“抗炎”缓释纳米微球修饰的PLLA载体对关节退变炎性环境的调控及其协同脐带干细胞修复骨关节炎关节面的研究

Reconstruction of joint surface by engineered osteochondral tissue is the forefront of treatment of osteoarthritis(OA), but little attention has been paid to apoptosis of seeded cells and degeneration of implanted tissue under the influence of inflammatory environment in osteoarthritic joints. In our previous studies, transplanted cell function was significantly impaired in OA inflammatory environment, which led to severe degeneration of repair tissue. Curcumin, a kind of plant extraction,was found to inhibit proliferation and induce apoptosis of inflammatory synovial cells,while tetrahydrocannabinol was noticed to promote migration and chondrogenic differentiation of mesenchymal stem cells(MSC). Our preliminary research also revealed that both curcumin and tetrahydrocannabinol could inhibit inflammatory factors-induced apopotosis of implanted cells. Based on these observations, we propose to construct "anti-inflammation/pro-differentiation" dual-effect PLLA nano-fibrous scaffold for OA joint surface repair.This new type of scaffold encloses curcumin and tetrahydrocannabinol into controlled-release PLGA nanospheres which are immobilized into the PLLA nano-fibrous scaffold. Potential "anti-inflammation/pro-differentiation" effect of the scaffold will be investigated both in in vitro co-culture system of umbilical cord MSC with inflammatory synovial cells or factors such as IL-1β or TNF-α,and also in rabbit knee osteoarthritis model for the repair of osteochondral defects. Our project will put forward a brand-new strategy in the reconstruction of OA articular surface. "Anti-inflammatory" engineered scaffold would enhance viability and proliferation of implanted seed cells, and improve the efficacy of engineered tissue in the repair of OA articular surface.

利用组织工程修复关节面是骨关节炎（OA）治疗的前沿，但目前研究对移植细胞和组织在OA关节内炎症环境中的凋亡及退变考虑较少。我们先期的研究发现移植的工程化组织细胞在OA炎性环境下功能退化，严重影响OA关节面修复的效果。据报道植物提取物姜黄素具有抑制关节炎滑膜细胞增殖，促进其凋亡的抗炎作用，而四氢大麻酚具有促进间充质干细胞(MSC)分化的功效。我们的初步研究结果表明两种药物均具有抑制炎症因子诱导干细胞凋亡的作用。本课题拟构建负载有缓释姜黄素和四氢大麻酚纳米微球的"抗炎-促分化"双效PLLA纳米纤维载体，考察其对OA滑膜细胞和移植干细胞存活﹑增殖及分化的影响；同时在兔膝OA模型中考察 "抗炎"载体与脐带MSC构建的新型"抗炎"工程化组织对OA关节面的修复作用。本项目是采用"抗炎"工程化组织修复OA关节面的崭新探索。研究结果有望增强移植细胞的存活与增殖，并显著改善工程化组织修复OA关节面的效果。

目的和方法： 骨关节炎（OA）的主要病理机理为关节内滑膜炎症，软骨合成代谢受抑，软骨降解产物进一步激活炎症反应，加剧软骨破坏。这一恶性循环是OA治疗和软骨修复的主要障碍。炎症环境对参与组织工程修复OA软骨的脐带组织间充质干细胞（UCMSC）生物活性的影响罕有报道。本项研究在离体实验中考察了炎症环境和抗炎措施对UCMSC来源软骨前体细胞的影响；构建聚乳酸羟基乙酸共聚物（PLGA ）姜黄素缓释纳米微球的“抗炎”组织工程载体；在兔OA模型中考察UCMSC和PLGA姜黄素缓释纳米微球对兔OA关节面的修复作用。旨在探索组织工程修复OA关节面的新策略。.结果： 研究结果证明炎症环境明显抑制hUCMSCs的细胞活性和成软骨分化能力。实施抗炎措施（和厚朴酚，姜黄素等）可以保护炎症环境下hUCMSCs的细胞活性，生存和成软骨分化能力，抑制细胞外基质降解。NF-κB通路是和厚朴酚保护hUC-MSCs的重要靶点。本实验采用改良的乳化-溶剂扩散法制备出PLGA姜黄素纳米缓释微球注射剂。通过关节腔注射木瓜蛋白酶诱导兔膝OA，继在疾病进展过程中行患膝前交叉韧带切断造成进展性OA模型。当给患膝关节腔单独注射姜黄素或PLGA姜黄素纳米缓释微球后，X线、大体观察、组织学特征、电镜及生化分子学综合评估显示姜黄素对重度OA具有治疗作用，可缓解骨关节软骨的破坏和OA的发展。相对于单独注射姜黄素组，PLGA姜黄素纳米微球组的抗骨关节炎作用更显著，关节软骨的破坏及炎性改变明显缓解。同时，采用关节腔内注射方法将体外分离培养HUCMSCs移植至兔OA膝关节内，可改善OA软骨及滑膜组织组织学病变，降低关节液以及血清中炎症因子水平，抑制OA软骨细胞凋亡，提高OA软骨组织中II型胶原表达水平。.结论： 炎症环境明显抑制hUCMSCs的细胞活性和成软骨分化能力。抗炎措施可保护hUCMSCs的细胞活性和成软骨分化能力，抑制细胞外基质降解。关节腔注射PLGA姜黄素纳米微球有明显抗骨关节炎作用，会利于干细胞修复关节软骨。研发脐带来源干细胞和抗炎药物的“抗炎”组织工程复合物将有可能改善OA的治疗效果。.关键词： 骨关节炎 姜黄素 脐带来源间充质干细胞 关节软骨修复