3D打印改性PCL/凝胶双相支架负载hWJ-MSC来源外泌体促进纤维环缺损修复及其机制研究

There is no efficacious annulus fibrosus (AF) repair strategy after discectomy. Tissue engineering of annulus fibrosus (AF), which is a promising treatment for AF defects, remains challenging due to the lack of the ideal AF scaffold and limited seed cell source. In our previous study funded by National Science Foundation of China, we developed a 3D printing technique for scaffold with complex structure, and fabricated high-strength hydrogels. Furthermore, we found that hWJ-MSC-derived exosomes can promote AF cell proliferation and inhibit its apoptosis. In this study, we will modify polycaprolactone (PCL) material by hydrophilic modification methods. A biomimetic PCL framework with ±30°angle-ply laminated structure would be fabricated based on FDM 3D printing technology. High-strength PEG/type I collagen hydrogel loaded with hWJ-MSC-derived exosomes would be inserted into the PCL framework. Then, the exosomes/scaffold complex would be implanted for repairing the sheep annulus fibrosus defects, moreover, inhibitors or activators of Akt signaling pathway would be injected into the repairing zone. The results would be obsessed by gross observation, imaging, histology, biochemistry, and biomechanical methods. The Influence and possible mechanism of Akt signaling pathway and exosomes/scaffold complex on the repairing AF defect would be explored. This study will provide new methods and theoretical basis for AF biological repair.

椎间盘切除术后纤维环（AF）缺损尚无理想的修复方法，组织工程技术为AF缺损修复带来希望，但目前缺乏理想的仿生AF支架并且种子细胞来源有限。我们前期在NSFC资助下具备了3D打印制备复杂组织结构的支架及高强度水凝胶的研究基础，发现人脐带间充质干细胞(hWJ-MSC)来源外泌体对AF细胞具有促进增殖及抑制凋亡作用。本研究拟对聚己内酯（PCL）材料进行亲水性改性，并基于FDM技术3D打印制备具备±30°斜交（angle-ply）叠层结构的仿生PCL支架，灌注负载hWJ-MSC来源外泌体的支化PEG/I型胶原水凝胶，将外泌体/支架复合物植入修复羊纤维环缺损，并在修复区注射Akt信号通路抑制剂/激活剂，通过大体、影像、组织学、生物化学、生物力学等动态评估，观察外泌体/支架复合体对AF缺损的修复情况，探讨Akt信号通路对外泌体/支架复合体修复AF缺损的影响及机制，为AF生物学修复提供新方法及理论依据。

目前，纤维环组织工程忽视了其结构成分及其力学特性的复杂性，所构建的纤维环支架缺乏天然仿生斜交叠层结构或叠层结构之间无法形成有效连接，致使细胞难以长入，无法重建胞外基质和生物学功能等问题，阻碍了纤维环组织工程的快速推进及临床应用。为了解决这些问题，本课题组成功构建了负载纤维环（AF）细胞仿生斜交叠层同心圆改性PCL纤维环支架；并进一步负载人脐带间充质干细胞（hWJ-MSC）来源外泌体进行生物学功能优化。随后论证hWJ-MSC来源外泌体对AF细胞增殖和凋亡的调控机制，重点探讨TGF-β/SMAD途径参与外泌体对AF细胞增殖和凋亡的分子机制。最后体内评估hWJ-MSC来源外泌体-纤维化支架复合体在体内修复纤维环的效应；结果表明：（1）负载纤维环（AF）细胞仿生斜交叠层同心圆改性PCL纤维环支架可为纤维环细胞提供适宜的生长环境，AF细胞能够沿改性PCL纤维取向进行Ⅰ型胶原沉积；（2）hWJ-MSC来源外泌体能够通过miR-320/SMAD3途径，促进细胞外基质合成，对抗细胞外基质降解以延缓椎间盘退变的进展；（3）聚丙交酯-丙二醇二甲基丙烯酸（PGLAMDA）-改性透明质酸（MeHA）光交联双相椎间盘支架可有效修复兔腰椎髓核-纤维环缺损。因此，本项目研究结论为纤维环仿生支架的构建、hWJ-MSC来源外泌体与纤维环生理修复组织工程学提供一定的理论与应用证据，为后续纤维环组织工程支架的临床转化应用奠定基础。