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.
Completion of this proposal will provide insight into the basic science of how mechanics influences cells during the production of highly-organized tissue such as the corneal stroma. The proposed work will also investigate the role of mechanical force in guiding the chemistry associated with the assembly of load-bearing tissue. Ultimately, we expect that the work will lead to better approaches to tissue engineering and a deeper understanding of the role that mechanics plays in the construction of vertebrate connective tissue.
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