Center of Biomedical Research Excellence in Matrix Biology Phase II

1.项目总结--低强度振动的细胞机械转导在调节中的作用 细胞外基质合成 1.1.总结家长奖的目标：生物医学研究中心的长期目标 卓越(Cobre)的基质生物学是建立、加强和积极推进多学科研究 中心致力于提高我们对细胞外基质在发育、健康、 和疾病，为预防、治疗和治愈高度优先的疾病作出贡献。具体的 Cobre Matrix Biology Parent奖的目标是：1)加强和发展研究人员的临界质量 围绕基质生物学的主题多学科重点建立，2)增强生物医学研究核心 能力，3)加强与现有计划的研究协作，4)加强研究培训 机遇。该项目将补充现有的科布雷基质生物学奖，以组成一个新的团队 调查员将来自Idea State的三名调查员聚集在一起，他们具有不同的视角和专业知识 解决复杂的基础、行为、临床和/或转化性研究问题 接近了。研究问题不重复父级奖项目前正在进行的问题，并且 显然受益于合作的集体努力。 1.2补充奖将解决的研究问题：工程生物物理信号前景 在破坏骨骼的条件下指导组织再生的非药物干预，例如 骨质疏松症、衰老、损伤、卧床休息或微重力。外加低强度振动(LIV)，a 类似肌肉活动的机械信号，提供了一种易于使用的技术来刺激间充质干细胞 组织工程和临床方法的细胞(MSC)合成代谢。LIV不会产生显著的 体内基质变形，因此排除了以前提出的大多数机械转导机制 高幅度和低频率的机械信号(例如，运动)。这就形成了一个巨大的差距 了解骨机械生物学知识，防止将LIV用作骨丢失的有效治疗方法。 MSC替代和恢复骨细胞群的能力是由两种动态机械力决定的 由日常活动(如肌肉活动)和细胞外基质(ECM)的质量决定。是1 相关蛋白(YAP)是一种转录共激活因子，可以激活基因的表达以响应 对机械力，包括ECM分子如结缔组织生长因子(CTGF)进行调节 细胞中的胶原蛋白的产生。组织工程和临床方法最终取得成功，原因是 有关LIV生成的高频信号如何被感知、转换并最终导致 核YAP的表达和ECM的产生是至关重要的。 这项建议旨在解决骨骼机械生物学的一个根本空白，通过机械地建立一个 细胞核对LIV产生的动态加速作出反应的机械感觉功能。使用我们的 新的团队方法，我们将测试LIV是否在细胞内产生细胞核的相对运动 加强核-细胞骨架支架，增加力诱导胞核内YAP信号转导 ECM生产。我们将通过三个子假设和具体目标来解决这一问题。 这项研究的目的是确定在活细胞中1)振动频率和加速度是否调制Liv. 诱导的核运动和由此导致的F-肌动蛋白重塑，2)Liv诱导的核周F-肌动蛋白重塑 增加细胞核上的细胞骨架张力，3)细胞核上细胞骨架张力的大小决定 YAP核进入和ECM生产的规模。 这些目标的完成将提供关于(1)如何提高基于LiV的再生效果的知识 (2)骨髓间充质干细胞的基本结构-功能关系。结果最终将使 以核-细胞骨架连接为目标的基于LiV的工程方法在许多领域的应用 包括但不限于组织再生、组织工程和老化。 1.3团队科学工作的好处：这项拟议的补充不能由任何单一的 调查人员，需要在不同领域工作的三名调查人员协调工作：CELL 机械生物学、机器学习和计算生物力学。联合项目负责人(CPL)Uzer将 致力于建立测量核运动、高保真细胞和生物结果的实验方法， ECM生产和核YAP水平。下士萨蒂奇将专注于开发机器学习算法，以 在活细胞中重建三维细胞核和细胞骨架几何形状以响应Liv。下士菲茨帕特里克将发展 用有限元(FE)模型来量化LIV治疗下细胞核上的细胞骨架力。成功后 完成这项工作后，我们的团队将首次为数据驱动、特定于单元的FE建立一条新的管道 用于理解细胞内力-函数关系的模型。这种新方法将使我们获得新的赠款。 提交研究细胞特异力在维持健康细胞功能和细胞外基质成分中的作用的意见书 以及为使用Liv在体外和体内延缓疾病进展的新的翻译研究提供信息。

1. PROJECT SUMMARY - Role of Cellular Mechanotransduction of Low Intensity Vibrations in Regulating Extracellular Matrix Synthesis 1.1. Summarize the goal of the parent award: The long-term goal of the Center of Biomedical Research Excellence (COBRE) in Matrix Biology is to establish, enhance, and actively advance a multidisciplinary research center focusing on improving our understanding of the role of the extracellular matrix in development, health, and disease, and contributing to the prevention, treatment, and cure for diseases of high priority. The specific aims of the COBRE Matrix Biology Parent award are: 1) enhance and grow upon the critical mass of investigators established around the thematic multidisciplinary focus of matrix biology, 2) enhance biomedical research core capabilities, 3) grow research collaborations with existing programs, and 4) enhance research training opportunities. This project will supplement the existing COBRE Matrix Biology award to form a new team of investigators that bring together three investigators from IDeA states with different perspectives and expertise to address complex basic, behavioral, clinical and/or translational research questions with complementary approaches. The research question does not duplicate those currently being pursued by the parent award and clearly benefits from the collective efforts of the collaboration. 