Bioengineering in vitro test beds to study fibrotic scar after spinal cord injury

项目摘要 本项目的总体目标是开发一个生理相关的体外纤维化瘢痕试验床， 脊髓损伤（SCI），并确定新的治疗靶点，以改善SCI治疗结果。SCI是一个 破坏性创伤状况，使受影响的个人永久性感觉运动缺陷， 社会经济负担。众所周知的SCI病理景观包括富含神经胶质的瘢痕， 由反应性星形胶质细胞沉积的抑制性硫酸软骨素蛋白聚糖（CSPG）。然而，很少有人注意到 尽管有文件证明，但已将其用于富含胶原蛋白和纤连蛋白的神经抑制性纤维化瘢痕。 现有的研究表明，血管周围的间充质细胞，如周细胞，是主要负责 沉积富含胶原的纤维化疤痕。在纤维化病理中，周细胞转变为肌成纤维细胞，肌成纤维细胞存款 纤维化疤痕然而，SCI后周细胞介导的纤维化瘢痕形成的详细机制尚不清楚。 该提案的一个具体重点是评估转化生长因子β（TGF-β 1） 以及周细胞对纤维化疤痕沉积的胶原纤维组装。TGF-β 1是一种众所周知的细胞因子， 周细胞-肌成纤维细胞转变，并且其水平在SCI后的时间点（损伤后5-7天）上调 这与纤维化疤痕沉积的增加相吻合。此外，病理性胶原纤维组织已被 记录在患者样本中。此外，PI最近表明，这种病理性胶原纤维组装可以 通过调节胶原水凝胶交联温度在体外模拟。病理性胶原纤维 组装通过Rho相关卷曲螺旋形成蛋白激酶（ROCK）驱动肌成纤维细胞分化- 介导的增强机械感应。然而，胶原纤维和TGF-β 1对周细胞、纤维化瘢痕 脊髓损伤后神经突的形成和随后的神经突生长仍不清楚。 为此，我们假设TGF-β 1和病理性胶原纤维网络促进周细胞 与血管系统分离和SCI后纤维化瘢痕沉积。为了验证我们的假设，我们将 生物工程三维（3D）SCI纤维化疤痕试验床通过脊髓脱细胞和温度- 控制胶原纤维组装方法。内皮细胞、周细胞和星形胶质细胞将在此培养。 3D测试床。胶原纤维组装体（Aim 1）和TGF-β 1（Aim 2）对周细胞-肌成纤维细胞的个体效应 将评估过渡、纤维化和神经胶质瘢痕沉积以及神经突浸润。的综合影响 将在目标3中测定TGF-β 1和胶原纤维。ROCK抑制剂Y27632和TGF-β 1受体抑制剂 SB 431542将分别用于破坏胶原纤维和TGF-β 1的作用。本研究的结果 将提供一个深入了解SCI的物理化学线索的作用，纤维化瘢痕沉积和星形胶质细胞 反应特别是，这项研究将突出了解纤维化瘢痕的重要性，而不仅仅是神经胶质 疤痕，SCI进展。此外，本科生和格拉德生参与这方面的研究将有助于 在基础和应用生物医学研究方面培养未来的科学家和工程师。

PROJECT SUMMARY The overall goal of this project is to develop a physiologically relevant in vitro test bed of fibrotic scar after spinal cord injury (SCI) and identify novel therapeutic targets to improve SCI treatment outcomes. SCI is a devastating traumatic condition that inflicts the affected individuals with permanent sensorimotor deficits and socioeconomic burdens. The well-known pathological landscape of SCI consists of glial scar rich in neuro- inhibitory chondroitin sulfate proteoglycans (CSPGs) deposited by reactive astrocytes. However, little attention has been given to the collagen and fibronectin-rich, neuro-inhibitory fibrotic scar despite documented evidences. Existing studies suggest that perivascular mesenchymal cells such as pericytes are primarily responsible for depositing collagen-rich fibrotic scar. In fibrotic pathologies, pericytes transition into myofibroblasts that deposit fibrotic scar. However, the detailed mechanism of pericyte-mediated fibrotic scar formation after SCI is unclear. A specific focus of this proposal is to evaluate the effects of transforming growth factor beta (TGF-1) and collagen fiber assembly on fibrotic scar deposition by pericytes. TGF-1 is a well-known cytokine behind pericyte-myofibroblast transition, and its levels are upregulated after SCI at a time point (5-7 days post injury) that coincides with increased fibrotic scar deposition. In addition, pathologic collagen fiber organization has been documented in patient samples. Further, PI has recently shown that this pathologic collagen fiber assembly can be mimicked in vitro by modulating collagen hydrogel crosslinking temperature. Pathologic collagen fiber assembly drives myofibroblast differentiation via Rho‑associated coiled‑coil‑forming protein kinase (ROCK)- mediated enhanced mechanosensing. However, the role of collagen fiber and TGF-1 on pericytes, fibrotic scar formation after SCI and subsequent neurite outgrowth remain unclear. To this end, we hypothesize that TGF-1 and pathological collagen fiber network promote pericyte dissociation from vasculature and fibrotic scar deposition after SCI. To test our hypothesis, we will bioengineer three-dimensional (3D) SCI fibrotic scar test beds via spinal cord decellularization and temperature- controlled collagen fiber assembly methods. Endothelial cells, pericytes and astrocytes will be cultured in this 3D test beds. Individual effects of collagen fiber assembly (Aim 1) and TGF-1 (Aim 2) on pericyte-myofibroblast transition, fibrotic and glial scars deposition, and neurite infiltration will be assessed. The combined effects of TGF-1 and collagen fibers will be determined in Aim 3. ROCK inhibitor Y27632 and TGF-1 receptor inhibitor SB431542 will be used to disrupt collagen fiber and TGF-1 effects, respectively. The outcomes of this study will provide an insight into the role of SCI physicochemical cues on fibrotic scar deposition and astrocyte response. In particular, this research will highlight the importance of understanding fibrotic scar, not just glial scar, on SCI progression. Further, undergrad and grad student participations in this line of research will help educate future scientists and engineers in fundamental and applied biomedical research.