已发表SCI收录论文5篇，申请国家发明专利7项，参加国际学术会议5次，培养硕士研究生4名，其中2名已取得学位，2名即将毕业。项目投入经费25万元，支出经费20.424739万元，剩余经费4.575261万元，剩余经费计划用于本项目后续3篇论文的出版和专利申请维护费用。