ARTICLE

Ocular Surface Reconstruction by Cultivated Epithelial Sheet Transplantation Yoshiyuki Satake, MD, PhD,* Takefumi Yamaguchi, MD,* Masatoshi Hirayama, MD, PhD,† Kazunari Higa, DVM, PhD,* Seika Shimazaki-Den, MD, PhD,* Murat Dogru, MD, PhD,* Tetsuya Kawakita, MD, PhD,† Motoko Kawashima, MD,† Shigeto Shimmura, MD,† Kazuo Tsubota, MD, PhD,† and Jun Shimazaki, MD, PhD*

Abstract: Recent advances in ocular surface reconstruction for patients with severe ocular surface diseases have significantly improved the prognosis of patients with vision-impairing corneal abnormalities. The history of cultivated epithelial sheet transplantation is short, and debate on the current approaches for these procedures is continuing. Limbal stem cell transplantation, including conjunctivolimbal autograft and keratolimbal allograft, has brought opportunities for vision improvement. In addition, the use of cultivated limbal epithelial transplantation from both allogeneic and autologous sources has provided further options for immediate postoperative epithelialization of the corneal surface. Finally, cultivated oral mucosal epithelial transplantation, which allows autologous transplantation for patients with bilateral limbal stem cell deficiency, has provided the best overall midterm and long-term results. Its biggest advantages are the absence of rejection reactions and the reduction of postoperative complications associated with steroid therapy. However, a solitary surgical approach is not sufficient for obtaining a good clinical outcome. To maximize the possibility of success using these procedures, it is important to preoperatively enhance aggressive treatment of the ocular surface, especially with factors that facilitate moisture retention. In this review article, we also discuss our clinical experience in relation to these surgical procedures. From the *Department of Ophthalmology, Tokyo Dental College, Chiba, Japan; and †Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan. J. Shimazaki is a consultant to Otsuka Pharmaceutical Co, and has received payment for lecturing from Otsuka Pharmaceutical Co, Santen Pharmaceutical Co, and Alcon Japan. Y. Satake received a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) (KAKENHI: 17791259); a grant from the Advanced and Innovational Research Program in Life Sciences of the Ministry of Education, Culture, Sports, Science, and Technology, Japan; a Grant-in-Aid for scientific research (C) from the Japan Society for the Promotion of Science (KAKENHI: 22591951); an Oral Health Science Center Grant hrc8 from Tokyo Dental College; and a grant from the Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, 2010 to 2012. M. Hirayama received funding from Keio University Research Grants for Life Science and Medicine, Keio University Medical Science Fund. J. Shimazaki received a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The other authors have no funding or conflicts of interest to disclose. Reprints: Yoshiyuki Satake, MD, PhD, Department of Ophthalmology, Tokyo Dental College, Sugano 5-11-13, Ichikawa, Chiba 272-8513, Japan (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins

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Key Words: ocular surface reconstruction, limbal stem cells, epithelial sheet transplantation, keratoplasty (Cornea 2014;33(Suppl):S42–S46)

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ntil the late 1980s, surgical treatment of ocular surface diseases, such as Stevens–Johnson syndrome, ocular cicatricial pemphigoid, and chemical or thermal injuries, was considered a contraindication for surgery because of the extremely poor prognosis of patients treated with conventional keratoplasty.1 The presence of corneal epithelial stem cells in the limbus2,3 and the usefulness of amniotic membrane as a substrate of epithelial cells4 have contributed to the progression of ocular surface reconstruction. Ocular surface reconstruction started with limbal stem cell transplantation (LSCT),5 and has progressed to the transplantation of ex vivo cultivated epithelial sheets owing to advances in tissue engineering in recent years.6 Our department started ocular surface reconstruction with LSCT in the early 1990s, and has expanded its surgical repertoire to the transplantation of ex vivo cultivated epithelial sheets, including cultivated limbal epithelial transplantation (CLET) and cultivated oral mucosal epithelial transplantation (COMET) over the past 15 years (Fig. 1). In recent years, COMET procedures have dominated in terms of number, and numbers of transplantation procedures with carrier-free sheets, especially, have increased.7 In this review, we focus on ocular surface reconstruction procedures based on our clinical experience.

