Resumen de Keratin-chitosan membranes as scaffold for tissue engineering of human cornea
Natalia Arruti Vázquez, Manuel Chacón, Alvaro Meana Infiesta, Yolanda Menendez Menendez, Amaia Ferrero Gutierrez, David Cereijo Martín, Miguel Naveiras, Jesús Merayo Lloves
Purpose: To study the attachment and growth of human corneal cells on keratin-chitosan membranes. The end goal is to develop a bioengineered cornea based on this material. Methods: Keratin-chitosan membranes were prepared as previously described by Tanabe et al., 2002. Briefly, 7.15 mg/cm2 of keratin dialysate was mixed with 10wt% chitosan solution and 20 wt% glycerol. The solution was cast into a silicone mold and dried at 50°C for 36 hours. Eyes were attained from a local eye bank after penetrant-keratoplastic surgery. Human epithelial, stromal and endothelial cells were obtained of the limbal, stromal and endothelial regions. Cells were cultured on keratin-chitosan membranes, as well as on plastic dishes as controls. When cultured cells reached confluence, they were fixed, incubated with primary antibodies (E-cadherin, cytokeratin high molecular weight (CK), vimentin and Na+/K+ ATPase) and visualized by indirect immunocytochemistry. Results: Epithelial, stromal and endothelial cells were able to attach and grow on keratin-chitosan membranes. All the cells maintained their morphology and cellular markers, both in the membrane and on the culture plate. Epithelial cells stained positively for CK and E-cadherin. A positive vimentin stain was observed in all stromal cells, while endothelial cells were positive for vimentin and Na+/K+ ATPase, but negative for E-cadherin. Conclusions: Keratin-chitosan membranes have been 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
