基于三维打印技术的胶原/丝素仿生微导管修复脊髓损伤研究

Recovery of neurological function after spinal cord injury (SCI) is still a worldwide problem. Difficulty in axonogenesis is the key point to inhibit the recovery of neurological function after SCI. Tissue-engineering spinal micro-conduit is one of the most potential therapies for axonogenesis. However, the selection of biomaterials and manufacturing process which are suitable for spinal cord needs to be further investigated. Our study is intended to improve the biological and mechanical properties of the spinal micro-conduit with the natural biomaterials of composite collagen/silk fibroin. We try to optimize the micro-structure of the spinal cord by finite element analysis according to the nerve tracts, then, we built the three dimensional CAD model of spinal cord and produce the bionic spinal micro-catheter by three-dimensional printing （3DP） technology. After that, the eighth thoracic (T8) spinal cord in SD rats is completely transected, and the 2 mm long spinal cord is removed. AT last, the bionic functional micro-conduit carrying neural stem cells labeled by eGFP and SPIO is transplanted into the injury site 1 week after SCI. The BBB score, SPIO-MRI and evoked potentials are examined in vivo. BDA anterograde labeling was used to evaluate the host axonogenesis. The survival, migration, differentiation, and synaptic formation of grafted stem cells are detected in vitro with immunofluorescence and immune electron microscopy. In this study, we propose a new concept to optimize the micro-structure of the spinal cord by finite element analysis according to the nerve tracts. We attempt to improve natural biological materials with complementary manner. We would like to produce a new and microstructure-simulating bionic spinal cord micro-conduit by 3DP technology, which could provide some preliminary basis for theoretical and translational research of spinal cord tissue engineering.

脊髓损伤的神经功能恢复仍是世界性的难题。轴突再生困难是阻碍功能恢复的关键。基于组织工程的微导管给轴突再生带来希望，但适宜制备脊髓微导管的生物材料和工艺形式，尚需进一步探索。本课题拟以复合胶原/丝素蛋白为材料，改良导管的生物和机械性能，以神经传导束为单位，有限元分析优化脊髓微结构，建立三维CAD数据模型，以三维打印技术为成型工艺，制备仿生脊髓微导管。将载神经干细胞(eGFP和SPIO标记)的仿生微导管，植入大鼠T8全横断脊髓损伤模型。活体BBB评分、诱发电位和MRI检测、BDA顺行示踪；离体采用免疫荧光组织化学和免疫电镜，观察移植细胞的存活、迁移、分化、突触形成及宿主轴突的再生。本课题首次提出以神经传导束为单位建立三维CAD模型，以优势互补的方式改良天然生物材料，基于三维打印技术制备一种新型的、模拟微结构的仿生脊髓微导管，为脊髓组织工程的理论研究以及临床转化提供一定的前期基础。

脊髓损伤（SCI）的神经功能恢复仍是世界性的难题。轴突再生困难是阻碍功能恢复的关键。基于组织工程的微导管给轴突再生带来希望，但适宜制备脊髓微导管的生物材料和工艺形式，尚需进一步探索。本课题以复合胶原/丝素蛋白为材料，改良导管的生物和机械性能，以神经传导束为单位，有限元分析优化脊髓微结构，建立三维CAD数据模型，以三维打印技术为成型工艺，制备仿生脊髓微导管，对其工艺研究、基本性能和生物相容性进行测试。将载有神经干细胞的胶原/丝素功能化支架植入大鼠T8全横断脊髓损伤模型，采用BBB评分、诱发电位和 MRI检测、BDA顺行示踪、HE染色和免疫荧光染色等方法，对微导管修复SCI神经功能进行评估。通过以上研究，课题组得出以下结果和结论：(1) 胶原和丝素蛋白二者均具有较好的生物相容性，通过调节二者的配比控制其生物降解性，可避免损伤修复后期材料瘢痕缩窄引起的脊髓压迫。(2) 已经建立了胶原/丝素蛋白质量标准，改良了提取丝素蛋白的工艺方法。(3) 快速成型三维打印技术集合了机械工程、CAD、逆向工程技术、分层制造技术和数控技术，精确度高，孔径可达15-150 μm，符合脊髓微导管要求。(4) 设计出以神经传导束为基础构架的脊髓微导管，更贴近脊髓组织的微结构，为SCI后轴突再生提供了仿生微环境。(5) 3D打印的功能化脊髓微导管，可促进大鼠SCI后轴突再生和神经功能的修复。. 由本基金项目和天津市科技支撑项目（基于三维打印技术功能化脊髓微导管材料的研发与应用，14ZCZDGX00005）的共同资助下，在完成大鼠SCI模型研究的基础上，课题组进行了32只大型动物比格犬SCI模型的实验研究和第1例脊髓损伤生物医用材料的临床研究。从目前的大动物研究及临床部分病例来看，可以部分恢复受损脊髓的神经功能。临床SCI患者神经功能恢复是目前国际研究的难点，也是我们本项目研究的突破点。 . 本项目共发表SCI期刊论文7篇，中文期刊论文9篇；申请专利3项；获武警部队科技进步二等奖2项；参编学术专著2部；培养博士生1名和硕士生2名。