LIMBAL STEM CELL TRANSPLANTATION

LSCT was the first reported procedure for the reconstruction of ocular surface diseases. In 1989, Kenyon and Tseng5 reported the successful transplantation of a conjunctivolimbal autograft (CLAU) obtained from a patient’s healthy eye for the treatment of severe unilateral ocular surface disease. For patients with bilateral diseases, Tsai and Tseng,8 followed by our group,9 reported the transplantation of keratolimbal allograft (KLAL) obtained from donor tissue. Modification of the technique using a preserved human amniotic membrane as a substrate replacement4 and eye drops containing autologous serum for tear replacement10 has provided Cornea  Volume 33, Number 11, Supplement, November 2014

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Ocular Surface Reconstruction

FIGURE 1. Numbers of surgical procedures involving ex vivo cultivated epithelial sheets administered by our department. AM, amniotic membrane.

FIGURE 3. Survival curve analysis of the stable ocular surface in the initial 40 eyes after COMET. Modified and reprinted from Satake et al25 with permission from Elsevier.

a new approach for the treatment of end-stage cicatricial diseases even with a complete loss of stem cells and tears.11 The principal purpose of LSCT is to maintain the stability of reconstructed corneal epithelium on the corneal surface. Ocular surface stability after KLAL can be successfully restored in approximately 50% of cases of bilateral ocular surface disease.12,13 A comparative study of the clinical outcomes between CLAU and KLAL in patients with chemical or thermal injury revealed that the postoperative corneal epithelialization stability of CLAU was significantly better than that of KLAL (Fig. 2) and that a decrease in the incidence of complications, such as glaucoma and repeated LSCT, was observed in CLAU.14 Stability of the ocular surface reconstructed by LSCT allows keratoplasty for visual improvement. Determination of the timing of keratoplasty is also critical. Postoperative corneal epithelialization and corneal clarity were more commonly attained in eyes treated with penetrating keratoplasty several months after LSCT, and numbers of postoperative complications, including endothelial rejection, persistent epithelial defects, glaucoma, and the need for repeat LSCT, were higher for patients treated with simultaneous penetrating keratoplasty.14 The surgical prognosis of ocular surface reconstruction in patients with severe

ocular surface diseases is greatly influenced by preoperative tear function.13 Proper evaluation and management of tears are also key to successful surgical results.

FIGURE 2. Kaplan–Meier survival analysis for corneal epithelialization. Modified and reprinted from Shimazaki et al14 with permission from Elsevier. Ó 2014 Lippincott Williams & Wilkins

CULTIVATED LIMBAL EPITHELIAL TRANSPLANTATION LSCT and amniotic membrane transplantation have substantially improved the surgical prognosis of patients with bilateral ocular surface diseases and increased opportunities for vision improvement in severe ocular surface diseases. However, the long-term success of KLAL remains approximately 50% in patients with bilateral ocular surface diseases.12,13 The most common cause of failure is the lack of proper regeneration of corneal epithelial cells from the transplanted limbal tissue. To overcome this epithelial problem after LSCT, CLET procedures were proposed as an alternative surgical approach. Initial reports using patient limbal tissues (autograft) were encouraging.6,15 Thereafter,

FIGURE 4. Kaplan–Meier analysis of the graft survival rate. Kaplan–Meier analysis revealed that the graft survival rate was significantly better in the carrier-free sheet group than in the amniotic membrane sheet group. AM, amniotic membrane. Modified and reprinted from Hirayama et al27 with permission from the Association for Research in Vision and Ophthalmology. www.corneajrnl.com |

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FIGURE 5. Anterior segment images of eyelid and eyelash abnormalities before (A) and after surgery (B). Without changing the eye drops before and after surgery, reduction of the ocular surface irritation resulted in reduced ocular surface inflammation, improvement of conjunctival hyperemia, and keratinization of corneal surface. Wettability of the ocular surface was also increased with a dramatic improvement in the status of the ocular surface even with eyelid surgery alone.

