title: Keratin-chitosan membranes as scaffold for tissue engineering of human cornea.
abstract: 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.
  
  
  
  Keratin-chitosan membranes were prepared as previously described by Tanabe et al., 2002. Briefly, 7.15 mg/cm2 of keratin dialysate was mixed with 10 wt% chitosan solution and 20 wt% glycerol. The solution was cast into a silicone mold and dried at 50ºC 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Eye Research Foundation - Instituto Oftalmológico Fernández-Vega, and University of Oviedo, Oviedo, Spain. natalia.vazquez@fio.as.
authors.forename: Natalia
authors.initials: N
authors.lastname: Vázquez
authors.affiliation: Eye Research Foundation - Instituto Oftalmológico Fernández-Vega, and University of Oviedo, Oviedo, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Chacón
authors.affiliation: Eye Research Foundation - Instituto Oftalmológico Fernández-Vega, University of Oviedo, and U714 Centre for Biomedical Network Research on Rare Diseases (CIBERER), Oviedo, Spain.
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Meana
authors.affiliation: Transplant and Cell Therapy Unit - Hospital Universitario Central de Asturias, Oviedo, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Menéndez-Menéndez
authors.affiliation: Transplant and Cell Therapy Unit - Hospital Universitario Central de Asturias, Oviedo, Spain.
authors.forename: Amaia
authors.initials: A
authors.lastname: Ferrero-Gutierrez
authors.affiliation: Prodintec Foundation, Gijón, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Cereijo-Martín
authors.affiliation: Eye Research Foundation - Instituto Oftalmológico Fernández-Vega, Oviedo, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Naveiras
authors.affiliation: Eye Research Foundation - Instituto Oftalmológico Fernández-Vega, and 3University of Oviedo, Oviedo, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Merayo-Lloves
mesh_terms: D001672:Biocompatible Materials; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D049109:Cell Proliferation; D002478:Cells, Cultured; D048271:Chitosan / Q000737:chemistry*; D003315:Cornea / Q000166:cytology / Q000378:metabolism; D003319:Corneal Stroma / Q000166:cytology / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000737:chemistry* / Q000378:metabolism; D008422:Materials Testing; D008567:Membranes, Artificial; D008855:Microscopy, Electron, Scanning; D008858:Microscopy, Phase-Contrast; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry*; D014746:Vimentin / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; D015820:Cadherins; D008567:Membranes, Artificial; D014746:Vimentin; D007633:Keratins; D048271:Chitosan; D000254:Sodium-Potassium-Exchanging ATPase