功能化ECM肽/miR-140复合水凝胶诱导软骨CPCs/CCs重编程增强OA早期软骨损伤修复的研究

Cartilage injury plays a key role in the pathogenesis of osteoarthritis (OA), and repair in early stage can help delay or even reverse OA progression. In recent years, in addition to mature chondrocytes (CCs), cartilage progenitor cells (CPCs) with multipotent differentiation potential have been found in cartilage, and the induction and activation of CPCs is a potential repair strategy for cartilage repair in early-stage of OA. MiR-140 exhibits cartilage specificity, and our previous studies have found that miR-140 can not only upregulate the percentage of CPCs in cartilage-derived cells, but also induce the CCs to “dedifferentiate” and exhibit CPCs’ phenotype, suggesting that miR-140 can induce reprogramming of CPCs/CCs and possess a potential to promote cartilage repair. However, directly intra-articular injection of miR-140 has problems such as low cartilage uptake and non-targeted aggregation. This project intends to construct a novel functionalized ECM peptide/miR-140 composite hydrogel with high cartilage-targeting and chondrogenic inducing activity based on cartilage extracellular matrix (ECM) properties through functional design strategies, and then its effects and mechanisms on CPCs/CCs reprogramming and cartilage repair in early-stage OA are investigated in vitro and in vivo, so as to to lay a foundation for the study of endogenous cartilage repair strategies.

软骨损伤在骨关节炎（OA）发病机制中起关键作用，早期修复有助于延缓甚至逆转OA进程。近年发现软骨中除成熟软骨细胞（CCs）外，还存在具有多向分化潜能的软骨前体细胞（CPCs），而诱导活化CPCs是OA早期软骨损伤的潜在修复策略。miR-140具有软骨特异性，前期发现miR-140不仅能上调软骨细胞中CPCs比例，还可诱导CCs“去分化”出现CPCs表型，提示miR-140具有诱导CPCs/CCs重编程及促软骨损伤修复的潜能。然而，直接关节内注射miR-140存在软骨摄取率低、非靶向聚集等问题。本项目拟基于软骨细胞外基质（ECM）特性，通过功能化设计策略构建新型具有高度软骨靶向性及成软骨诱导活性的功能化ECM肽/miR-140复合水凝胶，然后从体外、体内研究其对CPCs/CCs重编程及OA早期软骨损伤修复的调控作用和机制，为后续研究内源性软骨损伤修复策略奠定基础。

软骨损伤在骨关节炎（OA）发病机制中起关键作用，早期修复有助于延缓甚至逆转OA进程。近年发现软骨中除成熟软骨细胞（CCs）外，还存在具有多向分化潜能的软骨前体细胞（CPCs），而诱导活化CPCs是OA早期软骨损伤的潜在修复策略。miR-140具有软骨特异性，前期发现miR-140不仅能上调软骨细胞中CPCs比例，还可诱导CCs“去分化”出现CPCs表型，提示miR-140具有诱导CPCs/CCs重编程及促软骨损伤修复的潜能。然而，直接关节内注射miR-140存在软骨摄取率低、非靶向聚集等问题。综合文献资料及前期工作基础，提出科学假说：根据软骨ECM特性对自组装（SAP）支架材料进行功能化修饰后，材料本身就具有软骨靶向及一定成软骨诱导活性等功能，再搭载对CPCs/CCs重编程具有强诱导活化作用的miR-140，两者协同作用可进一步增强OA早期软骨损伤修复。在本项目中，首先通过功能化设计策略设计合成了一系列含有ECM功能肽段（纤连蛋白（FN）分子结构中的GGRGDSP肽段）和结合不同数量精氨酸（R）的功能化SAP材料，优化材料后通过静电作用装载miR-140获得功能化ECM肽/miR-140复合水凝胶（KLD2R-FN-miR-140），并证实了其具有良好的成胶、miR-140高载和体外缓释等特性。然后，分离纯化CPCs/CCs并与系列材料混合构建3D培养体系，从体外证实了功能化ECM肽/miR-140复合水凝胶可通过调控CPCs/CCs重编程增强成软骨分化。最后，在大鼠OA模型体内证实了功能化ECM肽/miR-140复合水凝胶可有效促进OA早期软骨损伤修复，其机制与Notch信号通路介导的CPCs/CCs重编程密切相关。本研究为后续基于miR和SAP的内源性软骨损伤修复策略研究和应用转化奠定了良好基础。