内置载诱向因子纤维和基因修饰雪旺细胞的导管用于长段周围神经缺损修复的研究

The repair of long-gap peripheral nerve defects has been a challenge in the treatment of peripheral nerve injuries for a long time. In the present project, a new solution based on neurochemotropic factor, factor-loaded fibrous luminal fillers, gene-modified Schwann cell and nerve guidance conduit has been proposed. A new type of biodegradable copolymers, chitosan-polydioxanone (CS-PDO), is synthesized by carefully controlling the chain structure and compositional proportion of CS-PDOs, and some optimal CS-PDOs are used as starting materials for building porous conduits using genipin as a crosslinker in considering that genipin is a type of non-cytotoxic and naturally occurring procudct. Some chitosan/silk fibroin composite fibers in the micron size range are prepared using a wet-electrospining technique, and they can be easily set into the conduit to serve as a physical matrix to induce the growth of newly grown axons. These composite fibers are also loaded with some neurochemotropic factors using a self-assembly method, and the entrapped factors are distributed along both longitudinal and radial direction of the fibers with a gradient characteristic. The factors are loaded in such a way that they can be released in a well-controlled manner without significant initial burst release over a prolonged period of time while functioning as chemical inducer for directing the growth of axons. Schwann cells are selected as seed cells but they are transfected with GDNF (glial cell line-derived neurotrophic factor) gene to improve its expression of GDNF before they can be seeded into the conduit. The achieved conduit complexes cotainning neurochemotropic factors fibers and Schwann cells would serve as a new type of implants for bridging long-gap peripheral nerve defects in the follow-up in vivo examinations. It can be expected that after being implanted, some optimized conduit complexes could significantly facilitate the directional induction regeneration of axons growth cone from the proximal end to the distal end of the nerve defect and greatly help to selectively establish reinnervation function of nerves due to the synergetic functions stemmed from neurochemotropic factors, fiber carriers, Schwann cells and the porous CS-PDO conduit. Based on the relationship between the conduit complexes and in vivo experimental results, the detailed mechanism for reconstructing long-gap peripheral nerves defects can be further clarified. The presently proposed solution would not only help to understand the micro-regulation mechanisms for the induction growth of axons during the regeneration of peripheral nerves but also possibly develop a new method for repairing long-gap peripheral nerve defects in the clinical treatment.

长段周围神经缺损修复中再生神经组织功能恢复率低一直是临床医学中的难题, 本项目提出了新的解决方法。优化合成可生物降解壳聚糖-聚对二氧环己酮共聚物并将其制备成孔状圆筒形导管; 用电湿纺方法制备壳聚糖/丝素蛋白复合物纤维,在设定长度且平行排列的纤维上用层-层自组装方法逐段组装加载诱向因子并使因子载量沿纤维轴向呈可控梯度分布; 对雪旺细胞进行基因修饰提高其对胶质细胞源神经营养因子的表达水平和时效性; 将平行排列的载诱向因子纤维束与基因修饰的雪旺细胞复合后内置于导管中组装成复合体。通过导管、纤维束、导管内呈浓度梯度分布诱向因子和雪旺细胞的协同作用，该导管复合体将协助新生轴突自长段神经缺损近端向远端方向定向生长并与靶器官建立正确连接，从而可望显著提高再生神经的功能恢复率。本项目如顺利实施不但有助于深入理解周围神经再生过程中调控轴突诱向生长的微观机制，还可能为临床修复长段周围神经缺损提供有效的新方法。

将活性因子加载于纤维束使得持续释放的因子沿纤维轴向呈浓度梯度分布并长期保持其活性。组装导管/内衬纤维复合体用于25mm或以上周围神经缺损进行修复或重建。合成壳聚糖-聚对二氧环己酮(CS-PDO)共聚物，将优化获得的CS-PDO制备成孔状导管使得导管壁具有可控的生物分子渗滤特性并可防止结缔组织内侵。另合成壳聚糖-聚乳酸(CS-PLA)共聚物并制备成表面亲水的长纤维。以海藻酸钠为涂层材料通过逐段分层组装方法将活性因子(NGF)加载到CS-PLA纤维束上。将NGF/CS-PLA纤维束复合雪旺细胞与CS-PDO导管复合, 由此发展出一类多功能NGF/CS-PLA/CS-PDO神经损伤修复导管。通过调节纤维直径和数量、涂层长度和厚度、NGF梯度分度及释放行为、导管力学和降解性质以及导管壁参数可综合控制该导管复合体的功能。动物体内长段神经缺损修复结果表明优化获得的多功能导管复合体能显著增强修复过程中新生神经轴突的诱向再生和明显提高新生神经组织的功能恢复。可能的促神经诱向再生机制涉及纤维支撑和导向、纤维之间微通道和NGF因子梯度生物化学诱向三者的协同效应。所获得的结果为后续转化应用研究打下了良好基础。