Long-term result of autologous cultivated oral mucosal epithelial transplantation for severe ocular surface disease

Abstract

The present study aimed to investigate the clinical outcomes of autologous cultivated oral mucosal epithelial transplantation (COMET) on human amniotic membrane (AM) for corneal limbal stem cell deficiency (LSCD). In this prospective, noncomparative case series, 20 eyes (18 patients) with bilateral severe ocular surface disease were chosen to undergo COMET on human AM. The primary outcome was clinical success, and the secondary outcomes were the best-corrected visual acuity difference, corneal opacification, symblepharon formation, and complications. The mean patient age was 48.2 ± 15.5 years. The mean follow-up time was 31.9 ± 12.1 months (range 8–50 months). All except one eye exhibited complete epithelialization within the first postoperative week. A successful clinical outcome, defined as a stable ocular surface without epithelial defects, a clear cornea without fibrovascular tissue invasion at the pupillary area, and no or mild ocular surface inflammation, was obtained in 15 of 20 eyes (75 %). The clinical success rate at 1 year was 79.3 %, and that at 4 years (end of follow-up) was 70.5 %. Fourteen of 20 (70 %) eyes exhibited improvement in visual acuity after COMET, and some required subsequent cataract surgery (2 eyes), penetrating keratoplasty (3 eyes), or keratoprosthesis implantation (1 eye). Preoperative symblepharon was eliminated in most eyes (8 of 13, 61.5 %) after COMET combined with eyelid reconstruction when needed. The only complication was corneal perforation (1 eye) induced by a severe eyelid abnormality; treatment with a tectonic corneal graft was successful. COMET can successfully restore ocular surface damage in most eyes with corneal LSCD.

Appendix: Reverse transcription–polymerase chain reaction (RT-PCR) (Table 3)

RT

Total RNA from cultured epithelium was used as a template for cDNA synthesis by reverse transcription using a SuperScript^® III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). The RNA template (500 ng) was mixed with 1 µl of 50-µM oligo (dT)₁₅ and 1 µl of 10-mM dNTP mix, and nuclease-free water was then added to a final volume of 10 µl. The mixture was incubated at 65°C for 5 min and then placed on ice for 1 min. The cDNA synthesis mix (2 µl of 10× RT buffer, 4 µl of 25 mM MgCl₂, 1 µl of 0.1 M DTT, 1 µl of 40-unit/μl RNaseOUT™, and 1 μl of 200-unit/μl SuperScript™ III RT) was gently added, mixed, and incubated at 50°C for 50 min and then at 85°C for 5 min to inactivate the enzyme activity. The cDNA was stored at −20 °C until PCR amplification.

PCR

The cDNA used as a template for each PCR amplification was amplified using reagents supplied in the Taq polymerase kit (Promega, Fitchburg, WI, USA). The PCR amplification reaction was performed using 30 cycles at 95 °C for 1 min, 53 °C for 1 min, and 72 °C for 3 min, with a final extension step of 72 °C for 10 min. The amplification cycle was performed in a thermal cycler (PE Applied Biosystems, Inc., Foster City, CA, USA). The PCR products were analyzed by 1.5 % DNA gel electrophoresis.

Agarose gel electrophoresis

The PCR products were mixed with 6X DNA loading buffer (10 µl of PCR product per 2 µl of 6× loading buffer). The band intensity was determined by electrophoresis using a 2 % agarose gel and visualized with a UV transilluminator after ethidium bromide staining.