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S. A. Pot, S. J. Liliensiek, E. Bentley, D. J. Brown, J. V. Jester, P. F. Nealey, C. J. Murphy; Substratum Topography Modulates Cell Shape, Orientation, Migration and SMA mRNA Expression of Rabbit Keratocytes and Myofibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1962. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The differentiation of keratocytes into myofibroblasts is a key factor in corneal wound healing. Fundamental cell behaviors, like differentiation, can be modulated by the surface topography of the extracellular matrix, which contains feature sizes in the nanometer range. Here, we report that nanoscale topographic cues modulate cell shape, alignment, migration and alpha smooth muscle actin (αSMA) mRNA expression in rabbit primary corneal keratocytes and myofibroblasts.
Polyurethane surfaces were patterned with six 2x2 mm areas. These areas contain anisotropic feature sizes, composed of grooves and ridges ranging in width from 200 nm to 2000 nm, separated by planar control surfaces. Groove depth is 300 nm. Primary rabbit corneal keratocytes and TGFß induced myofibroblasts were plated onto patterned surfaces. After a 24 hour incubation cells were either fixed in 4% paraformaldehyde and stained with TRITC-phalloidin and DAPI or collected for RT-PCR analysis to quantify αSMA mRNA. In parallel cultures images of each patterned surface were taken every 10 minutes for 12 hours and individual cell trajectories and migration rates were calculated.
Both keratocytes and myofibroblasts exhibit an alignment and elongation response of 40% on the larger scale (> 1 µm) topographic surfaces compared to 10% on the smaller scale (< 1 µm) and planar surfaces. Both cell types have a random migration trajectory on planar and small scale topographic features. On large scale topographic surfaces both cell types exhibit contact guidance, migrating parallel to the long axis of the ridges and grooves. Myofibroblasts migrate eight times faster than keratocytes, at an average rate of 0.3 µm/min. Myofibroblasts have a greater than five-fold decrease of αSMA mRNA expression on all of the patterned surfaces compared to planar.
Nanoscale topographic features modulate cell shape and orientation of rabbit keratocytes and myofibroblasts. Myofibroblast migration and αSMA mRNA expression are influenced as well. The native nanotopographic environment likely assists in stabilizing the keratocyte phenotype while pathologic alterations of the topographic environment may be permissive for transformation to the myofibroblast phenotype and the development of fibrosis. These findings also have relevance to the rational design of ocular prosthetics and cell and tissue culture surfaces.
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