不同三维结构的生物支架修复脊髓损伤的比较研究

Tissue engineering, as an emerging and rapidly growing field, has received extensive attention. For spinal cord injury, the ultimate goal of tissue engineering as a treatment concept is to restore the anatomic structure and function of injured spinal cord by combining scaffold, cells, and biologically active molecules. The only way to repair spinal cord injury is to build a bridge that spans the lesion gap with all the morphological, chemical, and biological cues that mimic the normal spinal cord. Of all the proposed designs for tissue-engineered neuronal bridging devices, the ones based upon the contact guidance concept using physical cues to guide neuronal outgrowth are among the most promising. To date, a variety of scaffolds made by biomaterials have been used in the repair of spinal cord injury. However, due to the anatomical reconstruction and functional recovery after spinal cord injury is a very complex and difficult problem, it is very important to build an three-dimensional scaffold in line with the anatomy and histology of the spinal cord. In this study, we construct two different scaffolds in three-dimensional structure with two different technique. And compare their effectiveness in the repair of spinal cord injury, in order to reveal whether the three-dimensional structure of the scaffolds affecting spinal cord repairing. The test will be divided into two groups, in Group 1, using nanotechnology to construct three-dimensional scaffold with poly(lactide-co-glycolide) acid (PLGA). The PLGA line is made of PLGA silk. And the line are weaved to small cylindrical structure with inner hollow tune, the inner diameter is 0.1mm. Then 50 small cylindrical structures are gathered to form a columnar scaffold, with a diameter of about 3.0mm; in Group 2, the PLGA is directly cast a cylindrical scaffold with a diameter of about 3.0mm, with 6 tunes of about 0.3mm diameter. Then, two scaffolds seeded with Schwann cells transferred with NT-3 gene are implanted into the spinal cord transection site. Axonal regeneration，functional recovery and scaffold degradation are detected and compared between the two groups.

在组织工程技术中，应用"接触导向概念"构建的桥接装置引导轴突生长是治疗脊髓损伤最有希望的方法。到目前为止有各种各样的组织工程材料制成生物支架，应用于脊髓损伤的修复。但由于脊髓损伤后解剖重建和功能恢复是一个十分复杂和棘手的问题，因此如何构建一个符合脊髓的解剖结构和组织特性的三维支架是至关重要的。本研究比较了两种不同三维结构的生物支架在修复脊髓损伤中的作用，以了解生物支架的三维结构对脊髓损伤修复的影响。实验分为两组，组1利用纳米技术将丙交酯乙交酯共聚物（PLGA）丝制成线后编织为中空外实的小柱状结构，其内径为0.1mm，再将50个小柱制成一大柱状结构，直径约3.0mm；组2直接将PLGA铸成一直径约3.0mm的圆柱状支架，内有6个孔径约0.3mm的孔腔。将两种支架分别植入脊髓横断损伤处，再将NT3基因修饰的雪旺细胞注入到支架的管腔上，然后比较两组的神经纤维再生、功能恢复及支架降解情况。

目的：比较两种不同三维结构PLGA神经导管移植于完全性脊髓损伤动物模型后对其功能恢复的影响。评估神经导管与两种协同作用的细胞联合的治疗策略在促进脊髓损伤后的轴突再生及功能恢复上的效果。方法：采用PLGA材料做成两种不同三维结构的神经导管，移植于完全性脊髓损伤大鼠模型上。术后通过运动功能恢复情况，空腔形成大小，轴突再生数量以及损伤区域神经元数量来比较两组间的差异。将载有激活态的雪旺细胞（ASC）与骨髓间充质干细胞（MSC)的3mm多空洞PLGA神经导管快速移植到切除3mm胸脊髓的成年大鼠完全性脊髓损伤动物模型上。结果：术后8周在促进运动功能恢复、减小空腔面积大小、增强轴突再生及神经元存活上导管1较导管2更有优势。体内移植4周以后，接受神经导管联合激活态的雪旺细胞与骨髓间充质干细胞移植的大鼠，在BBB评分和电生理检测上显示了良好的神经功能恢复。然而，接受空导管移植和完全性损伤对照组的大鼠未能检测到神经功能恢复。移植后4周和8周，免疫组织化学检测分析显示移植的BMC均匀的分布于移植损伤区域，并分化成神经元样细胞与宿主神经元连接。在ASC与BMC联合移植组，损伤区域观察到大量的再生纤维，经神经丝蛋白（NF）免疫组化染色确认这些再生纤维为神经轴突。此外，在移植损伤区域检查到髓鞘碱性蛋白（MBP）阳性表达的髓鞘结构，并通过电镜检查予以证实。很重要的一点是，在ASC与BMC联合移植大鼠的损伤区域发现，少量BMC来源的神经元样细胞除表达成熟神经元标志物外，还表达胆碱乙酰转移酶（ChAT）。结论：本研究的结果表明，采用PLGA材料做成内含微导管的神经导管移植于完全性脊髓损伤处，能良好的与宿主脊髓结合。ASC诱导的MSC来源的神经元样细胞与内源性神经元细胞相互接触及局部神经通路的形成，促进了严重脊髓损伤后的功能恢复。这种组织工程与两种协同作用的细胞联合治疗的策略可以作为一种新颖的治疗脊髓损伤的替代方法。