Dynamic Hydrogels for Probing Hepatic Stellate Cell Behavior During Fibrosis

描述（由申请人提供）：周细胞和成纤维细胞分化为纤维化肌成纤维细胞是许多组织（包括胰腺、肺、肾和肝）对损伤的常见反应。然而，肌成纤维细胞的持续存在导致过度的细胞外基质（ECM）沉积和收缩，导致纤维化和器官功能丧失。肝纤维化是一种严重的健康问题，发生在慢性侮辱，包括肝炎和过度饮酒。肝星状细胞（HSC）占总驻留肝细胞的约15%，也是纤维化期间肝肌成纤维细胞的主要来源。每年有超过170万例死亡归因于肝脏疾病，正在进行广泛的研究以更好地了解纤维化进展和HSC肌纤维母细胞分化。值得注意的是，人们越来越重视局部机械性能在调节HSC活化中的作用，这是拟议项目的重点。我建议，提高对调控HSC分化的微环境线索的理解对于开发对抗肝纤维化的疗法至关重要，并且具有受控生物物理特性的生物材料可以用作模型系统，以更好地探索局部力学对HSC行为的作用。虽然Burdick和威尔斯实验室的初步研究已经说明了静态材料力学对HSC分化的影响，但仍然非常需要动态材料系统来模拟天然组织的演变特性。因此，该提案的总体目标是开发基于透明质酸（HA）的机械动态材料系统，以模拟纤维化进展和消退期间的HSC表型变化。Burdick实验室在开发可调HA生物材料方面拥有丰富的经验，而威尔斯实验室在肝脏机械生物学和肝纤维化啮齿动物模型方面拥有专业知识;因此，我非常适合承担这里提出的工作。在目标1中，我将开发水凝胶，其随着时间的推移而老化，以使用二级自由基交联来模拟纤维化的进展，所述二级自由基交联将额外的交联引入到已经形成的水凝胶中。我假设硬化的速度将决定肌成纤维细胞分化的最终程度， 纤维化，更逐渐硬化导致中间表型。在目标2中，我将开发水凝胶，其通过交联的水解随时间软化以模拟纤维化的消退。我假设，软化材料将产生具有类似于在体内纤维化消退期间观察到的中间表型的HSC，并且软化速率将决定最终表型。总之，这里开发的动态底物将作为伤口愈合-纤维化-癌症三联体中其他疾病状态的简化模型和作为体外工具来鉴定治疗干预的潜在靶点是有用的。总而言之，除了宾夕法尼亚大学强大的合作环境外，拟议的研究和培训计划将指导我作为科学家的发展，并帮助我实现成为独立教师的职业目标。

DESCRIPTION (provided by applicant): The differentiation of pericytes and fibroblasts to fibrogenic myofibroblasts is a common response to injury in many tissues, including the pancreas, lung, kidney, and liver. However, the persistence of myofibroblasts leads to excessive extracellular matrix (ECM) deposition and contraction, resulting in fibrosis and loss of organ function. Liver fibrosis is a serious health problem occurring in response to chronic insults, including hepatitis and excessive alcohol consumption. Hepatic stellate cells (HSCs) comprise ~ 15% of total resident liver cells and are also the primary source of hepatic myofibroblasts during fibrosis. With over 1.7 million deaths attributed to liver disease annually, extensive research is underway to better understand fibrosis progression and HSC myofibroblastic differentiation. Notably, there is growing appreciation for the role of local mechanical properties in regulating HSC activation, which is the focus of the proposed project. I propose that improved understanding of the microenvironmental cues that regulate HSC differentiation is critical to the development of therapies to combat liver fibrosis, and that biomaterials with controlled biophysical properties can be used as model systems to better probe the role of local mechanics on HSC behavior. Although initial studies in the Burdick and Wells laboratories have illustrated the influence of static material mechanics on HSC differentiation, there is still a gret need for dynamic material systems to mimic the evolving properties of native tissues. Therefore, the general objective of this proposal is to develop mechanically dynamic material systems based on hyaluronic acid (HA) to model HSC phenotypic changes during the progression and regression of fibrosis. The Burdick lab has a wealth of experience in developing tunable HA biomaterials and the Wells lab has expertise in liver mechanobiology and rodent models of liver fibrosis; thus, I am ideally suited to undertake the work proposed here. In Aim 1 I will develop hydrogels that stiffen over time to model the progression of fibrosis using secondary radical crosslinking that introduces additional crosslinks into an already formed hydrogel. I hypothesize that the rate of stiffening will determine the ultimate degree of myofibroblast differentiation and fibrogenesis, with more gradual stiffening leading to intermediate phenotypes. In Aim 2 I will develop hydrogels that soften over time via hydrolysis of crosslinks to model regression of fibrosis. I hypothesize that softening materials will yield HSCs with an intermediate phenotype similar to that observed in vivo during fibrosis regression and that the rate of softening will determine the ultimate phenotype. Together, the dynamic substrates developed here will be useful as streamlined models of other disease states in the wound healing-fibrosis-cancer triad and as ex vivo tools to identify potential targets for therapeutic intervention. Together, the proposed research and training plans in addition to the strong collaborative environment at the University of Pennsylvania will guide my development as a scientist and help me meet my career goal of becoming an independent faculty member.