results of animal experiments and clinical trials using allografts obtained from cadaver eyes or patients living relatives have also been reported.16–18 CLET is expected to allow an immediate postoperative epithelialization of the corneal surface. However, in our experience, an initial epithelialization after allogeneic CLET was observed in 61.5% of eyes, and the final success rate was 46%. Severe cases do not always have a good final outcome.19 Therefore, in our experience, the allogeneic CLET procedure was no better than our previous approach of LSCT and amniotic membrane transplantation. Allogeneic cultivated limbal epithelial cells can become the target of immunological reactions, even when antigen-presenting cells are not present in the cultivated epithelial sheets. We reported that of 37 allogeneic CLET procedures, 3 rejection episodes in 2 eyes with chemical injury were associated with a linear epithelial defect at the host–graft junction, which occurred not only in allogeneic but also in living-related CLET while patients were being treated with systemic cyclosporine. Intensive steroid treatment was effective in controlling such rejections. Although the cause of these rejection reactions is not clear, careful evaluation of the host–graft junction in relation to the occurrence of a linear epithelial rejection defect and prudent management of tapering in relation to the timing and dose of steroids are critical for the control of CLET rejection.20 In autologous CLET, harvesting smaller amounts of limbal tissues is expected to reduce such effects in the donor eyes, which is a major advantage. However, in allogeneic transplantation, proper selection of surgical indications and

consideration of the potential risks of immunological rejection should be taken into account.

CULTIVATED ORAL MUCOSAL EPITHELIAL TRANSPLANTATION A major issue of allogeneic transplantation is the risk of immunological rejection. To overcome these issues, COMET was proposed as an alternative surgical procedure that allows autologous transplantation for bilateral ocular surface diseases with limbal stem cell deficiency.21–23 Favorable results were obtained even as midterm or long-term outcomes of COMET procedures (Fig. 3).24–26 The main causes of poor prognosis were persistent epithelial failure and fibrovascular tissue invasion onto the corneal surface. Persistent epithelial failure developed in the early postoperative period and was strongly correlated with preoperative persistent epithelial defects. However, gradual fibrovascular tissue invasion on the corneal surface was observed particularly in patients with ocular cicatricial pemphigoid. COMET was also effective for the reconstruction of a functional fornix or release of symblepharon. COMET, an autologous transplantation procedure, allows a reduction in instillation frequency and earlier cessation of topical steroid use for postoperative inflammation. Therefore, reduction of the postoperative complications of topical steroid therapy can be regarded as an advantage of this procedure. Postoperative complications include stromal melting or perforation after persistent epithelial defects, infectious keratitis, glaucoma, and neovascularization.25

FIGURE 6. Anterior segment photographs showing poor (A) and good (B) compliance of eye drop administration. The quality of eye drop compliance dramatically affects status of the ocular surface.

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The use of amniotic membranes as a substrate is another possibility as a standardized technique to prepare epithelial sheets. However, transplantation of carrier-free epithelial cell sheets, including temperature-responsive cellculture wells23 and enzymatic substrate degradation,7 is also clinically available. We used carrier-free epithelial sheets obtained by enzymatic substrate degradation and amniotic membrane-based sheets. A comparative immunohistochemical analysis by our group suggested that the amniotic membrane maintains epithelial cells in a less differentiated state.7 We also compared the clinical outcomes of COMET with these 2 types of sheets in retrospective and case–control studies.27 The graft survival rate in the carrier-free sheet group was significantly better than that in the amniotic membrane sheet group (Fig. 4). The postoperative mean best corrected visual acuity in the carrier-free sheet group was significantly better than in the amniotic membrane sheet group at all time points after COMET. Carrier-free epithelial sheets are in direct contact with the corneal stroma without carrier interference. This epithelial–stromal interaction may have resulted in better graft survival and better best corrected visual acuity in the carrier-free sheet group than in the amniotic membrane sheet group. Neovascularization underneath the transplanted epithelial sheets can cause visual disturbances. The incidence of postoperative neovascularization was decreased significantly in the carrier-free sheet group. Although previous reports suggested that oral mucosal epithelial cells secrete angiogenic factors that induce neovascularization underneath the transplanted epithelial sheets,28,29 keratocytes in the corneal stroma may limit peripheral vascularization by producing antiangiogenic factors.30 The space reduction caused by direct adhesion of the epithelial sheets may also influence postoperative neovascularization. Another major advantage of this approach is the surgical technique itself, which does not require the use of sutures for donor fixation. In addition to the aforementioned cultivation methods, the progression of tissue engineering enabled the creation of epithelial sheets without using xenogenic materials such as 3T3 feeder cells.31

