title: Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.
abstract: OBJECTIVE
  The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach.
  
  
  METHODS
  Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed.
  
  
  RESULTS
  Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1.
  
  
  CONCLUSIONS
  This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.
authors.affiliation: Centre LOEX de l'Université Laval, Génie tissulaire et régénération: LOEX - Centre de recherche FRSQ du Centre hospitalier affilié universitaire de Québec, and Département d’Oto-rhino-laryngologie et d’Ophtalmologie, Université Laval, Québec, QC, Canada.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Proulx
authors.forename: Jeanne
authors.initials: J
authors.lastname: d'Arc Uwamaliya
authors.forename: Patrick
authors.initials: P
authors.lastname: Carrier
authors.forename: Alexandre
authors.initials: A
authors.lastname: Deschambeault
authors.forename: Caroline
authors.initials: C
authors.lastname: Audet
authors.forename: Claude J
authors.initials: CJ
authors.lastname: Giasson
authors.forename: Sylvain L
authors.initials: SL
authors.lastname: Guérin
authors.forename: François A
authors.initials: FA
authors.lastname: Auger
authors.forename: Lucie
authors.initials: L
authors.lastname: Germain
mesh_terms: D000328:Adult; D000369:Aged, 80 and over; D001485:Basement Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D024042:Collagen Type I / Q000378:metabolism; D003315:Cornea / Q000166:cytology* / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology; D019573:Epithelium, Corneal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007223:Infant; D007633:Keratins / Q000378:metabolism; D008875:Middle Aged; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D024042:Collagen Type I; D007633:Keratins; D000254:Sodium-Potassium-Exchanging ATPase