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REVIEW URRENT C OPINION

Simple limbal epithelial transplantation Virender S. Sangwan and John A.H. Sharp

Purpose of review Simple limbal epithelial transplant (SLET) is a technique for addressing limbal stem cell deficiency. Limbal tissue from a donor eye, typically the patient’s fellow healthy eye, is transplanted onto an amniotic membrane attached to the surface of the diseased eye. SLET was developed to address limitations of other techniques. Specifically, the technical difficulty of ex-vivo expansion of cells required in some techniques and the larger amount of valuable limbal tissue harvested in techniques not relying on ex-vivo expansion. We described how the provision of this procedure adds to the armamentarium of techniques available to treat some of the many thousands of uniocular corneal blind around the world. Recent findings A total of 125 patients from a recent series from our centre and 68 from a multicentre study provide evidence for efficacy mainly in cases of unilateral corneal burn. Results were comparable to other stem cell techniques described in other papers. Numerous small case reports describe the use of SLET in other contexts including ocular surface squamous neoplasia and pterygium excision. Summary SLET offers a cheaper and perhaps safer alternative to other techniques. Further evaluation of clinical success against its most similar analogues of conjunctival limbal autograft and cultivated limbal autograft is required. Keywords limbal stem cell deficiency, simple limbal epithelial transplant, SLET

INTRODUCTION

INDICATIONS AND CONTRAINDICATIONS

The autologous technique of conjunctival–limbal autograft (CLAU) allows repair of damaged ocular surfaces in uniocular cases of limbal stem deficiency. However, a relatively large amount of tissue is required from the donor eye. This risks iatrogenic stem cell deficiency [1]. Cultivated limbal epithelial transplant (CLET) was first described in 1997 [2]. It addresses this problem by allowing smaller amounts of tissue to be taken. This is expanded as a sheet of cells ex vivo. However, the technique is relatively expensive because of the facilities required for cell culture. It also requires two surgeries, one for harvesting and one for seeding. Simple limbal epithelial transplant (SLET) addresses the two problems of large donor contribution requirement and expense by taking a smaller amount of tissue and expanding the cells in vivo on amniotic membrane fixed to the recipient eye. This was first described in 2012 by Sangwan et al. [3]. The present review describes indications for SLET and technical aspects. Published results and limitations are discussed. Finally, priorities for further evaluation are given.

Most reported uses of SLET have been for uniocular disease [4 ,5 ]. This is because tissue can be harvested from the healthy eye of the same patient. There is therefore no risk of immune rejection. Chemical and thermal burns make up the majority of cases treated because they form a large group of uniocular cases of stem cell deficiency. Use for surface reconstruction during pterygium [6] and ocular surface squamous neoplasia [7,8] excision has also been reported, along with limbal stem cell deficiency from a case of laryngo-onycho-cutaneous syndrome [9]. Some of these cases used ipsilateral tissue, others contralateral. Cadaveric SLET has been used for a case of bilateral alkali burn [10] and a case &

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LV Prasad Eye Institute, Banjara Hills, Hyderabad, India Correspondence to Virender S. Sangwan, MD, Dr Paul Dubord chair in Cornea, Tej Kohli Cornea Institute, Director, Centre for Ocular Regeneration, L V Prasad Eye Institute, Road 2, Banjara Hills, Hyderabad. Tel: +91 40 30612632; fax: +91 40 23548271; e-mail: [email protected] Curr Opin Ophthalmol 2017, 28:000–000 DOI:10.1097/ICU.0000000000000377

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KEY POINTS  Clinical results for SLET are similar to those reported for CLET and CLAU.  SLET employs a smaller transplant than in CLAU and therefore may be safer for the donor eye.  SLET does not require expensive ex-vivo expansion techniques.

of bilateral dry eye [11]. There is a risk of immune rejection when cadaveric tissue is used [10]. In addition, use of SLET in dry eye is considered by some to be a contraindication [4 ]. Autoimmune diseases, such as SJS and OCP, and aniridia are bilateral diseases and so are contraindications when SLET is used as an autograft. In addition, autoimmune disease is often associated with ongoing inflammation, dry eye and keratinization of tarsal conjunctiva [12], risking the survival of the transplanted cells. &

