3D打印NSCs水凝胶-胶原/丝素-分泌组微球“三丝成束”支架修复脊髓损伤的研究

The tissue engineering scaffold based on 3D printing technology has provided a new field for the study of neurological function reconstruction of spinal cord injury (SCI). In most researches, cells are not printed directly but planted onto the 3D-printed scaffolds, which exists the question of uncontrollable cell density and distribution. Although cells are printed directly in a few studies, the survival rate is very low in the injury microenvironment. In this study, we continue to use the natural biomaterials of collagen/silk fibroin to fit the elastic modulus of spinal cord soft tissue on the basis of 3D-printied scaffold in our Youth Fund. The CAD model was designed by the new concept based on three filaments bundle as the unit, and the scaffold with neural stem cells (NSCs) hydrogel, collagen/silk fibroin and secretome microspheres were fabricated by the extrusion 3D bioprinter with three nozzles. High density NSCs grow and stretch along the longitudinal axis of hydrogel stretch to bridge the broken axons, collagen/silk fibroin biomaterials provides mechanical support, and MSCs secretome microspheres construct factor network to improve the injury microenvironment and increase the survival rate of NSCs. T8 transection model of spinal cord was built in SD rats and 3D-printed scaffold is implanted into injury sites. The repair of neurological function, the recanalization of white matter tracts and the survival and differentiation of transplanted NSCs were observed. In this study, the three elements of the cell, the factor and the biological material can be controlled by 3D printing to achieve the time and space reorganization, providing the theoretical basis for the construction of tissue organ and the application of 3D printing technology in SCI.

基于3D打印的组织工程支架为脊髓损伤（SCI）神经功能重建开辟了研究新领域。多数3D打印并非一体成型，而是将细胞种植于已打印的支架，存在细胞密度和分布均不可控的问题；少数研究虽直接打印细胞，但细胞在损伤微环境的存活率较低。本课题拟在青年基金3D打印脊髓支架的基础上，沿用胶原/丝素天然生物材料以契合脊髓软组织的弹性模量，引入“三丝成束”的CAD设计新理念，采用挤压出丝型三喷嘴3D打印机制备NSCs水凝胶-胶原/丝素-分泌组微球支架：高密度NSCs沿纵轴水凝胶拉伸以桥接断裂轴突；胶原/丝素材料提供力学支撑；MSCs分泌组微球构建因子网络以改善损伤微环境和提高NSCs存活率。建立大鼠T8全横断SCI模型，植入3D打印支架，观察神经功能修复、白质纤维束再通及NSCs存活分化情况。本课题将细胞、因子和生物材料一体化3D打印以实现可控性时空重组，为组织器官构建及3D打印技术在SCI的应用提供理论基础。

脊髓损伤（SCI）现已成为世界范围内亟待解决的公共卫生问题。基于3D打印的组织工程支架为SCI神经功能重建开辟了研究新领域。因此，携脐带间充质干细胞分泌组的3D打印一体成型支架是治疗SCI神经功能缺损的有效治疗措施。本课题在青年基金3D打印脊髓支架的基础上，沿用胶原/丝素天然生物材料以契合脊髓软组织的弹性模量，采用挤压出丝型三喷嘴3D打印机制备NSCs水凝胶-胶原/丝素-分泌组支架：高密度NSCs沿纵轴水凝胶拉伸以桥接断裂轴突；胶原/丝素材料提供力学支撑；MSCs分泌组构建因子网络以改善损伤微环境和提高NSCs存活率。本课题还探讨了3D打印BDNF和胶原蛋白/壳聚糖支架对SCI后的神经再生的影响。建立大鼠T10全横断SCI模型，植入3D打印支架，观察神经功能修复、白质纤维束再通及NSCs存活分化情况。结果发现，3D打印NSCs水凝胶-胶原/丝素支架-分泌组支架和BDNF和胶原蛋白/壳聚糖支架促进SCI后神经纤维束的再生，加速突触连接的建立和髓鞘再生，改善SCI后的运动功能。本课题将细胞、因子和生物材料一体化3D打印以实现可控性时空重组，为3D打印技术在SCI的应用提供理论基础。