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N. Garagorri, S. Fermanian, G. Sexton, O. Schein, J. Elisseeff; Confocal imaging of corneal keratocytes encapsulated into photopolymerizing hydrogels . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3932.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To investigate if stromal keratocytes survive encapsulation in hydrogels formed by photopolymerization and to compare the cell morphology and matrix production to native keratocytes in tissue. To obtain 3D confocal images of cell morphology and ECM production and organization in the gels. Methods:Primary keratocytes from corneas of enucleated bovine eyes and keratocytes from pass 1 and 3 monolayer cultures were encapsulated in hydrogels made of PEODA, RGD–PEODA, Phos–PEG, CS–PEODA by photopolymerization (12,5 cell/ml and UV light λ= 365nm during 5 minutes) and incubated for 2 weeks and 1 month. The constructs were then incubated with a fluorescent probe to track the living cells. After fixing cells and blocking non–specific background, the constructs were incubated with primary antibodies against collagen I, collagen III, and keratan sulfate proteoglycans for 1 hour and 45 min with the fluorescent secondary antibody. The fluorescent signals were captured by confocal laser scanning microscopy. Results:Keratocytes encapsulated from pass 1 and 3 monolayer cultures were larger than native keratocytes (approximately 20 and 10µm respectively). Cells adopted mainly a round cell shape, except in RGD–PEODA polymer in which cells were more elongated. Although the dentritic morphology of native keratocytes was lost after encapsulation, cells continued living and secreting extracellular matrix components into the gels as in native tissue; the 3D scaffolds also helped cells from pass 1 to maintain collagen I and keratan sulfate proteoglycan production. Collagen III production increased in constructs containing passage 3 monolayer keratocytes that had been cultured for 2 weeks. The confocal images showed the secreted matrix components remained in the pericellular region and did not diffuse throughout the gels. Conclusions:Photopolymerization could be a good method to obtain 3D scaffolds for keratocytes because they survive encapsulation and produce stromal ECM components in photopolymerizing hydrogels. The hydrogels provide an environment that promotes maintenance of keratocyte phenotype. RGD peptide improves polymer adhesiveness, promoting cell attachment. The localization of ECM only around cells indicates that there is not enough space in the gels for their diffusion. Thus, modulating the polymer porosities will allow communication between keratocytes and could improve ECM production and distribution.
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