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ALVARO MEANA, Natalia Vázquez, Manuel Chacón, Yolanda Menéndez-Menéndez, Amaia Ferrero-Gutierrez, Jesus Merayo-Lloves; Keratin-chitosan membranes as scaffold for tissue engineering of human cornea. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1399.
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© ARVO (1962-2015); The Authors (2016-present)
To study the attachment and growth of epithelial, stromal and endothelial cells of human cornea on keratin-chitosan membranes. The end goal is to develop a bioengineered cornea based on this material.
1) Cells. Eyes were attained from a local eye bank after penetrant-keratoplastic surgery, and cells were taken from corneoscleral-ring tissue. Epithelial, stromal and endothelial cells were obtained from explants, 2-3 mm in diameter, of the limbal, stroma and descemet/endothelial regions. These cells were cultured in different media onto keratin-chitosan membranes, as well as on a plastic culture dish as control group. Cultures were examined by phase contrast microscopy. 2) Membranes. Keratin-chitosan membranes were prepared as previously described in Tanabe et. al., 2002. Briefly, 7,15 mg/cm2 of keratin dialysate was diluted in three volumes of glacial acetic acid, which was then mixed with 10 p/p chitosan solution (2,5mg/ml in 75% acetic acid solution) and 20 p/p glycerol. The solution was cast into a silicone mold and dried at 50 Celsius degrees for 36 hours. 3) Histological methods. When cultured cells reached confluence, they were fixed with 4% paraformaldehyde, permeabilized with Triton X-100 and incubated at 4 Celsius degrees overnight with primary antibodies (E-cadherin, Cytokeratin High Molecular Weight (CK), vimentin and N-cadherin). Immunolabeled cells were visualized by indirect immunocytochemistry.
Epithelial, stromal and endothelial cells were able to attach and grow onto keratin-chitosan membranes. All the cells maintained their morphology and cellular markers, both in the membrane and on the culture plate. Epithelial cells exhibited a positive stain to CK and E-cadherin. A positive vimentin stain was observed in all stromal cells, while in endothelial cells, a positive stain to vimentin and N-cadherin, but negative to E-cadherin was found.
Keratin-chitosan membranes have shown to be a good scaffold for culturing epithelial, stromal and endothelial corneal cells; therefore, future applications of keratin-chitosan membranes may be developed for reconstruction of the cornea.
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