TECHNIQUE Under local or topical anaesthetic a 2 to 3 mm length of limbus is excised from the healthy fellow eye. This is usually at the superior limbus. Vannas scissors are used to undermine a flap of conjunctiva approximately 1 mm peripheral to the superior limbus, continuing the dissection until the limbus is reached. At the limbus, a number 15 blade is used to dissect superficial limbus into the peripheral cornea approximately 1 mm. The flap is then excised and this donor tissue is kept in balanced salt solution. If significant symblepharon is present in the recipient eye the technique can be modified to take a larger area of conjunctiva peripheral to the limbus. This can be used to graft the bulbar sclera when the symblepharon is released from the bulbar surface. The recipient site is prepared by a 3608 peritomy and removal of pannus on the cornea by superficial keratectomy. If there is any healthy corneal epithelium this is left undisturbed on the cornea, as is normal limbal epithelium with associated conjunctiva. The recipient cornea is then covered with amniotic membrane. This is glued to the surface with fibrin glue, starting with thick component followed by thin. The amniotic membrane should be of sufficient size to cover cornea and sclera up to the extent of the peritomy. It is usually placed with the epithelial side facing upwards as this makes it easier to smooth it out over the surface of the cornea using a surgical instrument or cannula. 2

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The limbal graft is cut into approximately 10 pieces. This can be done with Vannas scissors, a blade on a cutting board, or using retinal microinstruments. The cut fragments of the transplant are then placed on the surface of the amniotic membrane. This is usually done just inside the limbus, distributed evenly around the area to be covered. Whether the orientation makes a difference in vivo is not known but intuition would suggest that maintaining normal orientation with respect to cell polarity is beneficial. A small amount of fibrin glue is dropped on top of the grafts to secure them. A bandage contact lens is placed after 1 min when the glue has polymerized. As an alternative to a bandage contact lens, one author has described use of another layer of amniotic membrane [13]. This data and in-vivo data [14] suggest that this can be of cryopreserved type. The technique can be viewed in a video file available online (see Video, Supplemental Digital Content 1, http://links.lww.com/COOP/A25) Simultaneous penetrating keratoplasty has been described for cases where it is not practical for the patient to undergo multiple procedures [4 ] or when extreme thinning of the stroma is found after removal of pannus with resultant perforation that cannot be sealed with tissue adhesive or a patch graft. However, whenever possible it is recommended to perform penetrating keratoplasty as an elective procedure at a later date. The contact lens is removed at 7 days. If healing has not occurred a bandage contact lens is placed for a further week. Topical ciprofloxacin 0.3% eye drops are used four times per day whilst the contact lens is in place. Topical prednisolone acetate 1% eye drops are used six times per day for 1 week and then tapered by one drop per day every week over 6 weeks. &

COMPLICATIONS Haemorrhage under the amniotic membrane should be avoided. Loss of the donor tissue during the early postoperative period, probably because of loss of the overlying contact lens, has been described and reduces chances of success [5 ]. Microbial keratitis and corneal melt have also been observed [5 ]. Repeat SLET may be required for cases that fail to reepithelialize. Immune rejection has been described in use of cadaveric SLET [10]. Failure is associated with acid burns rather than alkali burns, symblepharon presence on the cornea prior to surgery and combined penetrating keratoplasty and SLET [5 ]. &

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RESULTS In total, we found 244 cases for any indication reported as of 10 January 2017. Two case series Volume 28  Number 00  Month 2017

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included 125 [5 ] and 68 [4 ] eyes respectively, the larger derived solely from the authors’ group, the smaller from the authors’ group as well as other centres in India, Mexico and the United States. The remainder were single cases or case series of 10 or fewer eyes [3,6–10,13,15–23]. The two larger series were mainly for the indication of chemical or thermal burn. Definitions of success broadly relate to reversal of features of ocular surface failure. There is not complete clarity in the literature about the exact definition of ocular surface failure and how this relates to limbal stem cell deficiency. However, a triad of conjunctival epithelial ingrowth, corneal vascularization and delayed healing with recurrent erosion [1] is probably a reasonable description of the key results of limbal stem cell deficiency. In both the larger series [5 ,4 ] reversal of these features was the primary outcome measure defining success. Figure 1 illustrates a typical successful outcome. The definition of treatment failure included progressive conjunctivilization and occurrence of persistent epithelial defect. Other criteria for failure were occurrence of microbial keratitis and need for repeat surgery [5 ]. A secondary outcome was visual acuity improvement. Success on the primary measures was 76 and 83.8% at a minimum follow-up period of 1 year and 6 months in the respective studies. These outcomes were broadly in line with the other stem cell transplant techniques of CLAU and CLET. In CLAU the best reported results are 87% [24] and in CLET 77% [25]. Results for the paediatric population in SLET were the same as for adults. This appeared to be better than those that we found for CLET in a series published by our group [26], where success was achieved in 47% of cases. What underlies this improvement? Is it specific to SLET or is it merely a property of amniotic membrane grafting, removal of conjunctiva on the corneal surface, or preoperative recuperative eyelid &

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procedures? Two further questions are relevant to answering this question: Do transplanted cells expand in vivo in the SLET procedure and do they constitute a persisting cell population on the surface of the cornea?

