ASSESMENT OF CORNEAL FIBROBLAST BIOMECHANICAL BEHAVIOR

描述(申请人提供)：细胞通过施加机械力组织细胞外基质，推动发育形态发生、伤口愈合和生物工程组织的组织等基本过程。从历史上看，我们研究细胞力学行为的能力一直受到与测量3-D环境中细胞力产生的亚细胞模式和局部基质模式相关的技术挑战的限制。因此，我们对这些基本过程的了解是有限的，特别是在眼组织中。在过去的几年里，我们的研究旨在通过开发新的实验模型、使用新兴的成像技术和应用定量分析技术来应对这些挑战。在之前的资助期间进行的研究为小GTP酶Rho和Rac在调节力产生的亚细胞模式和三维胶原基质内的角膜成纤维细胞的细胞外基质重组中的作用以及它们对局部机械应力变化的反应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，这些空白与角膜创伤愈合过程直接相关：1)特定的生长因子如何调节角膜基质细胞的机械分化？2)角膜基质细胞如何对ECM力学特性的大规模变化做出反应？以及，3)在角膜基质细胞迁移过程中，是什么调节细胞和基质的模式？为了开始回答这些问题，我们建议：1)对特定的生长因子如何调节三维胶原基质内的角质细胞形态、细胞骨架组织、收缩力的产生和基质构型进行全面的评估，并确定Rho和Rac在介导这些影响中的作用；2)研究特定生长因子处理后基质硬度、密度和各向异性的变化如何调节角膜基质细胞的力学表型；以及3)研究Rho和Rac激活之间的相互作用以及ECM力学特性的变化如何调节三维胶原基质内角膜成纤维细胞迁移过程中的细胞和基质构型。这些特定目标的实现将为控制角膜成纤维细胞迁移、收缩和基质重组以响应特定生长因子的潜在生化和生物力学机制提供重要的新见解。这是使用标准的2-D文化模型无法获得的基本信息。公共卫生相关性：角膜基质细胞的机械行为在调节角膜对撕裂伤或屈光手术的反应中起着基础性作用。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基质细胞对生化和生物物理刺激的机械反应，这些刺激与角膜伤口愈合过程直接相关。

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