Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.

PURPOSE: 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.

Transplantation of a tissue-engineered corneal endothelium reconstructed on a devitalized carrier in the feline model.

PURPOSE: To evaluate the functional outcome of tissue-engineered corneal endothelium reconstructed on a devitalized carrier and transplanted in the living feline model. METHODS: Eighteen healthy adult cats underwent full-thickness corneal transplantation. In 11 animals, the donor cornea was reconstructed from cultured allogeneic feline corneal endothelial cells seeded on the denuded Descemet's membrane of a devitalized human cornea. The reconstructed corneal endothelium was cultured for 2 weeks before transplantation. Five control animals received autologous (n = 1), allogeneic (n = 3), or human xenogeneic (n = 1) native cornea. Two other control animals were grafted with the devitalized carrier only (no cells). Animals were observed daily by slit lamp until euthanatization on day 7. Postmortem analysis included optical coherence tomography (OCT), alizarin red staining, histology, fluorescence microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). RESULTS: Nine of the 11 reconstructed corneal endothelial grafts and all five native (autologous, allogeneic, xenogeneic) control grafts were clear and thin 7 days after grafting. In contrast, the two control grafts consisting of the carrier only (without endothelium) remained thick and opaque. Alizarin red staining, histology, SEM, and TEM showed that the transplanted reconstructed endothelium maintained a normal morphology and ultrastructure and expressed the function-related proteins Na(+)/K(+)-ATPase alpha1, Na(+)/HCO(3), and ZO-1. CONCLUSIONS: This study provides evidence for the short-term (7-day) anatomic and functional success of corneal transplantation with a tissue-engineered corneal endothelium reconstructed on a devitalized carrier.