DO CELLS EXPAND IN VIVO IN SLET? One study of four cases of SLET [17] imaged the growth of cells on amniotic membrane in vivo using fluorescein stain. The results of this study demonstrated the appearance of sheets of cells centred on individual transplants growing across the surface of the amniotic membrane. The growth was variable, with some transplants producing large sheets of cells and some appearing not to produce any. This lends biological plausibility to the idea that SLET produces beneficial effects beyond merely amniotic membrane transplantation.

DO CELLS DERIVED FROM SLET CONSTITUTE A PERSISTING CELL POPULATION ON THE SURFACE OF THE CORNEA? SLET is performed in cases of partial and total limbal stem cell deficiency and seems effective in both. In the larger case series at our centre we described evidence that the new epithelium of the cornea was indeed corneal in origin rather than conjunctival with cornea-like properties. This was in the form of immunohistochemistry demonstrating normal cornea epithelium. However, proving that new autologous transplanted cells have permanently integrated into an existing population of corneal epithelial stem cells is difficult because the cells are by definition identical to the eye’s own population of cells in autografts. However, in allografts this question has been explored. In this case the longterm persistence of donor cells on the recipient cornea is controversial [27]. This is despite the cases clinically improving. It would be expected that long

FIGURE 1. (A) The preoperative appearance of a unilateral case of total stem cell deficiency. (B) The appearance four days after surgery. The stem cell grafts can be seen on the surface of the amniotic membrane. There is some haemorrhage under the amniotic membrane. (C) The appearance four months after surgery. The stem cell grafts are just visible. 1040-8738 Copyright ß 2017 Wolters Kluwer Health, Inc. All rights reserved.

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term persistence of cells in allografts could be controversial because there may be immune response to allogenous cells. In our case series, we did however identify markers of stem cells in the areas of the cornea that had been grafted, lending support to a persisting population of SLET-derived cells on the cornea [5 ]. This may suggest that, at least in our follow-up period, SLET does indeed contribute to a new population of corneal epithelial cells. &

IS CLINICAL IMPROVEMENT A FUNCTION OF AMNION OR THE STEM CELL TRANSPLANT ITSELF? It has been suggested that in the case of partial deficiency the presence of amniotic membrane as well as the new population of cells acts mainly to improve the environment such that the existing population can expand or recover [28]. Assessment of the role of amnion alone has been performed in cases of partial limbal stem cell deficiency. In four cases of amniotic membrane graft for partial limbal stem cell deficiency all cases were judged to have been successfully treated [29]. Simple removal of conjunctival epithelium from the cornea by scraping has also been described in cases of partial limbal stem deficiency [30]. However, the long-term results of these conservative approaches have not been studied in comparison to SLET or indeed other stem cell procedures in a randomized manner. We do not therefore know for sure the relative benefits of removal of conjunctiva, amnion graft, small stem cell grafts such as SLET and larger stem cell grafts such as CLAU. We have previously discussed the practical difficulties with conducting such a study with adequate power to detect a difference in outcomes [5 ]. However, in our large case series we found that early displacement of stem cell transplants in SLET was associated with worse outcomes. This might provide support for a ‘dose response’ for the effect of SLET and therefore support its value beyond merely grafting amniotic membrane. However, it is confounded by the fact that early loss of stem cell transplants was associated with loss of the overlying bandage contact lens. The bandage contact lens could theoretically be providing a better environment for preexisting stem cells to expand, or even protecting the amniotic membrane from premature degeneration.

excision, the results of SLET have been encouraging. In a nonrandomized case series with historical controls at our centre [7] we found that primary use of SLET reduced the chances of limbal stem cell deficiency after removal of ocular surface squamous neoplasia (OSSN). For pterygium an observational study found zero recurrence at up to 8 months of follow-up in ten patients. A further study with a more adequate follow-up period was reportedly intended by the same authors [7].

CONCLUSION SLET holds out a practical solution to the many cases of uniocular corneal blindness, particularly in areas of the world where ex-vivo expansion is not practical. As mentioned above, although logistically difficult, there is a need for further comparison with other stem cell techniques. Outcomes that are more important to patients than pure anatomical outcomes might be evaluated as primary outcomes. These could include vision, comfort and visual success of a future lamellar or penetrating keratoplasty. Cost analysis should be included in any assessment. Long-term follow-up with sufficient power to assess damage to the healthy donor eye would be ideal to confirm safety of this technique. In addition, investigating what effect penetrating keratoplasty would have on SLET compared to CLAU and CLET would be helpful. In SLET the stem cells are transplanted typically within the area that might be excised during a penetrating keratoplasty. Would this affect the ability of the surface to maintain a healthy epithelial layer after surgery? Would CLAU and CLET fare better since their new stem cell population are probably located outside the trephined edge? [31]

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RESULTS IN CASES OF OCULAR SURFACE SQUAMOUS NEOPLASIA AND PTERYGIUM EXCISION For surface reconstruction after iatrogenic stem cell deficiency such as in OSSN [7] and pterygium [6] 4

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Acknowledgements None. Financial support and sponsorship None. Conflicts of interest There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Chen JJ, Tseng SC. Corneal epithelial wound healing in partial limbal deficiency. Invest Ophthalmol Vis Sci 1990; 31:1301–1314. 2. 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.

