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

研究脊髓损伤后纤维化疤痕的生物工程体外试验台

• 批准号: 10202272
• 负责人: Younghye Song
• 金额: $ 42.98万
• 依托单位: UNIVERSITY OF ARKANSAS AT FAYETTEVILLE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10202272
• 关键词: 3-Dimensional; Affect; Astrocytes; Attention; Basement membrane; Beds; Biomedical Engineering; Biomimetics; Blood Vessels; Cells; Chondroitin Sulfate Proteoglycan; Cicatrix; Coculture Techniques; Collagen; Collagen Fiber; Cues; Deposition; Dermal; Desmoplastic; Disease; Dissociation; Dose; Endothelial Cells; Engineering; Exhibits; Extracellular Matrix; Fibronectins; Fluorescence; Future; Gel; Goals; Harvest; Hydrogels; Image; Image Analysis; Immunofluorescence Immunologic; In Vitro; Individual; Infiltration; Injury; Investigation; Kidney; Lesion; Liver; Measures; Mediating; Mesenchymal; Methods; Molecular; Myofibroblast; Neurites; Normal tissue morphology; Outcome; Outcome Study; Pathologic; Pathology; Pathway interactions; Patients; Pericytes; Physiological; Protein Kinase; Rattus; Research; Role; Sampling; Scientist; Sensorimotor functions; Signal Transduction; Site; Spinal Cord; Spinal Ganglia; Spinal cord injury; TGFB1 gene; Tail; Temperature; Testing; Time; Tissues; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Treatment outcome; applied biomedical research; crosslink; cytokine; improved; in vitro testing; in vivo; inhibitor/antagonist; insight; malignant breast neoplasm; mechanotransduction; new therapeutic target; receptor; response; rho; scaffold; self assembly; socioeconomics; student participation; three dimensional cell culture; treatment strategy; undergraduate student

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.

项目总结 本项目的总体目标是开发一种具有生理相关性的纤维性瘢痕体外试验台。 并确定新的治疗靶点，以改善脊髓损伤的治疗结果。SCI是一种 毁灭性的创伤条件，使受影响的个人永久感觉运动障碍和 社会经济负担。众所周知，脊髓损伤的病理图景是由富含神经细胞的胶质瘢痕组成。 抑制反应性星形胶质细胞沉积的硫酸软骨素蛋白多糖(CSPGs)。然而，很少有人关注 已给予胶原和纤维连接蛋白丰富，神经抑制纤维瘢痕，尽管有文件证据。 现有的研究表明，血管周围间充质细胞，如周细胞，主要负责 沉积富含胶原的纤维性疤痕。在纤维化病理中，周细胞转化为肌成纤维细胞，然后沉积 纤维性疤痕。然而，SCI后周细胞介导的纤维性瘢痕形成的详细机制尚不清楚。 这项建议的一个具体焦点是评估转化生长因子β(转化生长因子-1)的效果。 胶原纤维聚集在周细胞形成的纤维性瘢痕沉积上。转化生长因子--1是已知的细胞因子 周细胞-肌成纤维细胞转化及其在脊髓损伤后某个时间点(损伤后5-7天)的水平上调 这与纤维性疤痕沉积增加不谋而合。此外，病理性胶原纤维的组织结构 记录在患者样本中。此外，PI最近表明，这种病理性的胶原纤维组装可以 通过调节胶原水凝胶的交联温度，在体外被模拟。病理性胶原纤维 组装通过Rho相关的卷曲形成蛋白激酶(ROCK)驱动肌成纤维细胞分化- 介导式增强型机械传感。然而，胶原纤维和转化生长因子-1对周细胞、纤维性瘢痕的作用 脊髓损伤后的形成和随后的轴突生长仍不清楚。 为此，我们假设转化生长因子-1和病理性胶原纤维网络促进周细胞 脊髓损伤后血管系统的解离和纤维性瘢痕沉积。为了检验我们的假设，我们将 生物工程三维(3D)脊髓纤维瘢痕试验床通过脊髓脱细胞和温度- 受控的胶原纤维组装方法。内皮细胞、周细胞和星形胶质细胞将在 3D试验台。胶原纤维组装(AIM 1)和转化生长因子-1(AIM 2)对周细胞-肌成纤维细胞的单独作用 将评估移行、纤维性和神经胶质瘢痕沉积以及轴突渗透。联合作用的结果 将在AIM 3中测定转化生长因子-1和胶原纤维。ROCK抑制剂Y27632和转化生长因子-1受体抑制剂 SB431542将分别用于阻断胶原纤维和转化生长因子--1的作用。这项研究的结果 将深入了解脊髓损伤的物理化学线索在纤维化瘢痕沉积和星形胶质细胞中的作用。 回应。特别是，这项研究将强调了解纤维性瘢痕的重要性，而不仅仅是胶质瘢痕 刀疤，在SCI进展中。此外，本科生和研究生参与这一领域的研究将有所帮助。 培养未来的科学家和工程师进行基础和应用生物医学研究。