HISTOLOGY AND HISTOPATHOLOGY

Cellular and Molecular Biology

 

Keratin-chitosan membranes as scaffold for tissue engineering of human cornea

Natalia Vázquez1,3,*, Manuel Chacón1,3,*, Álvaro Meana1,3,4, Yolanda Menéndez-Menéndez2, Amaia Ferrero-Gutierrez2, David Cereijo-Martín5, Miguel Naveiras1 and Jesús Merayo-Lloves1,3

1Eye Research Foundation - Instituto Oftalmológico Fernández-Vega, Oviedo, 2Transplant and Cell Therapy Unit - Hospital Universitario Central de Asturias, Oviedo, 3University of Oviedo, Oviedo, 4U714 Centre for Biomedical Network Research on Rare Diseases (CIBERER), Oviedo and 5Prodintec Foundation, Gijón, Spain
* These authors have contributed equally to this work

Offprint requests to: Natalia Vázquez, Eye Research Foundation, Instituto Oftalmológico Fernández-Vega, Avda. Dres. Fernández-Vega 34, 33012 Oviedo, Asturias, Spain. e-mail: natalia.vazquez@fio.as


Summary. 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. Histol Histopathol 30, 813-821 (2015)

Key words: Keratin-chitosan scaffold, Human limbal epithelial cells, Human corneal stromal cells, Human corneal endothelial cells

DOI: 10.14670/HH-11-585