缓释炎症诱导性外泌体仿生支架促进下颌髁突骨软骨缺损修复及机制研究

The incidence of osteoarthritis of temporomandibular joint is high which could induce osteochondral defects and dysfunctions of chewing and language. There are drawbacks in the clinical repair of the osteochondral defects. In recent years, the inflammation-stimulated stem cell-derived exosomes had great potential in acellular tissue engineering, but the immune mechanism to promote the repair of osteochondral is not clear. This study intended to build acellular osteochondral scaffolds with controlled realease inflammation-stimulated adipose-derived stem cells (ADSCs)-derived exosomes. Exsomes are seeded into this scaffold in vitro and implanted into osteochondral defects of rabbit mandibular condyle in vivo models. The realeased kinetics of exosomes from the scaffold was investigated. In this research, we aimed to study the effects of exosomes to promote bone mesenchymal stem cells poliferaton and the phenotype change of macrophages from M1 to M2. We supposed the inflammation-stimulated ADSCs-derived exosomes through COX-2/PGE2 induced JAK2/STAT3 to achieve macrophage polarization for attenuating inflammation and enhancing the efficiency of bone cartilage repair. This study will provide new strategy for accelular tissue engineering for osteochondral regeneration and explore its immune mechanism. Furthermore, this research will provide the experimental basis for clinical translation of exosomes to cure temporomandibular joint osteoarthritise.

颞下颌关节骨关节炎可导致髁突骨软骨缺损造成功能障碍，临床修复方法尚不足。无细胞组织工程研究表明，炎症刺激干细胞衍生外泌体具有更强的免疫调节功能并促进组织修复，但直接使用易流失、效率低，且其促进组织修复的免疫机制尚不清楚。本研究拟将炎症刺激脂肪干细胞衍生的外泌体（ADSCsE＋）负载到无细胞骨软骨支架上，构建缓释ADSCsE＋的骨软骨复合支架，研究新型支架ADSCsE＋释放动力学及其体内、外促进骨髓间充质干细胞增殖，促进巨噬细胞由促炎型向抗炎型转化作用；明确缓释ADSCsE＋骨软骨支架通过COX-2/PGE2介导JAK2/STAT3通路促进巨噬细胞极化发挥免疫调节作用的机制；运用组织工程手段评价此无细胞支架对骨软骨缺损动物模型的修复效能，有望获得兼有“骨软骨修复”及“免疫调节”作用的新型复合支架，为无细胞组织工程骨软骨修复提供新策略，为外泌体在颞下颌骨关节炎治疗临床转化提供基础研究证据。

细胞外囊泡（extracellular vesicles, EV）作为间充质干细胞（mesenchymal stem cells, MSCs）的分泌的主要成分之一，近年来已被广泛研究。有研究表明当MSCs暴露于炎症环境时，会释放更多的生长因子，外泌体以及趋化因子等。而EV内所携带的蛋白、核酸等物质会随着母细胞状态改变而随之发生变化。本研究中，通过诱导MSCs处于炎症状态，以改变EV内含物和功能来调节炎症微环境并促进组织再生。EV所携带的miRNA测序显示，一些有利于细胞功能的miRNA表达量有所上调。体外细胞实验表明，炎症刺激的脂肪来源间充质干细胞（adipose-derived mesenchymal stem cells, ADSCs）分泌的EV（IAE）和正常的ADSCs分泌的EV（AE）都可以促进细胞增殖；并且IAE能显著促进细胞迁移能力。在巨噬细胞调控方面，IAE组M2型巨噬细胞显著增加。巨噬细胞的RNA测序结果表明，IAE中高表达的miR27b-3p可能通过靶向CSF1，抑制PI3K-AKT信号通路来调节巨噬细胞表型。通过建立兔颞下颌关节（TMJ）髁突骨软骨缺损模型并进行治疗后可观察到，AE和IAE都促进了TMJ再生，其中IAE具有最显著的治疗效果。综上，将MSCs暴露于炎症环境中可以切实增强EV功能，并且修饰的EV能够起到更好的治疗效果，为细胞外囊泡临床转化应用于颞下颌关节骨关节炎所造成的下颌髁突骨质缺损修复提供理论研究基础。