经预神经化冻融干细胞修饰的仿生神经导管构建及其在完全性脊髓损伤修复中的作用及机制

Pre-neuralized biomimetic nerve conduits modified with freeze-thawed stem sells for spinal cord injury repair

中文关键词:  组织工程  脊髓损伤  仿生神经导管  干细胞  预神经化  微环境

英文关键词:Tissue engineering  Spinal cord injury  Biomimetic neural conduit  Stem cells  Pre-neuralization  Microenvironment

中文摘要:

  【摘要】 目的：构建一种经预神经化冻融干细胞裂解产物修饰的仿生神经导管（biomimetic nerve conduit modified with freeze-thawed mesenchymal stem cell lysates，BNC@F-MSCs），探讨其在完全性脊髓损伤（spinal cord injury，SCI）修复中的作用及机制。方法：采用定向冷冻铸造技术，以甲基丙烯酰化丝素蛋白（methacrylated silk fibroin，SFMA）和脊髓脱细胞外基质（decellularized extracellular matrix，d-ECM）为原料，制备具有径向取向微通道的仿生神经导管（biomimetic nerve conduit，BNC）。将骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）接种于导管内，体外培养7d使其预神经化，并经3次反复冻融处理使其裂解，最终制备成负载干细胞裂解产物的BNC@F-MSCs。通过体外实验评估其对BMSCs向成熟神经细胞分化以及促进其沿定向微通道排列的影响。建立大鼠T10节段脊髓完全横断模型，植入导管，饲养8周，在第1周时通过损伤部位组织学评估，第8周时通过损伤部位及膀胱组织学评估、膀胱自主功能检测、行为学测试、电生理检测等方法，评价其在体内的修复效果。结果：体外实验中，BNC@F-MSCs释放的细胞内成分使BMSCs的Nestin和Tuj1表达增强，细胞呈现伸长的神经元样突起，平均细胞长度显著增加，且长轴在0°~30°范围内沿微通道定向排列。体内实验中，植入BNC@F-MSCs后，损伤区域M2型巨噬细胞（CD163+）比例升高，A1型星形胶质细胞（C3+/GFAP+）比例为（12.32±2.3）%，A2型（S100A10+/GFAP+）比例为（60.62±8.50）%；损伤区空洞面积较对照组减少72.9%；8周时电生理检测显示神经传导振幅为166.20±18.21mV、不应期为2.72±0.60ms；行为学测试显示后肢足迹数量恢复至19.33±4.16，膀胱尿量为0.65±0.14mL。结论：BNC@F-MSCs通过调节损伤微环境和引导轴突有序再生的共同作用，在大鼠模型中展现出促进SCI后功能恢复的潜力。

英文摘要:

  【Abstract】 Objectives: This study aims to construct a pre-neuralized biomimetic nerve conduit modified with freeze-thawed mesenchymal stem cell lysates(BNC@F-MSCs) and to investigate its therapeutic effects and underlying mechanisms in repairing complete spinal cord injury(SCI). Methods: Biomimetic nerve conduits(BNCs) with radially aligned microchannels were fabricated via directional freeze-casting using methacrylated silk fibroin(SFMA) and decellularized spinal cord extracellular matrix(d-ECM). Bone marrow mesenchymal stem cells(BMSCs) were cultured for 7d to induce pre-neural differentiation after seeded into the conduits, and subjected to three repeated freeze-thaw cycles to obtain lysate-loaded BNC@F-MSCs. In vitro experiments evaluated their effects on promoting neural differentiation of MSCs and guiding cell alignment along the oriented microchannels. A complete T10 spinal cord transection model was established in rats. After conduit implantation, animals were maintained in an SPF environment for 8 weeks. Histological assessment of the injury site was performed at week 1, while repair efficacy at week 8 was comprehensively evaluated through histological analysis of the injury site and bladder, bladder autonomic function testing, behavioral assessments, and electrophysiological measurements. Results: In vitro, the intracellular components released from BNC@F-MSCs enhanced Nestin and Tuj1 expression in BMSCs, induced elongated neuron-like processes, significantly increased average cell length, and aligned cell long axes within 0°-30° along the microchannels. In vivo, implantation of BNC@F-MSCs increased the proportion of M2-type macrophages(CD163+), reduced A1-type astrocytes(C3+/GFAP+) to (12.32±2.3)%, and increased A2-type astrocytes(S100A10+/GFAP+) to (60.62±8.50)%; The cavity area at the injury site decreased by 72.9% compared with the control group; At 8 weeks, electrophysiological testing showed nerve conduction amplitude of 166.20±18.21mV and latency of 2.72±0.60ms; Behavioral tests showed hindlimb footprint count recovered to 19.33±4.16 and bladder urine volume to 0.65±0.14mL. Conclusions: By simultaneously modulating the injury microenvironment and guiding ordered axonal regeneration, BNC@F-MSCs exhibit strong potential for promoting functional recovery after complete SCI.