时间-空间可控生物活性分子释放的胶原支架用于脊髓损伤神经网络重建的研究

Neural stem cell (NSC) transplantation offer potential therapeutic strategies for spinal cord injury (SCI) repair. However, it will not be sufficient to achieve complete functional restoration. The poor axonal regeneration is due to the presence of axonal growth inhibitors and lack of temporal and spatial expression of neurotrophic factors. Functionalized scaffolds which can provide temporally and spatially controlled biomolecule delivery for NSC tranplantion are becoming increasingly attractive. We propose here a strategy based on gradient and sequential delivery of epidermal growth factor receptor (EGFR) antibody and brain-derived neurotrophic factor (BDNF) from collagen scaffolds. On the one hand, a sequential delivery of EGFR antibody and BDNF was supposed to reduce the activation of glial cells during the acute phase response, and further promote neuronal differentiation of NSCs during the long-term regeneration. On the other hand, a gradient delivery of EGFR antibody was supposed to attenuate spatially the effect of myelin associated inhibitors, and, BDNF gradient delivery was supposed to promote long-distance axonal regeneration. In order to achieve the gradient and sequential delivery, the EGFR antibody fused with a collagen-binding domain (CBD) derived from collagenase was electrosprayed gradiently on the three-dimensional collagen scaffolds incorporated with spatially distributed BDNF-poly (lactide-co-glycolide) (PLGA) microspheres. The present study will contribute to a better understanding of temporal and spatial regulation following NSC transplantation after SCI repair, and provide valuable information for its clinical use.

神经干细胞移植为脊髓损伤修复带来希望，但仍面临挑战。主要包括损伤急性期胶质细胞活化并分泌抑制因子，修复期缺乏空间分布的神经营养因子。因此，构建生物活性分子时间、空间协同释放的支架载体，才能满足脊髓损伤修复不同阶段、位置的需求。本项目拟将表皮生长因子受体(EGFR)抗体和脑源性神经营养因子(BDNF)的时空控释引入胶原支架，一方面，EGFR抗体、BDNF顺序释放抑制急性期胶质细胞活化，促进修复期神经干细胞向神经元分化；另一方面，EGFR抗体梯度释放阻断抑制因子作用，BDNF梯度释放诱导轴突长距离延伸。为此，我们采用融合胶原特异结合短肽(CBD)的EGFR抗体，利用CBD改造分子与微球担载因子释放速度不同实现顺序释放；最终通过CBD-EGFR抗体静电喷雾梯度涂覆和BDNF-PLGA微球梯度担载实现CBD-EGFR抗体/BDNF梯度、顺序释放。加深脊髓损伤修复时空调控的认识，为临床应用奠定基础。

神经干细胞修复脊髓损伤的挑战在于如何改善损伤区微环境，促进神经网络重建，提高修复效果，本项目通过组织工程的方法实现生物活性分子时间-空间释放来缓解损伤微环境，构建适宜神经干细胞迁移及向神经元分化的微环境，促进脊髓损伤修复。根据项目书，我们表达纯化了具有胶原特异结合短肽的CBD-EGFR抗体以构建胶原支架缓慢释放体系；构建了三维胶原支架梯度担载生物活性分子-微球体系，开发了胶原支架复合静电喷雾梯度涂层趋化因子梯度缓慢释放体系。实验结果表明，功能胶原支架可实现生物活性分子梯度或/和缓慢释放，可以缓解髓鞘蛋白等抑制因子的抑制作用，诱导神经干细胞定向迁移和/或向神经元细胞定向分化。将CBD-EGFR抗体修饰胶原支架复合神经干细胞移植到大鼠脊髓全横断损伤区，可促进神经干细胞向神经元细胞分化，促进神经纤维生长，提高脊髓损伤大鼠后肢运动功能恢复，为功能胶原材料的临床应用奠定基础。依托本项目，发表基金标注SCI论文9篇（影响因子均>5.0），申请国家发明专利5项。参加国内、国际会议4次，口头报告3次。