One of the most important and fundamental questions in vertebrate animal structure development remains unanswered: How are collagen molecules, once released by the cell, effectively incorporated into developing tissue matrix? Because collagen is the principal structural molecule in vertebrates, answering this question is critical to 1) our general understanding of how animal structure is initially produced; 2) understanding tissue remodeling and 3) informing attempts to engineer replacement structures dominated by collagen. In the corneal stroma, the dual functional requirements (mechanical stability and transparency) are satisfied by the incredibly precise arrangement and local alignment of collagen in the matrix. Unfortunately, our poor understanding of how collagen arrays are assembled has hampered our ability to recapitulate stromal tissue. Recently, we proposed a novel theory which suggests that forces applied by fibroblasts to the collagenous milieu immediately surrounding the cells are potentially direct drivers of collagen assembly and organization. Although mechanics are known to contribute to the development of many connective tissues, growth of the ocular globe and cornea are particularly sensitive to pressure during development. Further, it is known that disruption of the mechanical connections between fibroblastic cells and their ECM (e.g. by blocking integrins) severely retards ocular growth in a manner analogous to pressure loss, suggesting that local mechanical tension is also critical to proper ocular morphogenesis. In a series of publications, we have shown that cell-relevant forces regulate collagen fibril formation2 and stability via direct mechanochemical effects. However, precisely how local forces may act to control collagen during cell-mediated fibrillogenesis remains unknown. The working hypothesis for this proposal is that local extensional strain applied by the cells initiates fibril/lamellar assembly (neolamellogenesis) in the developing corneal stroma by accelerating collagen assembly kinetics via Flow-induced crystallization (FIC). Our long term goals are to use mechanics to promote (or prevent) matrix production by fibroblasts and to develop direct print methods produce corneal stromal matrix for tissue replacement or repair. The specific objectives of the proposed R21 investigation are to directly test the working hypothesis in our experimental model of human corneal neolamellogenesis and to attempt de novo assembly of individual corneal stromal lamellae. We plan to use our model of corneal stromal elaboration to test and translate a new hypothesis which directly couples locally applied forces to the molecular assembly of collagen during initial fibrillogenesis. If force drives collagen assembly, there are a myriad of mechanotherapeutic opportunities and our basic understanding of collagenous tissue formation will be fundamentally altered. The theory raises the possibility that mechanical stimulation is critical to the deposition of collagenous matrix and opens new opportunities for engineering tissues or treating fibrosis.
The purpose of the proposed work is to translate flow-induced crystallization (FIC) to directly print corneal stromal lamellae and to test the hypothesis that mechanical forces are used by primary human corneal fibroblasts (PHCFs) to initiate lamellar assembly. A sophisticated battery of microfluidic and mechanobiological tools will be employed to permit translation of FIC and to directly observe molecular kinetics during tissue assembly by PHCFs. The results have the potential to completely change our understanding of how load-bearing tissues are made and could accelerate corneal stromal regeneration on the benchtop.