梯度释放SDF1α的有序纤维支架诱导内源性神经干细胞迁移修复脊髓损伤的研究

For repair of spinal cord injury (SCI), recruiting endogenous neural stem cells (NSCs) into the lesion site has great potential clinical use. After SCI, the endogenous NSCs are activated and migrate from the central canal region towards the injury site. However, most of the endogenous NSCs centered in the rostral and caudal borders of the lesion site, and few cells were found in the central region. Therefore, repair of complete removal SCI will require enhanced migration of endogenous NSCs from the rostral and caudal borders to the central region to reestablish neural connectivity. In the previous studies, we have developed stromal-cell-derived factor-1α (SDF1α) gradient embedded in the electrospun mats, and it was found that the SDF1α gradient scaffolds directed and enhanced NSC migration significantly in vitro. Herein, microarrays with gradient patterns will be fabricated by using micromachining technology, and gradient fibrous scaffolds will be produced by electrospinning with the gradient microarray as the collector. Additionally, the SDF1α covalently functionalized collagen electrospun membrane will be combined with SDF1α incorporated poly (ε-caprolactone) (PCL) electrospun membrane, and the biphasic delivery will be achieved based on the different release profiles of collagen and PCL electrospun fibers. The aligned fibrous scaffolds with gradual release of SDF1α from the central region to the rostral and caudal borders will direct and enhance the endogenous NSC migration from the rostral and caudal ends to the central region of the lesion site. In addition, the biphasic delivery of SDF1α will induce fast direction of NSC migration from the central canal region, followed by long-term direction of NSC migration to the central region. The aligned fibrous scaffolds immobilized with SDF1α gradient would enhance the endogenous NSC migration to the center, and promote the functional recovery of the complete SCI rats. The present study will provide valuable information for directing endogenous NSC migration in SCI repair for their clinical use.

内源性神经干细胞修复脊髓损伤有重要临床价值。但内源性神经干细胞迁移能力有限，主要位于脊髓断端附近，损伤中心较少。诱导神经干细胞向损伤中心迁移，贯通缺损区是脊髓全横断损伤修复首要问题。前期研究制备了基质细胞衍生因子(SDF1α)梯度释放支架，可体外诱导神经干细胞定向迁移。在此基础上，本项目拟采用微加工技术制备硅基梯度分布微阵列，以此为接收装置，利用静电纺丝技术制备纤维密度梯度分布支架，实现SDF1α梯度担载；通过化学修饰SDF1α胶原膜与内部包载SDF1α聚己内酯膜释放速度不同实现速/缓双相释放。有序纤维支架担载SDF1α从中间向两侧梯度释放，诱导脊髓断端内源性神经干细胞向损伤中心定向迁移；速/缓双相释放体系早期快速梯度释放结合长期缓慢梯度释放，诱导神经干细胞更快、更多、更远迁移，到达损伤中心，贯通缺损区，促进脊髓全横断损伤大鼠运动功能恢复。为内源性神经干细胞修复脊髓损伤的临床应用奠定基础。

在脊髓损伤修复中内源性神经干细胞起着至关重要的作用，然而其主要位于脊髓断端附近，难以迁移到损伤中心，如何诱导神经干细胞迁移到损伤中心是脊髓全横断损伤修复面临的关键难题。本项目“梯度释放SDF1α的有序纤维支架诱导内源性神经干细胞迁移修复脊髓损伤的研究”旨在考察微阵列电场分布对纳米纤维聚集体结构梯度成型的影响，研究梯度纤维支架的因子梯度释放和速/缓释放规律及其对干细胞迁移、分化的协同作用，揭示梯度担载SDF1α纤维支架的促大鼠脊髓损伤修复的作用效果。项目执行期间主要开展了以下几个方面的研究工作：（1）通过研究微阵列电极分布对纺丝电场强度的影响规律，设计了程序化微阵列，得到了一系列不同梯度分布的微阵列电极接收装置，实现了纳米纤维聚集体结构的梯度有序成型；（2）分析了梯度纤维膜的聚集体结构在顶破、拉伸过程中的力学传递规律，探究了聚合物双网络交联体系与纤维支架力学性能的内在关联，研究了梯度纤维支架的SDF1α梯度释放和速/缓顺序释放规律；（3）阐明了SDF1α梯度纤维支架的梯度、速/缓顺序释放体系与干细胞迁移和分化的内在关联，深入探究了梯度释放与速/缓顺序释放对干细胞行为的协同作用效果；（4）建立大鼠全横断脊髓损伤模型，采用免疫组化染色检测损伤脊髓的神经干细胞迁移及分化效果，对修复后大鼠后肢运动功能进行了评估，探究梯度SDF1α纤维支架的梯度释放和速/缓顺序释放体系与大鼠脊髓损伤修复的内在关联，最终实现了促进体内神经干细胞迁移及全横断脊髓损伤大鼠运动功能恢复的目标。经过四年的研究，现已顺利完成任务书中规定的任务，项目执行期间在Adv. Funct. Mater.、Biomaterials、ACS Nano等杂志发表SCI论文11篇，申请国家发明专利3项，本项目的完成对实现梯度有序纳米纤维支架的可控制备及其在脊髓全横断损伤修复领域中的应用具有重要意义。