Project Summary The cornea and sclera are the principal load bearing members in the tough fibrous ocular tunic which we consider to be an integrated mechano-biological structure. During development, the shape of the eye has been tuned to conform to a specific mechanical environment through a long time-scale integration of its loading history with its initial genetic patterning. Although mechanics are known to contribute to the development of many connective tissues, the ocular globe is particularly sensitive to pressure (tensile wall stress) during the expansive phase of growth. Even in the mature ocular tunic, mechanical instabilities often manifest as conditions which disrupt vision (e.g. myopia, keratoconus, post-LASIK ectasia, tractional retinal detachments and glaucoma). While the underlying causes of tissue structural instabilities are poorly understood, we suspect that they are mechanobiological in nature and potentially reflect an imbalance in the tensional homeostasis that exists between mesenchymal cells, their local ECM and the global mechanical environment. We know that during development, disruption of the mechanical connection between fibroblastic cells and their ECM severely retards ocular growth in a manner analogous to pressure loss, suggesting that mechanical communication is critical to proper ocular morphogenesis. However, the effect of mechanical forces on the mechanisms which drive tissue formation and growth are not well characterized. It is remarkable that we still do not fully understand how the most important structural molecule in vertebrates, collagen, is efficiently assembled into highly-organized, functional, load-bearing tissues which are massively expanded into macroscale structures during growth. However, if we are able to uncover new mechanisms which control tissue formation and growth, we will have access to information which can inform therapies for a variety of pathological conditions including fibrosis, myopia, keratoconus and potentially, glaucoma. Additionally, if we understand how tissue is produced, then there will be implications for engineering corneas de novo and for improving approaches to regenerative corneal medicine. In the proposed work, we plan combine our human cell culture model of corneal stromal tissue elaboration with live-cell mechanodynamics imaging to directly observe single collagen molecules during their transition from solution to fibrils. We will thus directly test a new hypothesis which directly couples local and globally applied forces directly to molecular assembly of collagen during fibrillogenesis and growth. The working hypothesis for this proposal is that force causes corneal stromal ECM elaboration to regulate fibril assembly, remodeling and growth. If the hypothesis is correct, there are myriad mechanotherapeutic opportunities and more critically, our basic understanding of collagenous tissue formation and growth, will be fundamentally altered.

Project Narrative The purpose of the proposed work is to test the hypothesis that mechanical forces directly drive tissue assembly in corneal stromal matrix produced by human corneal cells. A sophisticated battery of mechanobiological tools will be combined to permit both local mechanical measurements and single molecule tracking while tissue is being elaborated in vitro. The results may change our understanding of how load-bearing tissues are made and could guide mechanotherapeutics for problems such as keratoconus, fibrosis, ectasia, myopia, glaucoma and even aid efforts at corneal stromal regeneration.