Integrating Biomaterials and Biophotonics to Assess How ECM Mechanics Regulate Cell Function in 3-D

ID：MPS/DMR/BMAT（7623）0805164 PI：Putnam，Andrew ORG：标题：整合生物材料和生物光子学评估ECM力学如何在3-D中调节细胞功能优点：细胞外基质（ECM）为细胞提供化学和机械线索，并且更好地理解这些线索如何在3-D中控制细胞功能对于设计仿生材料作为组织工程的形态发生指南至关重要。 在本提案中，PI计划利用基于聚（乙二醇）和纤维蛋白原（PEG-纤维蛋白原）的独特生物合成混合水凝胶明确检验局部基质机械特性影响3-D细胞表型的假设。将使用血管平滑肌细胞（SMC）作为生理学相关模型细胞系统来解决这一假设，不仅基于PI记录的该细胞类型的经验，而且基于这些细胞在体外和体内对静态和动态机械应力的众所周知的响应能力。 初步数据表明，PEG-纤维蛋白原水凝胶支持SMC粘附，扩散，活力，并在3-D分化标志物的表达在相对较长的培养期。这种材料还提供了独立于粘附配体密度和蛋白水解敏感性可预测地调节整体机械性能的手段，这是使用天然生物聚合物（例如，胶原蛋白、纤维蛋白）。PI还建议开发新的方法来测量PEG-纤维蛋白原凝胶的局部机械性能，利用co-PI的光镊专业知识来研究机械转导机制，以解决细胞-材料相互作用领域的一个主要未回答的问题。以下三个目标构成了所提出的研究：（1）使用PEG-纤维蛋白原设计、表征和开发新型生物合成混合水凝胶，并证明在保持纤维蛋白原浓度恒定的同时，可以可预测地操纵其整体机械性能。(2)询问PEG-纤维蛋白原凝胶的局部弹性和粘弹性，将测量值与先前目标中的批量测量值进行比较。(3)评估凝胶机械特性对SMC表型转换的影响（合成到收缩）在这种PEG-纤维蛋白原ECM类似物的背景下在3-D中培养，并量化这些细胞材料构建体的体积和局部机械性质如何随时间变化。完成这一建议的目标需要整合生物材料，生物光子学，以及细胞和分子生物学。 它将有助于开发材料系统和方法，以解决有关ECM化学和力学的基本问题的近期研究目标。这样的系统和方法将具有更广泛的影响，定义用于组织工程的生物材料的生物力学设计参数，并允许与发育生物学、心血管生理学、伤口愈合和肿瘤发生相关的基本力学问题在未来的提案中得到解决。 为了促进这里开发的方法的传播，它们将被整合到激光微束和医疗计划中，这是位于UCI校园贝克曼激光研究所的NIH生物医学技术资源中心。 除了培训研究生的项目，PI已经建立了一个工作关系，与加州联盟少数民族参与（CAMP）和数学，工程和科学成就（梅萨）计划在UCI校园涉及少数民族本科生和高中生的计划。

ID: MPS/DMR/BMAT(7623) 0805164 PI: Putnam, Andrew ORG: University of California-IrvineTitle: Integrating Biomaterials and Biophotonics to Assess How ECM Mechanics Regulate Cell Function in 3-DINTELLECTUAL MERIT: The extracellular matrix (ECM) provides both chemical and mechanical cues to cells, and an improved understanding of how these cues govern cell function in 3-D is critically important to design biomimetic materials as morphogenetic guides for tissue engineering. In this proposal the PI plans to utilize a unique biosynthetic hybrid hydrogel based on poly(ethylene glycol) and fibrinogen (PEG-fibrinogen) to explicitly test the hypothesis that local substrate mechanical properties influence cell phenotype in 3-D. This hypothesis will be addressed using vascular smooth muscle cells (SMCs) as a physiologically relevant model cell system, based not only on the PI's documented experience with this cell type but also these cells' well-known ability to respond to static and dynamic mechanical stresses both in vitro and in vivo. Preliminary data demonstrate that PEG-fibrinogen hydrogels support SMC adhesion, spreading, viability, and the expression of differentiation markers in 3-D over relatively long culture periods. This material also provides the means to predictably tune bulk mechanical properties independently from adhesion ligand density and proteolytic sensitivity, something which cannot be achieved using native biopolymers (e.g., collagen, fibrin). The PI also proposes to develop novel methodology to measure the local mechanical properties of PEG-fibrinogen gels, leveraging the co-PI's expertise with optical tweezers to investigate mechanotransduction mechanisms to address a major unanswered question in the area of cell-material interactions. The following three objectives constitute the proposed study: (1) Engineer, characterize, and develop novel biosynthetic hybrid hydrogels using PEG-fibrinogen and demonstrate that their bulk mechanical properties can be predictably manipulated while holding the concentration of fibrinogen constant. (2) Interrogate the local elastic and viscoelastic properties of the PEG-fibrinogen gels, comparing the measured values to the bulk measurements in the previous objective. (3) Assess the impact of gel mechanical properties on the phenotypic switch of SMCs (synthetic to contractile) cultured in 3-D in the context of this PEG-fibrinogen ECM analog, and quantify how the bulk and local mechanical properties of these cell-material constructs change over time.BROADER IMPACTS: Completion of the objectives of this proposal requires an integration of emerging principles from biomaterials, biophotonics, and cell and molecular biology. It will contribute to the near-term research goal of developing material systems and methods to address fundamental questions regarding ECM chemistry and mechanics. Such systems and methods will have a much broader impact defining biomechanical design parameters for biomaterials useful in tissue engineering, and allow fundamental mechanics questions relevant for developmental biology, cardiovascular physiology, wound healing, and tumorigenesis to be addressed in future proposals. To facilitate dissemination of the methods developed here they will be integrated into the Laser Microbeam and Medical Program, an NIH Biomedical Technology Resource Center at the Beckman Laser Institute on the UCI campus. In addition to training graduate students on the project, the PIs have established a working relationship with the California Alliance for Minority Participation (CAMP) and the Mathematics, Engineering, and Science Achievement (MESA) programs on the UCI campus to involve minority undergraduate and high school students in the program.