Mechanobiology of Matrix Production by Corneal Fibroblasts

描述（由申请人提供）：受控制的成纤维细胞定向运动、自组织和合成能力是结缔组织发育、生长、重塑和修复的基础。然而，驱动成纤维细胞行为这些方面的信号知之甚少。越来越清楚的是，间充质组织中的成纤维细胞具有内在的模式和合成潜力，并且在适当的条件下，可以用来修复或再生高度组织的结缔组织，如角膜基质。在哺乳动物间质发育过程中，神经嵴来源的间充质细胞在眼压升高的情况下侵入前瞻性角膜间隙，自我组织，然后产生、扩展并随后维持基质细胞外基质（ECM）结构。我们对这一过程的理解非常有限，特别是关于1)初始角膜间充质/成纤维细胞模式的驱动因素，2)基质沉积的局部和全局控制机制，以及3)在机械力作用下指导组织扩展（生长）的机制。我们对控制基质沉积、保留和吸收的因素的有限认识延伸到组织再生，包括基质伤口愈合、疤痕消退和植入物重塑。人们一直认为，在基质中发现的高度组织的胶原阵列难以原位再生（特别是在人类中）。我们的长期基础科学目标是确定高度组织化的结缔组织是如何在生长、维持、修复和重塑过程中构建、扩展的。我们的长期转化目标是利用这些知识制定角膜伤口修复和再生的新方法。为了实现长期目标，有必要首先确定在基质产生过程中是什么因素控制成纤维细胞的组织行为，并确定它们通过什么机制控制沉积胶原的取向和保留。本提案的具体目的是研究机械信号对胶原基质的组织、合成和保留的影响。为了实现这一目标，我们将利用我们在活的、动态的细胞成像、生物反应器设计、机械生物学和机械化学方面的专业知识。我们的中心假设是，机械信号为角膜成纤维细胞提供了一个强大而持久的引导线索，它能够1)控制细胞的整体组织，2)指导胶原的沉积和优先保留，3)控制组织生长。为了检验这些假设，我们将利用我们最近开发的机械生物反应器，该反应器允许在组织生产过程中对成纤维细胞进行活的、长期的成像。将不同大小的负载放在培养系统上，细胞的输出将按分钟记录。拟议的调查结果不仅应充分检验中心假设，而且应提供有关在基质生产过程中刺激和控制PCFs行为所需的力水平的定量细节。

DESCRIPTION (provided by applicant): Controlled fibroblast directional motility, self-organization and synthetic capacity are fundamental to connective tissue development, growth, remodeling and repair. However, the signals which drive these aspects of fibroblast behavior are poorly understood. It is becoming increasingly clear that fibroblasts in mesenchymal tissue possess intrinsic patterning and synthetic potential and, under appropriate conditions, could be harnessed to repair or regenerate highly-organized connective tissues such as the corneal stroma. In mammalian stromal development, neural crest-derived mesenchymal cells invade the prospective corneal space, organize themselves, then produce, extend and subsequently maintain stromal extracellular matrix (ECM) architecture under increasing intraocular pressure. Our understanding of this process is quite limited, particularly with respect to 1) drivers of inital corneal mesenchymal/fibroblast cell patterning, 2) mechanisms of local and global control of matrix deposition and 3) mechanisms which guide tissue extension (growth) under mechanical force. Our limited appreciation of factors which control matrix deposition, retention and resorption extends to tissue regeneration including stromal wound healing, scar resolution and implant remodeling. It has been a dogmatic belief that the highly-organized collagenous arrays found in the stroma are refractory to in situ regeneration (particularly in humans). Our long-term basic science goals are to determine how highly-organized connective tissue is constructed, extended during growth, maintained, repaired and remodeled. Our long-term translational goals are to utilize this knowledge to formulate new approaches to corneal wound repair and regeneration. To accomplish the long term goals, it is necessary to first determine what factors control fibroblast organizational behavior during matrix production and to determine by what mechanism they control the orientation and retention of deposited collagen. The specific objective of this proposal is to investigate the effect of mechanical signaling on the organization synthesis and retention of collagenous matrix. To accomplish this objective, we will utilize our expertise in live, dynamic cell imaging, bioreactor design, mechanobiology and mechanochemistry. Our central hypothesis for this proposal is that mechanical signaling provides a robust and persistent guidance cue to corneal fibroblasts which is capable of 1) controlling the global organization of the cells, 2) guiding the deposition and preferential retention of collagen and 3) controlling tissue growth. To examine these hypotheses we will utilize our recently developed mechanobioreactor which permits live, long-term imaging of fibroblasts during the production of tissue. Loads of varying magnitude will be placed on a culture system and the output of the cells will be recorded on a minute-to-minute basis. The results of the proposed investigations should not only fully test the central hypothesis, but provide quantitative details about the levels of force necessary to stimulate and control the behavior of PCFs during matrix production.