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D. Karamichos, W. M. Petroll; The Role of Matrix Anisotropy in Regulation of Corneal Fibroblast Morphology and Mechanical Behavior in 3-D Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1869. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Patterning of cells and extracellular matrix during development and wound healing are thought to depend on both biochemical and biomechanical signals. The purpose of this study was to investigate how extracellular matrix mechanical anisotropy influences corneal fibroblast morphology, orientation, cytoskeletal organization and matrix remodeling in 3-D culture.
Human corneal fibroblasts were seeded within 45 x 20 mm collagen matrices that were either: 1) unconstrained (UN); 2) fully constrained (FC) along the long axis by attaching the short ends of the construct to two immobilized plastic bars; or, 3) partially constrained (PC) by allowing linear elastic displacement of one of the two bars. After 24 hours, constructs were fixed and cells labeled with AlexaFluor 488 phalloidin. Fluorescent (for f-actin) and reflected light (for collagen) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and cell morphology and local collagen density were measured using MetaMorph.
Corneal fibroblasts within all of the constructs generally had a bipolar morphology, however cell length was significantly greater in constrained matrices (FC>PC>UN, p<0.05, ANOVA). Cells were aligned nearly parallel to the long axis of the construct in constrained matrices, whereas cells in unconstrained matrices showed no preferential alignment (FC=PC>UN, p<0.05). At the ends of cells, both the local collagen density and the degree of cell/collagen coalignment were higher in constrained matrices (FC>PC>UN, p < 0.005). Furthermore, when cells in constrained matrices were in close proximity to each other, additional bands of compacted and aligned collagen were often observed spanning between them.
Overall the data suggests that cell spreading, alignment and contractile force generation are directly influenced by the mechanical properties of the extracellular matrix. In anisotropic matrices, isolated corneal fibroblasts generally align and compact collagen parallel to the axis of greatest stiffness; however, mechanical cross-talk between cells at higher density can result in additional, more complex matrix patterning.
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