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C. Zhou, L. Ma, W. M. Petroll; Rho Kinase Regulation of Corneal Fibroblast Migration Through 3-D Collagen Matrices. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6221.
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To investigate the role of Rho kinase in modulating the mechanics of fibroblast migration through fibrillar collagen extracellular matrix (ECM).
Studies were performed using human corneal fibroblasts (HTK cells). 6 ml of type I collagen (final concentration 2 mg/ml) containing HTK cells was poured into a mould and polymerized. Matrices were then compacted using external compression to form a thin sheet of high collagen concentration, similar to the in vivo cornea. 6 mm diameter buttons were punched out of this tissue equivalent sheet, placed inside acellular uncompressed collagen matrices, and overlaid with media containing either 1% fetal bovine serum (FBS), or 1% FBS plus the Rho kinase inhibitor Y-27632. Using a Nikon microscope with an environmental chamber, time-lapse DIC imaging was performed for up to 72 hrs using a 20X objective. A 3-D stack of images was collected every 25 minutes to produce a 4-D dataset. Cell migration and matrix displacement were measured from these datasets using MetaMorph. In additional experiments, confocal reflection imaging was used to further assess cell-induced matrix reorganization.
In 1% FBS condition, cells extended pseudopodial processes into the outer matrices beginning as early as 6 hours after plating. By 24 hours, many cells had completely escaped the inner matrix; most cells maintained a bipolar fibroblast morphology. Significant tractional forces were generated during migration, as indicated by inward displacement and reorganization of the collagen ECM at the leading edge of cells. When Rho kinase was inhibited, cells became more elongated, and extended thinner dendritic processes into the outer matrix. While both the rate of cell migration and the amount of collagen displacement decreased following Rho kinase inhibition, cell migration was more dramatically impaired.
Rho kinase modulates both corneal fibroblast morphology and migratory mechanics in this 3-D culture model. Interestingly, cells are still able to generate significant tractional forces as they extend thin dendritic processes into the outer matrix following Rho kinase inhibition. However, the amount of cell translocation is more substantially reduced, suggesting that the contractile forces needed to overcome cell/matrix adhesions and pull the cell body forward during migration are Rho kinase-dependent.
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