Assessment of Corneal Fibroblast Biomechanical Behavior

项目总结 细胞和细胞外基质(ECM)之间的机械相互作用推动基本过程，如 生物工程组织的形态发生、伤口愈合和组织。我们的研究重点是如何 这些相互作用通过发展模拟角膜基质细胞的培养模型来调节角膜基质细胞的行为。 3-D组织环境，以及使用体外、原位和体内的多维成像方法。 在上一个资助期，我们使用这些工具对 准分子激光屈光性角膜切削术后角膜基质细胞的分化和图案化 兔子。这些研究为天然基质和天然基质之间过渡的本质提供了新的见解 纤维组织，以及细胞如何使用胶原层作为组织重塑和再生的模板。 联合浅表激光治疗性角膜切削术(PTK)和紫外线交联术(CXL)的后续研究 结果表明，CXL诱导了基质内正常细胞模式的破坏，并似乎阻断了 间质顶部出现纤维化。这些研究突显了变化的深刻影响 角膜结构和硬度会对整体角膜伤口愈合反应产生影响。然而， 标准CXL程序的大尺寸和整体对称性延长了正常的时间进程 PTK后的愈合，限制了生物力学研究的范围。 使用我们的体外3D培养模型，我们还进行了关于细胞如何扩散和 集体迁移在纤维蛋白基质中受到调节，并确定了纤维连接蛋白、51整合素和 在这一过程中，局部细胞诱导的基质重组。我们还第一次证明了，抑制 波形蛋白细丝的组织改变了角膜成纤维细胞在三维基质中的扩散、形态和运动性。 在体内，波形蛋白与角膜基质细胞的肌成纤维细胞转化有关，并且 最近对其他系统的研究表明，它可以在调节细胞的关键方面发挥核心作用 力学行为，包括机械传感、极化和定向迁移。 基于这些和其他已发表和试验的数据，我们现在建议：1)研究组织的影响 特异地应用紫外线交联剂对PTK术后角膜伤口愈合的影响 从有限元建模模拟中确定的模式，2)建立细胞分化的时间进程 以及在全层纤维化的发展和消退(重塑)过程中的细胞/基质图案 并确定了调整创口边界条件和机械性能的效果 3)应用我们已建立的3-D文化模型和工程设计的2-D 研究波形蛋白在调节角膜成纤维细胞分化、图案化和 机械行为。鉴于细胞力学在细胞生物学的众多领域中的普遍重要性， 这些发现可能具有广泛的意义。

PROJECT SUMMARY Mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as morphogenesis, wound healing, and organization of bioengineered tissues. Our research focuses on how these interactions regulate corneal keratocyte behavior through development of culture models that mimic the 3-D tissue environment, and use of multi-dimensional imaging approaches in vitro, in situ, and in vivo. In the previous funding period, we used these tools to perform a comprehensive assessment of the differentiation and patterning of corneal keratocytes following photorefractive keratectomy (PRK) surgery in the rabbit. These studies provided novel insights into the nature of the transition between native stromal and fibrotic tissue, and how cells use the collagen lamellae as a template for tissue remodeling and regeneration. Subsequent studies combining superficial phototherapeutic keratectomy (PTK) and UV cross-linking (CXL) showed that CXL induces a disruption in normal cell patterning within the stroma, and appears to block the development of fibrosis on top of the stroma. These studies highlight the profound impact that changes in corneal structure and stiffness can have on overall corneal wound healing responses. However, the large size and overall symmetry of standard CXL procedures extends the normal time course of healing after PTK, and limits the scope of biomechanical insights that can be made. Using our in vitro 3D culture models, we also performed mechanistic studies on how cell spreading and collective migration are regulated in fibrin matrices, and identified key roles for fibronectin, 51 integrin, and local cell-induced matrix reorganization in this process. We also demonstrated for the first time, that inhibition of vimentin filament organization alters corneal fibroblast spreading, morphology and motility in 3-D matrices. Vimentin has been associated with myofibroblast transformation of corneal keratocytes in vivo, and recent studies in other systems suggest it can play a central role in regulating key aspects of cell mechanical behavior, including mechanosensing, polarization, and directional migration. Based on these and other published and pilot data, we now propose to: 1) Investigate the effects of tissue stiffness and anisotropy on corneal wound healing following PTK, by applying UV cross-linking in specific patterns determined from finite element modeling simulations, 2) Establish the time course of cell differentiation and cell/matrix patterning during the development and resolution (remodeling) of fibrosis following full thickness incisional injury, and determine the effects of modulating the wound boundary conditions and mechanical environment on these processes, and 3) Apply our established 3-D culture models and engineered 2-D substrates to investigate the role of vimentin in regulating corneal fibroblast differentiation, patterning and mechanical behavior. Given the general importance of cell mechanics in numerous fields of cell biology, these findings could have broad significance.