多相ECM支架微环境调控BMSCs外泌体在肩袖腱骨愈合中的作用及其机制研究

Rotator cuff injury is the most common cause of shoulder pain and dysfunction. Non-restoration of the gradient transitional tissue at the tendon-bone interface is the leading cause of high failure rates after rotator cuff repair. Our previous study which published in Biomaterials, has shown that combined multiphase extracellular matrix (ECM) scaffold and bone marrow mesenchymal stem cells (BMSCs) augmentation promotes the rotator cuff tendon-bone interface regeneration, but the underlying mechanism is not known. At present, it is widely accepted that MSCs-derived exosomes play an important role in tissue regeneration and can be regulated by the parent cell microenvironment. However, to date the relationship between MSCs-derived exosomes and tendon-bone interface regeneration has not been studied. Therefore, we speculate that the microenvironment of multiphase ECM scaffold can regulate BMSCs-derived exosomes to promote the regeneration of rotator cuff tendon-bone interface. In this study, we will evaluate the regulation of BMSCs-derived exosomes by the ECM scaffold microenvironment, explore the effects of BMSCs-derived exosomes on the host BMSCs and identify the signaling pathway at molecular, cellular, tissue levels and in animal models. We will also construct a novel multiphase ECM scaffold immobilized with BMSCs-derived exosomes. From a new perspective, this study will reveal the relationship between BMSCs-derived exosomes and the regeneration of transitional structure at the tendon-bone interface, providing new approaches for effective intervention of tendon-to-bone healing. The constructed novel bioscaffold has a high potential for clinical translation to reduce the failure rate of rotator cuff repair.

肩袖损伤是肩关节疼痛和功能障碍的最常见原因。肩袖修复高失败率主要归因于腱骨界面梯度移行结构的缺失。前期研究中我们证实多相细胞外基质(ECM)支架联合BMSCs能够促进肩袖腱骨界面再生，相关成果已发表于Biomaterials，但其确切机制还未阐明。目前已知MSCs外泌体受母细胞微环境调控并参与组织再生，然而其与腱骨界面再生的关系，迄今国内外尚无报道。据此我们推测多相ECM支架微环境通过调控BMSCs外泌体介导肩袖腱骨界面再生。本课题拟从分子、细胞、组织和动物水平多层次明确ECM支架微环境对BMSCs外泌体的影响，探讨BMSCs外泌体对内源性BMSCs的作用及其信号传导通路。同时构建一种新型负载BMSCs外泌体的多相ECM支架。本研究将从新视角揭示BMSCs外泌体与腱骨界面梯度移行结构再生的关系及机制，为有效干预和优化腱骨愈合提供新手段，所构建的新型生物支架有望临床转化以降低肩袖修复失败率。

肩袖损伤是临床上引起肩关节疼痛和功能障碍的最常见原因，其修复术后较高的失败率主要归因于无法恢复腱骨界面特征性的移行结构。前期研究我们已证实多相细胞外基质(ECM)支架能够诱导骨髓间充质干细胞定向分化（BMSCs），鉴于MSCs外泌体受母细胞微环境调控并参与组织再生，本课题拟从分子、细胞、组织和动物水平多层次明确ECM支架微环境对BMSCs外泌体的影响，同时构建一种有望临床转化的新型仿生多相ECM支架。本研究取得的主要成果及意义如下：1. 结合反复冻融技术和核酸酶处理，成功构建了兔去细胞化腱骨界面生物支架（TFBC），采用组织学，DNA、GAG和胶原含量检测，扫描电镜和原子力显微镜观察，证实该支架保留了与天然腱骨界面相近的ECM微环境；2. 成功构建人BMSCs细胞膜片，将其置于普通培养皿和TFBC支架上培养7天后，两组所获外泌体的外观及大小无显著差异。qRT-PCR示TFBC支架培养组外泌体中Ihh通路关键分子Ihh, Ptch1和Gli1的基因水平较普通培养组显著上调，说明微环境参与外泌体成分的调控；3. 普通培养组和TFBC支架培养组外泌体对兔BMSCs的活性无明显影响，但TFBC支架培养组外泌体能显著促进兔BMSCs的增殖和移行能力，并显著上调兔BMSCs纤维软骨标志物表达水平；4. 成功构建负载BMSCs膜片的仿生支架，通过建立兔急性肩袖损伤模型，随机选择一侧采用常规修复、单纯TFBC支架修复和仿生支架修复。12周后组织学观察示仿生支架组较单纯TFBC修复组的腱骨界面出现更多Sharpey 纤维连接、较成熟的软骨细胞和新生纤维软骨，而常规修复组的腱骨界面由疤痕组织填充。仿生支架组的组织学评分显著优于常规修复组和单纯TFBC支架组。力学测试显示TFBC支架和仿生支架组的最大失效载荷较常规修复组有增加趋势，但没有显著性差异。综上，仿生支架能够有效促进肩袖修复后的腱骨界面愈合及再生，为有效干预和优化腱骨愈合提供了新的手段，该新型仿生支架有望转化于临床以降低肩袖修复后的再撕裂率。.在项目执行期间，发表高水平SCI论文2篇；参编英文专著1部；获第十一届湖南省青年科技奖；培养硕士研究生2名。