Abstract
Purpose
To evaluate the results of cultivated limbal epithelial and oral mucosal epithelial transplantation (CLET and COMET) in limbal stem cell deficiency (LSCD)-induced rabbit model.
Materials and methods
Six New Zealand white rabbits were divided into two groups of three rabbits each. Limbal tissue was harvested from the first group, and oral mucosal biopsy was obtained from the second group. The tissues were cultured using an explant technique with amniotic membrane as a substrate and co-culture with the 3T3 fibroblast and air-lifting method. The right eye of each rabbit was induced to have LSCD using alkali burns. After three weeks, the LSCD-induced rabbit eyes were transplanted with the cultivated limbal and oral mucosal epithelial sheet in the first and second group, respectively. The transplanted eye was evaluated weekly post-operation. After 2 months, all transplanted eyes were enucleated and the epithelial morphology and phenotype of ocular surfaces were studied and compared with normal corneal and oral mucosal tissue.
Results
At 2-month post-transplantation, the eyes of four animals recovered with corneal transparency, one partially recovered, and one failed. The histology of the majority of transplanted eyes was stratified layers of corneal epithelia similar to normal rabbit cornea with some different findings such as goblet cells in the limbal region. Corneal epithelial thickening and stromal vascularization in two animals were observed. Phenotypic characterization of transplanted eyes showed a similar pattern of marker expression with the absence of p63 expression in the limbal or corneal epithelium in the COMET group.
Conclusions
The histology and phenotype of transplanted eyes after CLET and COMET were most likely to have similar characteristics as a normal healthy rabbit eye even though the COMET eyes have some inferior characteristics to the CLET eyes.
Similar content being viewed by others
References
Kenyon KR, Tseng SC (1989) Limbal autograft transplantation for ocular surface disorders. Ophthalmology 96:709–722
Dua HS (1998) The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol 82:1407–1411
Dua HS, Azuara-Blanco A (1999) Allo-limbal transplantation in patients with limbal stem cell deficiency. Br J Ophthalmol 83:414–419
Dua HS, Azuara-Blanco A (2000) Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency. Br J Ophthalmol 84:273–278
Tsubota K, Shimmura S, Shinozaki N, Holland EJ, Shimazaki J (2002) Clinical application of living-related conjunctival-limbal allograft. Am J Ophthalmol 133:134–135
Rao SK, Rajagopal R, Sitalakshmi G, Padmanabhan P (1999) Limbal allografting from related live donors for corneal surface reconstruction. Ophthalmology 106:822–828
Holland EJ, Schwartz GS (2000) Changing concepts in the management of severe ocular surface disease over twenty-five years. Cornea 19:688–698
Holland EJ, Djalilian AR, Schwartz GS (2003) Management of aniridic keratopathy with keratolimbal allograft: a limbal stem cell transplantation technique. Ophthalmology 110:125–130
Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M (1997) Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349:990–993
Tsai RJ, Li LM, Chen JK (2000) Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 343:86–93
Koizumi N, Inatomi T, Suzuki T, Sotozono C, Kinoshita S (2001) Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 108:1569–1574
Rama P, Bonini S, Lambiase A, Golisano O, Paterna P, De Luca M, Pellegrini G (2001) Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 72:1478–1485
Nishida K (2003) Tissue engineering of the cornea. Cornea 22:S28–S34
Liang L, Sheha H, Tseng SC (2009) Long-term outcomes of keratolimbal allograft for total limbal stem cell deficiency using combined immunosuppressive agents and correction of ocular surface deficits. Arch Ophthalmol 127:1428–1434
Ma DH, Kuo MT, Tsai YJ, Chen HC, Chen XL, Wang SF, Li L, Hsiao CH, Lin KK (2009) Transplantation of cultivated oral mucosal epithelial cells for severe corneal burn. Eye 3:1442–1450
