ASSESMENT OF CORNEAL FIBROBLAST BIOMECHANICAL BEHAVIOR

描述（由申请人提供）：细胞通过机械力的作用来组织细胞外基质，驱动基本过程，如发育形态发生、伤口愈合和生物工程组织的组织。从历史上看，我们研究细胞力学行为的能力受到与测量细胞力产生的亚细胞模式和三维环境中局部矩阵模式相关的技术挑战的限制。因此，我们对这些基本过程的理解是有限的，特别是在眼部组织中。在过去的几年里，我们的研究一直致力于通过开发新的实验模型、使用新兴成像技术和应用定量分析技术来解决这些挑战。在先前的资助期内进行的研究为小gtpase Rho和Rac在调节三维胶原基质中角膜成纤维细胞的力产生和细胞外基质重组的亚细胞模式以及它们对局部机械应力变化的响应中的作用提供了重要的新见解。我们现在建议进一步应用和扩展我们的实验模型，以解决我们对角膜成纤维细胞力学行为的理解中的三个关键空白，这些行为与角膜创面愈合过程直接相关：1)特定生长因子如何调节角膜角质细胞的力学分化？2)角膜上皮细胞对ECM力学特性的大规模改变有何反应？3)在角膜形成细胞迁移过程中，是什么调控了细胞和基质模式？为了回答这些问题，我们提议：1)全面评估特定生长因子如何在三维胶原基质中调节角质细胞形态、细胞骨架组织、收缩力产生和基质模式，并确定Rho和Rac在介导这些效应中的作用；2)研究特定生长因子处理后基质刚度、密度和各向异性的改变如何调节角膜角质细胞的机械表型；3)研究Rho和Rac活化和ECM力学特性改变之间的相互作用如何调节角膜成纤维细胞在三维胶原基质内迁移过程中的细胞和基质模式。完成这些特定的目标将为控制角膜成纤维细胞迁移、收缩和基质重组的潜在生化和生物力学机制提供重要的新见解。这是使用标准二维培养模型无法获得的基本信息。公共卫生相关性：角膜角质细胞的力学行为在调节角膜对撕裂伤或屈光手术的反应中起着重要作用。我们建议使用创新的三维培养模型和定量成像技术来评估角膜细胞对与角膜伤口愈合过程直接相关的生化和生物物理刺激的机械反应

DESCRIPTION (provided by applicant): The organization of extracellular matrices by cells through the exertion of mechanical forces drives fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. Historically, our ability to investigate cell mechanical behavior has been limited by the technical challenges associated with measuring the sub-cellular pattern of cellular force generation and local matrix patterning in a 3-D environment. Thus our understanding of these fundamental processes is limited, especially in ocular tissues. Over the last several years, our research has been aimed at addressing these challenges through the development of new experimental models, use of emerging imaging technologies, and the application of quantitative analysis techniques. Research conducted in the prior grant period has provided important new insights into the roles of the small GTPases Rho and Rac in regulating the sub-cellular pattern of force generation and extracellular matrix reorganization by corneal fibroblasts within 3-D collagen matrices, as well as their response to local changes in mechanical stress. We now propose to further apply and expand our experimental models to address three key gaps in our understanding of corneal fibroblast mechanical behavior which have direct relevance to the process of corneal wound healing: 1) How do specific growth factors modulate the mechanical differentiation of corneal keratocytes?, 2) How do corneal keratocytes respond to large scale alterations in ECM mechanical properties?; and, 3) What regulates cell and matrix patterning during corneal keratocyte migration? To begin to answer these questions, we propose to: 1) perform a comprehensive assessment of how specific growth factors modulate keratocyte morphology, cytoskeletal organization, contractile force generation and matrix patterning within 3-D collagen matrices, and determine the role of Rho and Rac in mediated these effects, 2) investigate how alterations in matrix stiffness, density and anisotropy modulate the mechanical phenotype of corneal keratocytes following treatment with specific growth factors, and 3) Investigate how the interplay between Rho and Rac activation and alterations in ECM mechanical properties modulate cell and matrix patterning during corneal fibroblast migration within 3-D collagen matrices. Accomplishing these Specific Aims should provide important new insights into the underlying biochemical and biomechanical mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization in response to specific growth factors. This is fundamental information which can not be obtained using standard 2-D culture models. PUBLIC HEALTH RELVANCE: The mechanical behavior of corneal keratocytes plays a fundamental role in regulating the corneal response to lacerating injury or refractive surgery. We propose to use innovative 3-D culture models and quantitative imaging techniques to assess the mechanical response of keratocytes to biochemical and biophysical stimuli which have direct relevance to the process of corneal wound healing