Sequential development of intercellular junctions in bioengineered human corneas.

We have carried out a sequential study of intercellular junction formation and differentiation on human corneal substitutes consisting of an artificial corneal stroma and a corneal epithelium, developed by tissue engineering. To generate these artificial human corneas, we developed a corneal stroma substitute, using fibrin and agarose scaffolds with human keratocytes immersed within, then cultured the human corneal epithelium on top. Electron microscopy and immunofluorescence analyses revealed that artificial corneas with one or two epithelial cell layers did not show any formation of intercellular junctions. In contrast, several types of cell-cell junction, especially desmosomes, were found in multilayered mature corneal substitutes. Concomitantly, the expression of genes encoding for plakoglobin 3 (PKG3), desmoglein 3 (DSG3) and desmoplakin (DSP), zonula occludens 1 (ZO-1) and 2 (ZO-2) and connexin 37 (Cx37) was higher in multilayered artificial corneas than in immature artificial corneas, as shown by both microarray and immunofluorescence. Although expression of ZO-1, ZO-2 and Cx37 proteins was homogeneous, PKG3, DSG3 and DSP expression was restricted to the most apical cell layers in artificial corneas submerged in culture medium at all times, whereas expression was higher in intermediate cell layers, similar to normal human control corneas, when corneal substitutes are submitted to air-liquid culture techniques. These results suggest that cultured corneal substitutes submitted to air-liquid culture technique tend to form a well-developed epithelium that is very similar to the epithelium of human native corneas, suggesting that these artificial corneas could eventually be used for clinical or in vitro purposes.