Demonstration of mechanochemical force-structure causality during human corneal development using live-cell dynamic imaging and fluorescent collagen tracking

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During tissue development, cells create and organize the extracellular matrix (ECM), which is the acellular component of tissues comprising macromolecules and mechanosensitive proteins, to create a functional and complex structure. However, the process of ECM formation is not fully understood. It has been shown by Paten et al. (2016) in our extracellular research laboratory that collagen molecules - the most abundant protein in vertebrates - can assemble into fibrils, in vitro and in a cell free system, through flow-induced crystallization. Here, we hypothesize that cell-generated mechanical strain can induce extracellular matrix formation through flow-induced crystallization (FIC) or aggregation (FIA).To investigate extracellular matrix formation due to cell-generated strain, we cultured primary human corneal fibroblasts, which have been shown by Guo et al. (2007) to produce organized, collagen-dense, corneal stroma-like matrix. We used differential interference contrast microscopy to perform live-cell imaging with high temporal and spatial resolution over a four-day period. Additionally, we used confocal and fluorescent microscopy to track fluorescently labeled collagen within the culture over a period of 48 hours. In our live-cell videos, we observed five types of rapid cellular pulling events that formed persistent, extracellular filaments: flat cell process pull, thick cell process pull, thin cell process pull, ultrafine cell process pull, cell surface pull. We analyzed these events frame-by-frame to determine their velocity and extensional strain rate profiles. In four of these pull types, the average maximum extensional strain rate was between 0.1-0.33 sec-1, which is large enough to induce the crystallization or aggregation of mechanosensitive biopolymers as suggested by Paten et al. (2016). In our confocal and fluorescent microscopy photos, we visualized our fluorescent labeled collagen probe associating with the cells' extant extracellular network when added in a subthreshold concentration, or self-polymerizing when added in a suprathreshold concentration to form a matrix that is reorganized by the cells. These results suggest that corneal fibroblasts finely control ECM development, and that the rapid pulling of their cell membranes and processes forms extracellular filaments. We hypothesize that the mechanical force from cellular pulls lowers the activation energy required for FIC or FIA, which allows these persistent filaments to form. This proposed mechanism for tissue development has the potential to revolutionize our understanding of ECM assembly, informing future tissue engineering and medical research. In summary, this data supports a mechanochemical mechanism of matrix assembly, whereby cell-generated forces produce extensional strains that initiate and sustain the growth of mechanosensitive extracellular filaments directly in the path of the force that created them.--Author's abstract