碱性成纤维生长因子修饰脱细胞脊髓支架结合载NTP温敏凝胶构建仿生脊髓修复大鼠脊髓损伤的实验研究

For complications such as motor function damage and neuropathic pain caused by spinal cord injuries, currently routine treatments just focus on recovery of motor function but neglect treatments for long-term pain, lacking comprehensive treatment approaches. Furthermore, as the traditional single spinal cord scaffold cannot really simulate macroenvironment and microenvironment of the spinal cord, it hard to repair spinal cord injuries simply by using routine scaffolds for tissue engineering. Based on the above-mentioned reasons and jobs by this research group, we combined advantages of acellular spinal cord scaffold and temperature-sensitive gel and modified the acellular spinal cord scaffold with bFGF so that the scaffold could continuously release exogenous bFGF at spinal cord injuries. Meanwhile, bionic spinal cords were constructed based on characteristics of body temperature gel state of NTP temperature-sensitive gel in low temperature liquid state to induce long-term release of NTP so as to inhibit neuropathic pain and promote regeneration of spinal cord tissue. At the same time, we transplanted the autologous activated Schwann cells to transplantation areas of bionic spinal cords to supplement the missing trophic factors and neurons. The inhibiting effect on neuropathic pain was judged via determining pain thresholds of rats to mechanical and cold stimuli. Behavior function recovery of rats in different groups were assessed by BBB scoring and other methods. Regeneration of damaged axons was researched using GAP43, BDA tracing and other methods so as to provide clinical treatments for spinal cord injuries with new ideas.

脊髓损伤后导致运动功能受损及神经病理性疼痛等并发症，目前常规治疗只关注运动功能恢复忽视了对长期疼痛的治疗，缺乏综合性治疗的手段，而且由于传统单一类型脊髓支架无法真实模拟脊髓的宏观及微观环境，所以单纯采用常规组织工程支架修复脊髓损伤比较困难。基于上述原因及本课题组工作基础，本课题设计了一种结合脱细胞脊髓支架及温敏凝胶共同优势，并利用bFGF修饰脱细胞脊髓支架，使其可以在脊髓损伤局部持续释放外源性bFGF，同时载NTP温敏凝胶低温液态体温凝胶态的特点构建仿生脊髓，诱导移植到损伤后长期缓释NTP抑制神经病理性疼痛并促进脊髓组织再生，同时将自体激活雪旺细胞移植到仿生脊髓移植区以补充缺少的营养因子和神经元，通过大鼠机械痛阈及冷痛阈测定，判断神经病理性疼痛的抑制作用，通过BBB评分等方法评估不同组间大鼠行为功能恢复，采用GAP43、BDA示踪等方法研究损伤轴突的再生，进而为临床治疗脊髓损伤提供新方法。

背景：.脊髓损伤是指损伤平面以下不可逆的感觉和运动功能障碍，胶质瘢痕、抑制分子和炎症反应进一步限制神经元和轴突再生。本研究旨在模拟脊髓组织的空间结构和生理功能，利用增材制造技术制备仿生神经支架，通过理化性质检测、分子生物学分析、组织学染色、免疫荧光染色、运动功能评价等实验方法，探讨3D打印仿生神经支架联合骨髓间充质干细胞和雪旺细胞移植修复脊髓损伤的有效性研究。.方法：.使用3D打印技术制备支架，并对仿生支架制备和理化性质的检测。通过对仿生支架体外生物学行为的测定，评估支架的生物安全性。通过对大鼠脊髓半切损伤动物模型进行细胞支架的移植，通过大鼠运动功能恢复，来判断支架移植后的有效性。.结果：.1.本研究制备了一种具有水平轴单方向孔径支架，且具有良好的刚度，弹性应变量，支架的内部疏松网状结构和材料表面亲水性便于细胞粘附与物质交换，支架具有良好的理化性质，且导电性好，利于脊髓内神经电信号传导。2. 支架搭载干细胞的存活率达 95%以上，促进 BMSC大量增殖，同时维持其分化潜能，具有良好的生物相容性，并且排异反应小；支架具备良好的生物降解性与缓释功能，通过控制细胞空间分布与支架孔径大小，引导细胞增殖分化。3.通过免疫荧光染色可见支架内部可观察到神经元样细胞和多形态细胞；并观察到缺损区域的组织再生趋势。免疫荧光染色显示支架填充损伤区域可见大量骨髓间充质干细胞、雪旺细胞、少突胶质细胞和极少量星形胶质细胞，并发现再生神经元和轴突大致沿单方向孔径延伸，支架内部区域可见髓鞘包绕神经元。.结论：.1.通过3D打印技术，模拟脊髓空间结构和功能，制备的仿生支架具有良好的理化性质和生物学功能，为搭载干细胞提供仿生环境。2.聚焦相对空间位置促进干细胞定向分化的研究，有效促进向目标细胞分化；调控孔径参数，引导再生轴突定向延伸。3.聚焦修复效果，仿生支架填充空洞，维持脊髓轮廓，重塑有髓神经纤维结构，促进运动功能恢复，为进一步临床转化奠定理论础。

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