Liu J, Sheha H, Fu Y, Giegengack M, Tseng SC (2011) Oral mucosal graft with amniotic membrane transplantation for total limbal stem cell deficiency. Am J Ophthalmol 152:739e1–747e1
Nakamura T, Endo K, Cooper LJ, Fullwood NJ, Tanifuji N, Tsuzuki M, Koizumi N, Inatomi T, Sano Y, Kinoshita S (2003) The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci 44:106–116
Kinoshita S, Koizumi N, Nakamura T (2004) Transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction. Exp Eye Res 78:483–491
Inatomi T, Nakamura T, Koizumi N, Sotozono C, Kinoshita S (2005) Current concepts and challenges in ocular surface reconstruction using cultivated mucosal epithelial transplantation. Cornea 24:S32–S38
Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, Nagai S, Kikuchi A, Maeda N, Watanabe H, Okano T, Tano Y (2004) Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 351:1187–1196
Promprasit D, Bumroongkit K, Tocharus C, Mevatee U, Tananuvat N (2015) Cultivation and phenotypic characterization of rabbit epithelial cells expanded ex vivo from fresh and cryopreserved limbal and oral mucosal explants. Curr Eye Res 40:274–281
Guide for the Care and Use of Laboratory Animals. Washington DC: National Academy of Sciences.; 2011
Avila M, Espana M, Moreno C, Pena C (2001) Reconstruction of ocular surface with heterologous limbal epithelium and amniotic membrane in a rabbit model. Cornea 20:414–420
Ti SE, Anderson D, Touhami A, Kim C, Tseng SC (2002) Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits. Invest Ophthalmol Vis Sci 43:2584–2592
Sekiyama E, Nakamura T, Kawasaki S, Sogabe H, Kinoshita S (2006) Different expression of angiogenesis-related factors between human cultivated corneal and oral epithelial sheets. Exp Eye Res 83:741–746
Kanayama S, Nishida K, Yamato M, Hayashi R, Sugiyama H, Soma T, Maeda N, Okano T, Tano Y (2007) Analysis of angiogenesis induced by cultured corneal and oral mucosal epithelial cell sheets in vitro. Exp Eye Res 85:772–781
Nakamura T, Inatomi T, Cooper LJ, Rigby H, Fullwood NJ, Kinoshita S (2007) Phenotypic investigation of human eyes with transplanted autologous cultivated oral mucosal epithelial sheets for severe ocular surface diseases. Ophthalmology 114:1080–1088
Chen HC, Chen HL, Lai JY, Chen CC, Tsai YJ, Kuo MT, Chu PH, Sun CC, Chen JK, Ma DH (2009) Persistence of transplanted oral mucosal epithelial cells in human cornea. Invest Ophthalmol Vis Sci 50:4660–4668
Davanger M, Evensen A (1971) Role of the pericorneal papillary structure in renewal of corneal epithelium. Nature 229:560–561
Gipson IK (1989) The epithelial basement membrane zone of the limbus. Eye 3:132–140
Shortt AJ, Secker GA, Munro PM, Khaw PT, Tuft SJ, Daniels JT (2007) Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells. Stem Cells 25:1402–1409
Daya SM, Watson A, Sharpe JR, Giledi O, Rowe A, Martin R, James SE (2005) Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction. Ophthalmology 112:470–477
Kawasaki S, Tanioka H, Yamasaki K, Yokoi N, Komuro A, Kinoshita S (2006) Clusters of corneal epithelial cells reside ectopically in human conjunctival epithelium. Invest Ophthalmol Vis Sci 47:1359–1367
Pauklin M, Steuhl KP, Meller D (2009) Characterization of the corneal surface in limbal stem cell deficiency and after transplantation of cultivated limbal epithelium. Ophthalmology 116:1048–1056
Acknowledgements
This study was granted by the Faculty of Medicine Endowment Fund, Faculty of Medicine, Chiang Mai University (Grant Number 25/2555). The authors would like to thank Prof. Nipon Chattipakorn, M.D, PhD. for his kind support in the animal laboratory enabling tissue biopsy and transplantation procedures in the rabbits.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
None.
Rights and permissions
About this article
Cite this article
Tananuvat, N., Bumroongkit, K., Tocharusa, C. et al. Limbal stem cell and oral mucosal epithelial transplantation from ex vivo cultivation in LSCD-induced rabbits: histology and immunologic study of the transplant epithelial sheet. Int Ophthalmol 37, 1289–1298 (2017). https://doi.org/10.1007/s10792-016-0402-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10792-016-0402-5