仿生斜交叠层结构的纳米PCL/SF纤维环支架复合人脐带间充质干细胞修复纤维环缺损

Intervertebral disc herniation is very common and discectomy is a standard surgical treatment for serious herniation. However, defect of disc is left after discectomy; that may lead to further degeneration and reoccurrence of herniation, which indicates the need of repair of defect. Nowadays, there is no ideal repair strategy for disc defect after discectomy. Tissue engineering provides a revolutionary opportunity for biological repair of disc defects, for which, a key point is the repair of annulus fibrous (AF), that is still very difficult owing to the lack of ideal scaffold and seed cell. In our previous study funded by NSFC, AF scaffolds were constructed and various mechanical stmulations were performed, and it was found that specific mechanical stimulations were benifical to functional construction. In this study, we will fabricate oriented nanofibrous membrane with polycaprolactone/silk fibroin by means of electrospun technology, during which holes are produced and regulated by electrospraying-generated PEO microparticles. Bionic nano AF scaffold is fabricated with (+30degree/-30degree) angle-ply lamellate membranes, with good biocompatibility and biomechanics property. Human umbilical cord mesenchymal stem cells (hWJ-MSCs) are seeded into scaffold to construct a engineered composite. In vitro repair of AF defect with the composite is studied with organ culture model under dynamic mechanical stimulation, and the effect of stimulation on repair is evaluated according to microstructure, biochemical component and gene expression. In vivo repair of AF defect with the PCL/SF-hWJ-MSCs composite is studied in goat, and result is evaluated on gross observation, imaging, histology, biomechanics, especially on interface integration and the revolve of hWJ-MSCs. In this project, theoretical and experimental evidences are expected to obtain for biological repair of disc defect.

椎间盘切除后尚无理想的修复方法，组织工程技术为椎间盘修复带来希望，其中修复纤维环缺损是关键环节，但目前缺乏理想的纤维环支架及种子细胞。我们前期在NSFC资助下研究了纤维环支架及力学刺激对构建的影响，发现特定的力学刺激有利于纤维环功能化构建。本研究拟采用聚己内酯/丝素蛋白为原料，以静电纺丝技术制备取向性纤维膜，通过电喷微球致孔法调控孔径，最终制备具有取向斜交叠层（+30度/-30度）结构、良好力学特性和生物相容性的纳米纤维环支架，接种人脐带间充质干细胞（hWJ-MSCs），体外修复纤维环部分缺损并在力学刺激下器官培养，从微观结构、生化成分、基因表达等方面研究力学刺激和hWJ-MSCs移植对修复纤维环缺损的影响；将细胞-支架复合体植入羊体内修复纤维环部分缺损，从大体、组织学、影像、生物力学等动态评估修复效果，重点观察界面整合情况和hWJ-MSCs转归，为椎间盘生物学修复提供理论基础和实验依据。

椎间盘切除术治疗椎间盘退行性疾病的常用手术方法。但切除后遗留的椎间盘缺损会加速椎间盘的退变，可能导致再次复发。椎间盘缺乏血运，并处在复杂的力学环境中，自身修复能力非常有限，因而椎间盘切除后一直没有理想的修复重建方法。椎间盘切除后内固定融合会丧失活动节段、引起相邻节段退变；人工假体和非融合技术也存在内植物材料疲劳失败、再手术等诸多问题，因此椎间盘手术缺损的修复重建是临床上亟需解决的难题。. 我们课题组针对椎间盘切除术后缺损开展了以下两项系列研究：1）构建山羊髓核摘除术后缺损模型，分别将富集自体骨髓血的明胶海绵颗粒和体外扩增培养的自体BMSCs复合明胶海绵颗粒植入山羊椎间盘缺损处，体内修复三个月后，通过影像学、生物力学及组织学染色切片评估显示，两组均能够有效延缓椎间盘退变，促进髓核细胞增殖和细胞外基质的分泌，有利于维持椎间盘高度及生物力学性能；与自体BMSCs培养组相比，虽然富集器组的修复效果较弱，但其操作简单、安全、经济，提供了一种临床可行的椎间盘修复策略。2）以生物相容性和生物可降解的材料，采用熔融纺丝法制备了既具有仿生斜交结构、又具备良好生物相容性和生物降解的聚己内酯/聚乳酸聚乙醇酸共聚物（PCL/PLGA）纤维环支架，并接种BMSCs，在绵羊体内修复纤维环部分缺损，从大体、影像学（X线/MRI）、组织学的初步结果观察发现，PCL/PLGA纤维环支架可以阻止间盘突发复发，延缓间盘退变，具有良好的应用转化前景。. 另外，椎间盘退变晚期往往退变较重，多存在纤维环-髓核的同时退变。针对晚期椎间盘退变，课题组成功构建了具有定向交叉微通道结构的纤维环以及多孔海绵状结构髓核的一体化椎间盘仿生支架，两部分结构均高度模拟天然椎间盘的微观结构。行鼠尾椎间盘置换原位修复，3个月观察结果表明该仿生椎间盘支架可以与大鼠原生组织良好的融合生长，并且有助于恢复椎间盘高度和部分力学性能，为退变椎间盘再生修复提供一种有潜力的方法。