1.2 Research question to be addressed by the supplement award: Engineering biophysical signals promises non-pharmacologic interventions to direct tissue regeneration in conditions that devastate bone such as osteoporosis, aging, injury, bedrest, or microgravity. Externally applied Low-Intensity Vibrations (LIV), a mechanical signal similar to muscle activity, offers a readily usable technology to stimulate Mesenchymal Stem Cell (MSC) anabolism for both tissue engineering and clinical approaches. LIV does not generate significant matrix deformations in vivo, thus excluding most mechano-transduction mechanisms previously proposed for high-magnitude and low-frequency mechanical signals (e.g., exercise). This presents a significant gap knowledge about bone mechanobiology and prevents utilization of LIV as an effective treatment for bone loss. MSC’s ability to replace and rejuvenate bone cell populations are informed by both dynamic mechanical forces generated during daily activities (e.g. muscle activity) and by the quality of the Extracellular Matrix (ECM). Yes1 Associated Protein (YAP) is a transcriptional co-activator that can activate the expression of genes in response to mechanical force, including ECM molecules such as Connective Tissue Growth Factor (CTGF) to regulate collagen production in cells. For tissue engineering and clinical approaches to ultimately succeed, causative information on how high-frequency signals generated by LIV are sensed, transduced, and eventually lead to nuclear YAP expression and ECM production is critical. This proposal aims to address a fundamental gap in bone mechanobiology by mechanistically establishing a mechanosensory function of the cell nucleus to respond to dynamic accelerations produced by LIV. Using our novel team approach, we will test whether LIV generates relative motions of the nucleus within a cell to strengthen nucleo-cytoskeletal scaffolding and to increase force-induced YAP signaling in the cell nuclei to elicit ECM production. We will address this through three sub-hypotheses and specific aims. The aims of this study are to determine in live cells if 1) vibration frequency and acceleration modulate the LIV- induced nuclear motions and resulting F-actin remodeling, 2) LIV-induced perinuclear F-actin remodeling will increase cytoskeletal tension on the nucleus, 3) the magnitude of cytoskeletal tension on the nucleus determines the magnitude of YAP nuclear entry and ECM production. Completion of these aims will provide knowledge on (1) how to enhance the efficacy of LIV-based regenerative modalities in clinic, and (2) foundational structure-function relationships in MSCs. Results will ultimately enable engineering LIV-based approaches that target nucleo-cytoskeletal connectivity with application in many areas including, but not limited to, tissue regeneration, tissue engineering, and aging. 1.3 Benefit of team science effort: This proposed supplement cannot be accomplished by any single investigator and requires an orchestrated effort by three investigators working in different fields: cell mechanobiology, machine learning, and computational biomechanics. Co-Project Lead (CPL) Uzer will work on establishing experimental methods to measure nuclear motion, high-fidelity cell and biological outcomes, ECM production, and nuclear YAP levels. CPL Satici will focus on developing machine learning algorithms to reconstruct 3D nuclear and cytoskeletal geometries in response to LIV in live cells. CPL Fitzpatrick will develop finite element (FE) models to quantify cytoskeletal forces on the nucleus under LIV treatment. Upon successful completion of this work, our team will establish, for the first time, a novel pipeline for data-driven, cell-specific FE models for understanding force-function relationships in cells. This novel method will lead us to new grant submissions to study the role of cell-specific forces in maintaining healthy cell function and ECM composition as well as informing new translational studies that use LIV to attenuate disease progression both in vitro and in vivo.