仿生支架增强BMSC旁分泌调控免疫炎症反应促进椎间盘修复

Intervertebral disc herniation is a very common disease with high morbidity and disability rate. As a standard surgical treatment for disc herniation, discectomy procedures remove herniated tissue fragments but unrepaired annulus fibrosus (AF) defect remain allowing reherniation or progressive degeneration. At present, there is no ideal repair strategy or reconstructive approach for the treatment of AF defect. Tissue engineering strategy provides a new avenue to repair of AF defect, in which the scaffold is a key component. Scaffold structure affects stem cell growth and differentiation through specific cell-material interactions, but it is not clear whether it affects stem cells paracrine function of MSCs and participates in the process of immunomodulation to promote AF repair. In this study, we will fabricate a silk protein-based angle-ply scaffold by infusing SF solutions into the polycaprolactone microfiber sacrificed templates under vacuum condition, freeze-dry and sacrificial template-leaching process. We will firstly study the effect of biomimetic microchannel scaffolds on the paracrine function of seeded bone marrow mesenchemal stem cells (BMSCs) and elucidate the role of BMSCs paracrine on macrophage polarization in vitro. Then, an engineered BMSCs-scaffold composite was constructed to repair AF defect in a goat model. The relationship between the repair effect of AF defect and inflammatory reaction was systematically evaluated. The effects of orientated micro-channel scaffolds enhance BMSCs paracrine to modulate the inflammatory response for the repair of AF defect was clarified. This project will provide a new strategy for biological repair of disc degeneration disease. This study also provides new ideas for tissue regeneration and functional integration, and has a strong research and application value.

椎间盘突出症发病率和致残率较高，髓核摘除术虽可改善症状，但会造成纤维环缺损，导致突出复发及退变加剧，目前尚无理想的修复措施。组织工程技术为其提供了新的修复策略。支架结构可调控干细胞生长分化，但能否调控干细胞的旁分泌，进而改善免疫微环境促进纤维环修复尚需进一步研究。本项目拟采用丝素蛋白材料，以聚己内酯纤维为致孔模板，经浇铸、冻干、洗脱沥滤等步骤制得仿生定向交叉结构的微通道支架。首先体外研究仿生微通道支架对骨髓间充质干细胞（BMSCs）旁分泌作用的影响，同时阐明BMSCs旁分泌作用对巨噬细胞表型极化的影响。然后将BMSCs-支架复合物植入羊纤维环缺损模型中，评价纤维环缺损修复效果与炎症反应的关系，阐明仿生支架增强BMSCs旁分泌作用进而调控免疫炎症反应促进椎间盘纤维环缺损修复的效果，为椎间盘再生修复提供新策略。本研究可为组织再生修复提供新思路，具有较强的研究和应用价值。

针对椎间盘退变往往需纤维环-髓核联合修复的问题，通过组织工程技术构建仿生结构的一体化纤维环-髓核支架，采用熔融纺丝技术制备定向交叉微米PCL纤维环致孔模板，中央填充石蜡球作为髓核致孔模板，然后灌注丝素溶液进行冷冻干燥，最后利用洗脱沥滤的方法去除PCL和石蜡材料从而反向构建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。细胞实验证实仿生支架的生物相容性较好；皮下埋植实验证实仿生支架可降低免疫排斥反应；体内实验证实定向交叉微通道丝素蛋白一体化椎间盘支架可促进鼠尾椎间盘修复。. 针对椎间盘切除术后遗留椎间盘纤维环缺损进而导致退变加重、突出复发的问题，采用组织工程技术，通过构建模拟天然纤维环微观结构的支架引导纤维环拟天然再生，修补椎间盘纤维环缺损。本项目通过洗脱沥滤PCL模板，反向制备获得定向交叉微通道丝素支架，通道取向性好，孔径均匀，支架孔隙率合适。对照组支架规整，无取向孔道结构，孔隙率稍差。两组支架的生物相容性表现均良好，但细胞生长增殖能力，细胞外基质分泌能力取向微通道组表现更为突出，且细胞长入支架内部更能发挥其生物活性。体内实验结果显示取向微通道支架组不仅几乎完美的封闭了纤维环的缺损，保护了椎间盘的密闭性不受破坏，支架内还发现了成簇的软骨样细胞，提示其可能招募体内干/祖细胞并诱导其定向分化。