Comparison of Cultivated Limbal Epithelial Sheets With and Without Substrates

Abstract

Abstract: : Purpose: To study histological differences between cultivated limbal epithelial sheets with and without substrates. Efficacy of transplantation of the epithelial sheets to rabbit corneas was also examined. Methods: Rabbit limbal epithelial cells were cultivated on human amniotic membrane (AM group) or fibrin–coated dishes (fibrin group) with mytomicin C–treated 3T3 feeder cells. In the fibrin group, the epithelial sheet was dislodged from the underlying plastic dishes by discontinuing aprotinin, a proteinase inhibitor in the culture medium. Limbal epithelial sheets were subjected to histological examinations. Expression of cornea–specific keratin (AE5), p63, and basement membrane markers including laminin and type IV collagen were examined by immunohistochemistry. Transplantation of AM and fibrin epithelial sheets was studied using a rabbit limbal deficiency model (n=3 in each group). Results: Both types of epithelial sheets demonstrated stratified epithelial cells with positive staining for AE5 and p63. While the cell sheet in the AM group showed positive staining for basement membrane markers between the basal cells and AM, there were no such staining in the fibrin group, suggesting that the epithelial sheet in the latter group detached immediately beneath the basal epithelium. Both types of epithelial sheets attached well on the rabbit corneas after mechanically removing total corneal and limbal epithelium. The corneas that had sham surgery showed delayed epithelialization with conjunctival invasion. While suturing with 10–0 nylon was needed in the AM group, no sutures were necessary in the fibrin group. The corneas in the fibrin group had complete epithelialization and clear visibility of the iris compared with the AM group by 1 week following surgery. Some corneas in the AM group had localized epithelial defects associated with loose attachment of the sheet to the underlying stroma that persisted for over a week. Histological examination demonstrated that the epithelium remained well organized in the both groups. Subconjunctival inflammation was more intense in the AM group than the fibrin group. Conclusions: Both types of the cultivated limbal epithelial sheet showed histological integrity and efficacy in the rabbit model. Sheets without a substrate may be superior to those with substrates in terms of clarity and attachment to the corneal stroma.

Keywords: cornea: epithelium • regeneration • transplantation