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A.H. Dahlmann, M. Eastwood, M. Bailly, P.T. Khaw; Force Generation by Ocular Fibroblasts and Simultaneous Behavioral Imaging in a Dynamic 3D Environment . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2437.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study 1. cell morphology and cell–matrix interactions during the generation of force by ocular fibroblasts and 2. their response to external tension by real–time microscopy and simultaneous force measurement. Methods: We used human scleral, corneal, and Tenon's fibroblasts in a standard fibroblast–populated collagen matrix. Using a novel device, the micro–culture force monitor, we measured the endogenous tension generated by cells over the first 20 hours after matrix preparation, as well as their reactive behavior to externally applied matrix tension. We simultaneously imaged cell morphology and matrix remodeling with phase, differential interference, and confocal reflection timelapse microscopy. Openlab and Volocity software packages were used to analyse the 3 and 4D reconstructed timelapse series. Results: Corneal and scleral fibroblasts contract the collagen matrix with a force of 40–60 dyne per million cells. Force generation is greatest during initial cell spreading. During this phase, cells extend and retract numerous protrusions before assuming their characteristic spindle shape with few, polarized extensions. The endogenous force reaches a plateau when cell protrusive activity diminishes. Cell migration across the matrix is absent in the first and rare in the second phase. Stretching the matrix with an additional force equivalent to a third of the endogenous force does not induce measurable changes in cell shape. Cell–generated tension transiently relaxes, but quickly returns to its original level. Conclusions: The cells used in this study are involved in a variety of ocular conditions including glaucoma, axial myopia, corneal scarring after injury or surgery, and subconjunctival scarring after glaucoma surgery. Intraocular pressure and cellular responsiveness to mechanical stimulation have been implicated in all these conditions, yet the exact mechanisms are unknown. This is the first report of simultaneous live cell imaging and measurement of force generated by ocular fibroblasts in a physiological 3D environment. The unique setup presented here is also the first to allow analysis and comparison of the response of ocular cells to mechanical stimulation transmitted by the matrix. It will give us important insights into the exciting new field of cellular signaling in response to mechanical stimulation.
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