Preventive and Therapeutic Effects of Bone Marrow Stem Cell-Derived Exosomes on Spine Dural Fibrosis

项目总结/摘要 腰椎手术失败综合征（FBSS）是指腰椎手术后脊髓和神经根出现的一系列症状。 脊柱手术，特别是椎板切除术和椎间盘切除术，高达40%的患者经历过慢性 神经根痛和下肢无力。硬脑膜受压是FBSS的主要原因， 脊神经根由于硬脊膜纤维化，包括硬膜外纤维化和硬膜外粘连。多重因素导致 硬脑膜纤维化是手术创伤后的炎症反应和过度的基质沉积， 激活肌成纤维细胞。硬脊膜纤维化，如果不适当的保守治疗，一般可以 恶化，可能需要手术治疗。然而，这些手术干预的成功率仅为30- 35%， 高达20%的患者病情更严重。因此，迫切需要防止和 治疗硬脊膜纤维化。外泌体由于其作为硬脑膜炎抑制剂的巨大潜力而引起人们的关注 纤维化受损的组织通过外泌体的旁分泌信号而不是直接增殖来修复 这种旁分泌效应意味着外泌体疗法比干细胞疗法具有临床优势 移植在免疫应答和肿瘤发生方面。间充质基质/干细胞（MSC）是 外泌体的有希望的来源，并且MSC衍生的外泌体显示出肝纤维化的明显减少。 和肺 长期目标是开发一种微创抗纤维化治疗脊柱硬脊膜纤维化。实现 为此，我们建议评估外泌体对α平滑肌肌动蛋白（α-SMA）减少的影响， I型胶原和人肌成纤维细胞的收缩活性以及大鼠体内的预防作用 椎板切除模型。我们的中心假设是：外泌体有可能减轻肌成纤维细胞 活性，并且水凝胶将允许外泌体的持续释放并限制成纤维细胞迁移作为物理抑制剂。 硬脑膜和后部组织之间的屏障。这些假设是根据我们的 初步数据显示，MSC衍生的外泌体显示出预防和治疗潜力， 转化生长因子β 1（TGF-β1）诱导的牛肌成纤维细胞。减少 α-SMA表达作为肌成纤维细胞标志物在TGF-β1处理前和处理后的外泌体中显著。 在拟议的R21项目完成时，我们的预期成果是确定治疗潜力 外泌体用于预防硬膜外纤维化，并鉴定可调节抗纤维化的microRNA（miRNA）。 方面的影响.这些结果将为我们未来的R 01提供初步数据，从而产生非常重要的积极影响 资助申请，这将是评估MSC衍生的外泌体治疗后的体内治疗效果。 诱导硬脑膜纤维化和合成靶向miRNA以避免干细胞培养作为外泌体的手段 生产合成的miRNAs将被装载在工程化的外泌体中用于体内递送，从而取代MSC 培养作为外来体生产的潜在手段。

PROJECT SUMMARY/ABSTRACT Failed back surgery syndrome (FBSS) refers to a cluster of symptoms in spinal cord and nerve roots after spinal surgery, especially laminectomy and discectomy, and up to 40% of patients have experienced chronic nerve radicular pain and lower extremity weakness. The main cause of FBSS is the compression of dura and nerve roots due to spinal dural fibrosis, including epidural fibrosis and peridural adhesion. Multiple factors leading to dural fibrosis are the inflammatory response post-surgical trauma and excessive matrix deposition by highly activated myofibroblasts. Spinal dural fibrosis, if not properly treated by conservative management, can generally worsen, which may require surgical treatments. However, success of these surgical interventions is only 30-35%, and up to 20% of patients have more serious condition. Thus, there is a critical and urgent need to prevent and treat spinal dural fibrosis. Exosomes have attracted attention due to their great potential as an inhibitor of dural fibrosis. Damaged tissues are repaired by the paracrine signaling of exosomes rather than direct proliferation and differentiation and this paracrine effect implies that exosome therapy has a clinical advantage over stem cell transplantation in terms of immune response and tumorigenesis. Mesenchymal stromal/stem cells (MSCs) are promising sources of exosomes, and MSC-derived exosomes showed an evident reduction of fibrosis in liver and lung. The long-term goal is to develop a minimally invasive anti-fibrosis therapy for spine dural fibrosis. To achieve that goal, we propose to evaluate the effects of exosomes on reduction of alpha smooth muscle actin (α-SMA), type I collagen, and contractile activities in human myofibroblasts and the in vivo preventive effects in a rat laminectomy model. Our central hypotheses are that: exosomes have the potential to alleviate myofibroblast activities, and hydrogel will allow sustained release of exosomes and restrict fibroblast migration as a physical barrier between the dura mater and posterior tissue. The hypotheses have been devised on the basis of our preliminary data, which revealed that MSC-derived exosomes showed both preventive and therapeutic potential in exogenously induced bovine myofibroblasts by transforming growth factor beta 1 (TGF-β1). The reduction of α-SMA expression as a myofibroblast marker was notable in pre- and post-TGF-β1 treatment with exosomes. At the completion of the proposed R21 project, our expected outcomes are to define the therapeutic potential of exosomes for prevention of peridural fibrosis and to identify microRNAs (miRNA) that may regulate anti-fibrotic effects. These results will have a very important positive impact by providing preliminary data for our future R01 grant application, which will be to evaluate in vivo therapeutic effects of MSC-derived exosomes treated after induction of dural fibrosis and to synthesize target miRNAs to avoid stem cell culture as a means of exosome production. Synthetic miRNAs will be loaded in engineered exosomes for in vivo delivery, thereby replacing MSC culture as a potential means of exosome production.