Lung Microvascular Development During Dynamic Mechanical Deformation in Matrix Bioblocks

PI: Lewis H RomerProposal ID: 1264184Intellectual Merit：在三维细胞外基质的复杂编织中，组织形态发生是由机械和生化信号的相互作用指导的。本提案解决了基质机械化学耦合的组合机制，在哺乳动物生命至关重要的发育组织-肺微血管的特定背景下。该项目旨在应用软物质微加工方法来开发一个动态机械测试平台，并利用它来研究机械变形对基质组装和血管生成的重要步骤的影响。例如，生物块或不同形状和大小的三维基质结构将通过在微结构PDMS井中生长的肺成纤维细胞产生，并在静态条件或周期性应力下，在这些材料中有无流体流动的情况下进行研究。期望系统地构建一个关于机械力（如振幅、频率、矩阵刚度和流动）对组织组织和发育的影响的知识体系。这项计划中的研究代表了生物医学工程和发育生物学的重大进步，它提供了一个平台，在这个平台上，施加在三维（3D）组织特异性基质上的动态机械力可以被精确控制，从而可以测量机械扰动的下游后果，作为细胞信号传导和形态发生的终点。本研究将为组织形态发生过程中机械化学耦合机制的研究提供新的基础见解。在第一个目标中，精确结构的3D合成生物块将用于设计和研究人类胎儿肺成纤维细胞的几何形状和信号。具有生长或脱细胞的天然基质的生物块将受到动态拉伸的模式，再现胎儿呼吸的第二个目标。第三个目标将集中在内皮信号和形态发生在这个动态的3D矩阵。总之，这些研究将阐明机械变形对基质和血管的发育影响。该项目由跨学科协同推动：PI Romer在基质生物学，细胞骨架信号传导和细胞粘附领域做出了重大贡献，而PI Gracias在软材料和微流体系统的微图案方面具有相当的专业知识。该提案的变革特征涉及在一个平台上研究组织形态发生，该平台精确地模拟了三维动态体内环境。这些特性是通过将微结构凹槽与表面修饰、图案各向异性、动态机械测试和微流体相结合而实现的。广泛影响：系统地剖析指导组织生长和发育的机械和化学刺激之间的复杂关系，将导致从发育生物学到群体感应等许多领域的新知识体系。这项计划将巩固两位杰出的科学教育家的合作，使学生学习如何将工程工具应用于生物学和医学的重要问题。PI Romer在研究生科学教育方面的工作体现在课程开发和研究伦理和病理生理过程的分子机制方面。他的网站提供免费下载用于研究细胞牵引力的新工具，这表明他致力于分享科学研究成果。2007年，PI Gracias被马里兰州教育委员会授予教育成就奖（由州参议员Ulysses Currie颁发），以表彰K-12的推广工作。如果这项提案得到资助，它将进一步推动我们的努力，让本科生、K-12学生和公众接触到生物学和工程学的前沿。

PI: Lewis H RomerProposal ID: 1264184Intellectual Merit: Tissue morphogenesis is directed by the interplay of mechanical and biochemical signals in the complex weave of three-dimensional extracellular matrix. This proposal addresses the combinatorial mechanisms of matrix mechanochemical coupling in the specific context of a developing tissue that is essential for mammalian life--the lung microvasculature. This project seeks to apply soft-matter microfabrication methodologies to develop a dynamic mechanical testing platform and use it to investigate the impact of mechanical deformation on the vital steps in matrix assembly and vasculogenesis. i.e. bioblocks, or three-dimensional matrix constructs of different shapes and sizes will be produced by growing lung fibroblasts within microstructured PDMS wells and studied during static conditions or periodic stresses, with and without fluid flow in those materials. It is expected to systematically construct a body of knowledge on the effect of mechanical forces such as amplitude, frequency, matrix stiffness and flow on tissue organization and development.The research planned represents a significant advance for biomedical engineering and developmental biology by providing a platform in which the dynamic mechanical forces exerted on three dimensional (3D) tissue-specific matrix can be accurately controlled so that the downstream consequences of mechanical perturbations can be measured as cell signaling and morphogenesis endpoints. The proposed research will provide new fundamental insights into mechanisms of mechanochemical coupling during tissue morphogenesis. In the first aim, precisely structured 3D synthetic bioblocks will be used to engineer and study the geometry and signaling of human fetal lung fibroblasts. Bioblocks with growing or decellularized natural matrices will be subjected to patterns of dynamic stretch that recapitulate fetal breathing in the second aim. The third aim will focus on endothelial signaling and morphogenesis in this dynamic 3D matrix. Together, these investigations will elucidate the developmental impact of mechanical deformation on matrix and blood vessels. This project is catalyzed by interdisciplinary synergy: PI Romer has made major contributions to the fields of matrix biology, cytoskeletal signaling, and cell adhesion, while PI Gracias has considerable expertise in the micropatterning of soft materials and microfluidic systems. The transformative features of the proposal involve the study of tissue morphogenesis in a platform that accurately mimics the 3D dynamic in vivo environment. These features are enabled by the integration of microstructured recesses coupled with surface modifications, patterned anisotropy, dynamic mechanical testing and microfluidics.Broad Impact: Systematic dissection of the complex relationships between the mechanical and chemical stimuli that direct tissue growth and development will lead to a new body of knowledge in a number of fields ranging from developmental biology to quorum sensing. This project will solidify the collaboration of two outstanding science educators, enabling students to learn how to apply engineering tools to important problems in biology and medicine. PI Romer's work in graduate science education is exemplified by curriculum development and actuation in research ethics and molecular mechanisms of pathophysiologic processes. His website that offers free downloads of novel tools for the study of cell traction forces demonstrates his commitment to sharing the fruits of scientific investigation. In 2007, PI Gracias was awarded an education accomplishment citation from the Maryland State Board of Education (given by state senator Ulysses Currie) for K-12 outreach efforts. If this proposal is funded, it will further our efforts to expose undergraduates, K-12 students and the general public to frontiers of biology and engineering.