Tunable Synthetic ECMs for Investigating Stellate Cell Activation and Migration

摘要 肝纤维化是慢性肝损伤的异常愈合反应，使患者容易患肝硬化， 这是全世界的主要死因。虽然纤维化有许多病因，但它仅发生在门静脉高压症之后。 成纤维细胞和/或肝星状细胞（HSC）分化为肌成纤维细胞，这一过程称为激活。 HSC是纤维化和慢性肝损伤中肌成纤维细胞群的主要贡献者， 持续的HSC激活、组织收缩和过量的细胞外基质（ECM）沉积，导致 纤维化和器官衰竭。因此，了解导致纤维化进展的因素非常重要 和HSC肌成纤维细胞分化等物理微环境。迄今为止，体外研究已经 显示基质弹性是肌成纤维细胞分化的关键介质，使用不同的水凝胶， 力学这被认为是模拟肝纤维化过程中发生的肝硬度变化;然而， 肝脏存在于纤维状3D ECM中，其不能通过在光滑水凝胶上接种而重现。 重要的是，纤维材料可以被细胞操纵，并且具有非线性力学，使得能够长时间地生长。 通过光纤对准实现力的范围传输。因此，本提案的目的是开发可调 基于透明质酸的合成纤维材料模拟肝脏的ECM，以研究静态 动态纤维力学影响HSC的活化和迁移。这些建议的纤维系统是 与难以定制的天然ECM和典型的刚性合成纤维不同。的首要目标 该提议将是开发具有一系列机械性能的细胞培养基质。 假设较硬的纳米纤维将限制细胞纤维重塑和活化，而较软的纳米纤维将限制细胞纤维重塑和活化。 允许纤维重塑/募集和在纤维化刺激物TGF β 1下活化。本提案的目标2 将是纳米纤维材料的发展，可以局部硬化周围的HSC使用光触发 化学反应来模拟纤维化过程中发生的ECM的动态交联。它是假设 HSC将基于局部纤维机械性质定向迁移。总之，开发的材料 这里将提供新的平台来研究纤维化和愈合，激发新的纤维化疗法的发展， 并为申请人提供新材料和HSC生物学方面的培训。

ABSTRACT Liver fibrosis is an abnormal healing response to chronic liver injury and predisposes patients to cirrhosis, which is a major cause of death worldwide. Although fibrosis has many etiologies, it only occurs after portal fibroblasts and or hepatic stellate cells (HSCs) differentiate into myofibroblasts, a process termed activation. HSCs are the major contributors to the myofibroblast population in fibrosis and chronic liver damage stimulates continuous HSC activation, tissue contraction and excessive extracellular matrix (ECM) deposition, resulting in fibrosis and organ failure. Thus, it is important to understand the factors that contribute to fibrosis progression and HSC myofibroblast differentiation, such as the physical microenvironment. To date, in vitro studies have shown that substrate elasticity is a critical mediator of myofibroblast differentiation, using hydrogels of varied mechanics. This is thought to mimic the changes in liver stiffness that occurs during fibrosis; however, cells in the liver reside in a fibrous 3D ECM, which may not be recapitulated with seeding atop smooth hydrogels. Importantly, fibrous materials may be manipulated by cells and have non-linear mechanics that enable long- range force transmission through fiber alignment. Thus, the objective of this proposal is to develop tunable synthetic fibrous materials based on hyaluronic acid that mimic the ECM of the liver to investigate how static and dynamic fiber mechanics influence HSC activation and migration. These proposed fibrous systems are unique from both natural ECMs that are difficult to tailor and from typically rigid synthetic fibers. The first Aim of this proposal will be the development of nanofiber cell culture substrates with a range of mechanical properties. It is hypothesized that stiffer nanofibers will limit cell fiber remodeling and activation, while softer nanofibers will permit fiber remodeling/recruitment and activation under the fibrogenic stimulus TGFb1. Aim 2 of this proposal will be the development of nanofibrous materials that can be locally stiffened around HSCs using light triggered chemical reactions to mimic the dynamic crosslinking of ECM that occurs during fibrosis. It is hypothesized that HSCs will migrate directionally based on local fiber mechanical properties. Together, the materials developed here will provide new platforms to study fibrosis and healing, inspire the development of new fibrosis therapies, and provide training within new materials and HSC biology to the applicant.