Abstract
Purpose: :
Transplantation of ex vivo cultivated epithelial cell sheets has brought a new era for ocular surface reconstruction. Although histological studies showed restoration of cornea-type ocular surface epithelia, functional property of the sheets has not been investigated. The aim of the study is to investigate the barrier function and expression of tight junction (TJ)-related proteins in the cultivated epithelial sheets.
Methods: :
Cultivated limbal epithelial sheets (CLES) and oral mucosal epithelial sheets (COMES) were subjected to immunohistochemistry for ZO-1, occludin, connexin 43, and E-cadherin, and they were compared with those in corneal and oral mucosal epithelium. Transmission electron microscopy (TEM) was performed to study cell-cell junctional complex. TJ integrity was assayed by a surface biotinylation method using EZ-Link Sulfo-NHS-LC-Biotin (LC biotin). After applying LC biotin on the epithelial side of the sheets, barrier function was evaluated by intercellular penetration of the tracer. Transepithelial electrical resistance (TER) was measured to study barrier function using an epithelial voltmeter (EVOM, World Precision Instruments). Rabbit limbal dysfunction models were created by superficial keratectomy involving limbal area. Two weeks after the rabbits had CLES transplantation, and barrier function was assessed by fluorophotometry (DR-1, Kowa Co.) before and after transplantation (n=4).
Results: :
ZO-1 and occludin was localized in the apical area of CLES and corneal epithelium. They were diffusely distributed in the oral mucosal epithelium, but were localized in the apical area in COMES. Connexin 43 and E-cadherin were distributed to cell-cell junction in all layers of each sample. TJ-like structure was observed by TEM in both CLES and COMES. While COMES showed excellent epithelial barrier function by LC-biotin assay, CLES showed variable integrity. Electrical resistance was greater in COMES than CLES (11.8±5.8Ω vs. 198.4±73.9Ω). Corneal surface was covered by conjunctival epithelium with neovascularization after superficial keratectomy in rabbits, which showed marked regression after CLES. Fluorometric values were 268.4±179.1 ng/ml and 2237.8±11575 ng/ml before and 2 weeks after keratectomy, and 657.0±233.7 ng/ml and 965.0±583.5 ng/ml 5 and 10 weeks after CLES, respectively.
Conclusions: :
Both CLES and COMES showed expression of TJ-related proteins. Reconstruction of barrier function was observed in both CLES and COMES, with the latter seemed to have better integrity. Evaluation of barrier function may be useful for non-invasive assessment of cultivated epithelial sheets.
Keywords: cell adhesions/cell junctions • cornea: epithelium • transplantation