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W. M. Petroll, A. Kim; Effects of Collagen Matrix Structure on Corneal Keratocyte Migration Mechanics. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6220.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate how the structure of collagen matrices impacts the pattern and amount of keratocyte migration, as well as protease dependency under different culture conditions.
Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-). A mixture of cells and rat tail collagen (2 mg/ml) was poured into wells and polymerized for 30 minutes. Matrices were compacted using external compression, and 6mm diameter buttons were punched out and nested within acellular uncompressed outer matrices (2.5mg/ml) consisting of either pepsin-extracted bovine dermal type I collagen or rat tail type I collagen (not pepsin-extracted). Media was supplemented with 10% FBS, PDGF (50ng/ml) or no growth factor (control) with or without the broad spectrum matrix metalloproteinase (MMP) inhibitor GM6001 (10 µM). After 4 days, constructs were labeled with AlexaFluor 546 Phalloidin and TOTO-3, and imaged using confocal fluorescent and reflection (for collagen) microscopy. In other experiments, time-lapse DIC imaging of cell migration through the outer collagen matrices was performed.
Rat tail collagen matrices had smaller pores and thinner fibrils than bovine dermal collagen. In both matrices: 1) PDGF and 10% FBS stimulated increased cell migration into the outer matrix as compared to S-; 2) cells in 10% FBS assumed a bipolar morphology and induced significant matrix reorganization during migration; and, 3) PDGF induced a more dendritic cell morphology with limited cellular force generation. With bovine dermal collagen, GM6001 induced a 35% reduction in the number of cells in the outer matrix following culture in 10% FBS, but had no significant effect on migration in PDGF. With rat tail collagen, MMP inhibition reduced cell numbers in the outer matrix by 74% following culture in 10% FBS; whereas the number of cells increased by 98% under PDGF.
Pepsin treatment reductively cleaves cross-link mediating telopeptides from collagen monomers, which alters the structural and mechanical properties of re-assembled collagen matrices. Importantly however, the morphological, migratory and mechanical responses of corneal keratocytes to different culture conditions were remarkably similar for these two matrix types, with serum inducing a contractile fibroblastic phenotype and PDGF inducing a non-contractile dendritic phenotype in both cases. By contrast, the dependency of migration on MMPs appears to vary depending upon both the collagen structure and the expressed mechanical phenotype of the cells.
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