Regulation of Tendon Repair with Stem Cell-Derived Extracellular Vesicles

项目总结/摘要 肌腱和韧带损伤是最常见和最具挑战性的骨科疾病之一。许多患者 遭受长期疼痛和受伤后功能下降。大量证据表明，这些 不令人满意的结果主要是由于过度和/或持续的不平衡的愈合反应， 炎症和肌腱再生不足。我们发现，细胞外囊泡产生的 炎症引发的脂肪源性干细胞（iEV）可以通过减弱炎症， 由巨噬细胞介导的炎症和刺激肌腱细胞驱动的再生。然而，散装iEV 是异质的。不同产生的iEV携带不同的mRNA和microRNA分子， 确定iEV功能。有强有力的证据表明，只有一小部分iEV携带活性分子， 平衡巨噬细胞和肌腱细胞的损伤反应，而许多其他iEV可能 损害肌腱愈合或引起不希望的副作用。因此，有必要对iEV进行识别 亚群和活性货物分子特异性用于肌腱修复，并使用它们来开发疾病特异性 治疗，以大大提高肌腱修复。为了实现这一目标，批量iEV将按层次分类为 基于囊泡生成和确定囊泡货物的货物分类机器的有组织的亚组 混合物.特异性增强肌腱愈合的亚群和活性货物分子将被 通过其调节腱细胞和巨噬细胞活性和功能的效力来鉴定。治疗 将在临床前小鼠跟腱中检查鉴定的iEV和货物分子用于肌腱修复的功效 肌腱损伤修复模型。我们预计，去除功能上非特异性的iEV或选择性的iEV， 功能特异性货物分子在本体iEV中的富集将大大增加治疗功效， 避免用于肌腱损伤治疗的散装iEV的潜在不良副作用。这项研究是一个关键的证据- 基于细胞外囊泡（EV）的肌腱和韧带治疗的临床应用的概念分析 这将使数百万患者受益。本研究还将揭示iEV作用机制， 肌腱损伤，以及肌腱细胞和巨噬细胞活性的分子调节机制 和功能获得有关iEV的活性分子、表面标志物和生物起源的知识 肌腱修复特异性亚群将用于增加亲本的这些iEV的产量和释放 细胞，并通过基于免疫亲和性的方法扩大iEV生产。我们建立的方法可以用于 未来开发基于EV的和针对其他疾病的特定疗法， 改善更多患者的生活质量和预后。

PROJECT SUMMARY/ABSTRACT Tendon and ligament injuries are among the most common and challenging orthopedic conditions. Many patients suffer from long-term pain and reduced function after injury. Substantial evidence indicates that these unsatisfactory outcomes result primarily from an imbalanced healing response with excessive and/or sustained inflammation and inadequate tendon regeneration. We discovered that extracellular vesicles produced by inflammation-primed adipose-derived stem cells (iEV) could potentially restore balanced healing by attenuating inflammation mediated by macrophages and stimulating regeneration driven by tendon cells. However, bulk iEV are heterogeneous. Differentially generated iEV carry different mRNA and microRNA molecules, which determine iEV functions. There is strong evidence that only a fraction of iEV carry the active molecules that balance the injury responses of macrophages and tendon cells, whereas many other iEV could potentially compromise tendon healing or cause unwanted side effects. Therefore, it is necessary to identify the iEV subpopulation and active cargo molecules specific for tendon repair and use them to develop disease-specific therapy to greatly enhance tendon repair. To achieve this goal, bulk iEV will be sorted into hierarchically organized subgroups based on vesicle generation and cargo sorting machineries that determine vesicle cargo composition. The subpopulation and active cargo molecules that specifically enhance tendon healing will be identified by their potency in regulating tendon cell and macrophage activities and functions. The therapeutic efficacy of identified iEV and cargo molecules for tendon repair will be examined in a preclinical mouse Achilles tendon injury and repair model. We anticipate that removal of functionally non-specific iEV or selective enrichment of functionally specific cargo molecules in bulk iEV will greatly increase the therapeutic efficacy and avoid potential adverse side effects of bulk iEV for tendon injury treatments. This study is a critical proof-of- concept analysis toward clinical application of extracellular vesicle (EV)-based therapy for tendon and ligament injuries, which will benefit millions of patients. This study also will discover the mechanism of iEV action for tendon injuries, and mechanisms underlying the molecular regulation of tendon cell and macrophage activities and functions. Knowledge gained about the active molecules, surface markers, and biogenesis of the iEV subpopulation specific for tendon repair will be used to increase the yield and release of these iEV by parent cells and scale up iEV production via immunoaffinity-based method. Our established approaches can be used for the future development of EV-based and disease-specific therapies for other medical conditions, which will improve the quality of life and outcomes of more patients.