KERATOPLASTY FOR VISUAL RECOVERY In patients with stromal opacity, keratoplasty for visual improvement is necessary. A stable ocular surface after ocular surface reconstruction procedures may allow keratoplasty for visual recovery. Based on our experience, it seems to be safer to perform ocular surface reconstruction first, and then keratoplasty several months later, to reduce postoperative complications.14 In cases maintaining their endothelial functions, lamellar keratoplasty is safer than penetrating keratoplasty when postoperative complications are considered. Keratoprosthesis is an alternative procedure to conventional corneal transplantation. The Boston keratoprosthesis (Boston KPro) type 1 has an indication for a broad range of ocular surface disorders.32–34 In most severe cases with keratinizing dry eyes that cannot be treated with the ocular surface reconstructions described above, the Boston KPro type 2 device32–34 and osteo-odonto-keratoprosthesis35,36 represent surgical alternatives. Ó 2014 Lippincott Williams & Wilkins

Ocular Surface Reconstruction

DISCUSSION LSCT has increased opportunities for visual improvement in severe ocular surface diseases considered a contraindication for keratoplasty. During the past 2 decades, the procedures of ocular surface reconstruction have progressed with fewer disadvantages associated with each new surgical procedure. The surgical procedures of ocular surface reconstruction at our institution have shifted from LSCT to cultured epithelial sheet transplantation. However, a solitary surgical approach is not sufficient for a good clinical outcome. From our clinical experience in .200 CLET and COMET procedures, preoperative ocular surface status is strongly related to postoperative corneal surface stability.8,16 Nakamura et al26 have also reported that clinical outcomes of COMET for eyes with conjunctivalization of the corneal surface were fairly favorable. This report does not conflict with our previous report.25 To obtain a better clinical prognosis following ocular surface reconstructions, aggressive treatment of the prognostic factors affecting postoperative ocular surface status should be considered preoperatively. In particular, moisture retention of the ocular surface is critical for both the promotion of epithelialization and its stability, and for creating an antiinflammatory environment at the ocular surface, which leads to stability of ocular surface epithelium. In cases with tear deficiency, artificial-tear eye drops, autoserum eye drops, and punctual occlusion are the firstchoice therapies. The surgical correction of lid abnormalities also improves tear dynamics and moisture retention of the ocular surface and protection of the ocular surface from microtrauma (Fig. 5). Equally important is compliance with eye drop administration, which dramatically influences the status of the ocular surface (Fig. 6). Thus, improvement of the microenvironment at the ocular surface before surgery and its maintenance are critical to obtain a better prognosis.

ACKNOWLEDGMENTS The authors thank M. Aiba, F. Morito, and S. Yamaguchi for assisting with the culture procedures. REFERENCES 1. Buxton JN, Buxton DF, Westphalen JA. Indications and contraindications. In: Brightbill FS, ed. Corneal Surgery: Theory, Technique, and Tissue. St Louis, MO: Mosby; 1993:77–88. 2. Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;103:49–62. 3. Cotsarelis G, Cheng SZ, Dong G, et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell. 1989;57:201–209. 4. Kim JC, Tseng SC. Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea. 1995;14:473–484. 5. Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology. 1989;96:709–722; discussion 722–723. 6. Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349:990–993. 7. Higa K, Shimmura S, Kato N, et al. Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier. Invest Ophthalmol Vis Sci. 2007;48: 597–604.

