Tuesday, April 23, 2024
4/23/2024
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.
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