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8. Tsai RJ, Tseng SC. Human allograft limbal transplantation for corneal surface reconstruction. Cornea. 1994;13:389–400. 9. Tsubota K, Toda I, Saito H, et al. Reconstruction of the corneal epithelium by limbal allograft transplantation for severe ocular surface disorders. Ophthalmology. 1995;102:1486–1496. 10. Tsubota K, Satake Y, Ohyama M, et al. Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens– Johnson syndrome. Am J Ophthalmol. 1996;122:38–52. 11. Tsubota K, Satake Y, Shimazaki J. Treatment of severe dry eye. Lancet. 1996;348:123. 12. Tsubota K, Satake Y, Kaido M, et al. Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N Engl J Med. 1999;340:1697–1703. 13. Shimazaki J, Shimmura S, Fujishima H, et al. Association of preoperative tear function with surgical outcome in severe Stevens–Johnson syndrome. Ophthalmology. 2000;107:1518–1523. 14. Shimazaki J, Shimmura S, Tsubota K. Donor source affects the outcome of ocular surface reconstruction in chemical or thermal burns of the cornea. Ophthalmology. 2004;111:38–44. 15. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2000;343:86–93. 16. Koizumi N, Inatomi T, Quantock AJ, et al. Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea. 2000;19:65–71. 17. Schwab IR. Cultured corneal epithelia for ocular surface disease. Trans Am Ophthalmol Soc. 1999;97:891–986. 18. He YG, Alizadeh H, Kinoshita K, et al. Experimental transplantation of cultured human limbal and amniotic epithelial cells onto the corneal surface. Cornea. 1999;18:570–579. 19. Shimazaki J, Aiba M, Goto E, et al. Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders. Ophthalmology. 2002;109:1285–1290. 20. Satake Y, Dogru M, Yamaguchi T, et al. Immunological rejection following allogeneic cultivated limbal epithelial transplantation. JAMA Ophthalmol. 2013;131:920–922. 21. Nakamura T, Endo K, Cooper LJ, et al. The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci. 2003;44:106–116. 22. Nakamura T, Inatomi T, Sotozono C, et al. Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders. Br J Ophthalmol. 2004;88:1280–1284.

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23. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med. 2004;351:1187–1196. 24. Inatomi T, Nakamura T, Koizumi N, et al. Midterm results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial transplantation. Am J Ophthalmol. 2006;141:267–275. 25. Satake Y, Higa K, Tsubota K, et al. Long-term outcome of cultivated oral mucosal epithelial sheet transplantation in treatment of total limbal stem cell deficiency. Ophthalmology. 2011;118:1524–1530. 26. Nakamura T, Takeda K, Inatomi T, et al. Long-term results of autologous cultivated oral mucosal epithelial transplantation in the scar phase of severe ocular surface disorders. Br J Ophthalmol. 2011;95: 942–946. 27. Hirayama M, Satake Y, Higa K, et al. Transplantation of cultivated oral mucosal epithelium prepared in fibrin-coated culture dishes. Invest Ophthalmol Vis Sci. 2012;53:1602–1609. 28. Sekiyama E, Nakamura T, Kawasaki S, et al. Different expression of angiogenesis-related factors between human cultivated corneal and oral epithelial sheets. Exp Eye Res. 2006;83:741–746. 29. Kanayama S, Nishida K, Yamato M, et al. Analysis of angiogenesis induced by cultured corneal and oral mucosal epithelial cell sheets in vitro. Exp Eye Res. 2007;85:772–781. 30. Hiscott P, Sorokin L, Nagy ZZ, et al. Keratocytes produce thrombospondin 1: evidence for cell phenotype-associated synthesis. Exp Cell Res. 1996;226:140–146. 31. Kolli S, Ahmad S, Mudhar HS, et al. Successful application of ex vivo expanded human autologous oral mucosal epithelium for the treatment of total bilateral limbal stem cell deficiency. Stem Cells. 2014;32: 2135–2146. 32. Dohlman CH, Schneider HA, Doane MG. Prosthokeratoplasty. Am J Ophthalmol. 1974;77:694–700. 33. Yaghouti F, Nouri M, Abad JC, et al. Keratoprosthesis: preoperative prognostic categories. Cornea. 2001;20:19–23. 34. Sayegh RR, Ang LP, Foster CS, et al. The Boston keratoprosthesis in Stevens–Johnson syndrome. Am J Ophthalmol. 2008;145:438–444. 35. Strampelli B. Osteo-odontokeratoprosthesis. Ann Ottalmol Clin Ocul. 1963;89:1039–1044. 36. Falcinelli G, Falsini B, Taloni M, et al. Modified osteo-odontokeratoprosthesis for treatment of corneal blindness: long-term anatomical and functional outcomes in 181 cases. Arch Ophthalmol. 2005;123: 1319–1329.

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Ocular surface reconstruction by cultivated epithelial sheet transplantation.

Recent advances in ocular surface reconstruction for patients with severe ocular surface diseases have significantly improved the prognosis of patient...
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