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Simple limbal epithelial transplantation Sangwan and Sharp 3. Sangwan VS, Basu S, MacNeil S, Balasubramanian D. Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency. Br J Ophthalmol 2012; 96:931–934. 4. Vazirani J, Ali MH, Sharma N, et al. Autologous simple limbal epithelial & transplantation for unilateral limbal stem cell deficiency: multicentre results. Br J Ophthalmol 2016; 100:1416–1420. Sixty-eight cases are described giving results that are similar to other limbal stem cell transplant techniques. 5. Basu S, Sureka SP, Shanbhag SS, et al. Simple limbal epithelial transplanta& tion: long-term clinical outcomes in 125 cases of unilateral chronic ocular surface burns. Ophthalmology 2016; 123:1000–1010. This largest case series so far provides both clinical and immunohistochemical evidence of efficacy. 6. Herna´ndez-Bogantes E, Amescua G, Navas A, et al. Minor ipsilateral simple limbal epithelial transplantation (mini-SLET) for pterygium treatment. Br J Ophthalmol 2015; 99:1598–1600. 7. Kaliki S, Mohammad FA, Tahiliani P, Sangwan VS. Concomitant simple limbal epithelial transplantation after surgical excision of ocular surface squamous neoplasia. Am J Ophthalmol 2017; 174:68–75. 8. Mittal V, Narang P, Menon V, et al. Primary simple limbal epithelial transplantation along with excisional biopsy in the management of extensive ocular surface squamous neoplasia. Cornea 2016; 35:1650–1652. 9. Mohamed A, Sangwan VS. Ocular surface reconstruction in laryngo-onychocutaneous syndrome. Ocul Immunol Inflamm 2016; 1–3. [Epub ahead of print] 10. Bhalekar S, Basu S, Sangwan VS. Successful management of immunological rejection following allogeneic simple limbal epithelial transplantation (SLET) for bilateral ocular burns. BMJ Case Rep 2013. doi: 10.1136/bcr-2013009051. 11. Arya SK, Bhatti A, Raj A, Bamotra RK. Simple limbal epithelial transplantation in acid injury and severe dry eye. J Clin Diagn Res 2016; 10:ND06–ND07. 12. De Rojas MV, Dart JK, Saw VP. The natural history of Stevens–Johnson syndrome: patterns of chronic ocular disease and the role of systemic immunosuppressive therapy. Br J Ophthalmol 2007; 91:1048–1053. 13. Amescua G, Atallah M, Nikpoor N, et al. Modified simple limbal epithelial transplantation using cryopreserved amniotic membrane for unilateral limbal stem cell deficiency. Am J Ophthalmol 2014; 158:469–475. 14. Shortt AJ, Secker GA, Lomas RJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells. Biomaterials 2009; 30:1056–1065. 15. Mittal V, Jain R, Mittal R, et al. Successful management of severe unilateral chemical burns in children using simple limbal epithelial transplantation (SLET). Br J Ophthalmol 2016; 100:1102–1108. 16. Nair D, Mohamed A, Sangwan VS. Outcome of cataract surgery following simple limbal epithelial transplantation for lime injury-induced limbal stem cell deficiency. BMJ Case Rep 2015. doi: 10.1136/bcr-2015-212613.

17. Mittal V, Jain R, Mittal R. Ocular surface epithelialization pattern after simple limbal epithelial transplantation: an in vivo observational study. Cornea 2015; 34:1227–1232. 18. Queiroz AG, Barbosa MM, Santos MS, et al. Assessment of surgical outcomes of limbal transplantation using simple limbal epithelial transplantation technique in patients with total unilateral limbal deficiency. Arq Bras Oftalmol 2016; 79:116–118. 19. Das S, Basu S, Sangwan V. Molten metal ocular burn: long-term outcome using simple limbal epithelial transplantation. BMJ Case Rep 2015. doi: 10.1136/bcr-2014-209272. 20. Vazirani J, Lal I, Sangwan V. Customised simple limbal epithelial transplantation for recurrent limbal stem cell deficiency. BMJ Case Rep 2015. doi: 10.1136/bcr-2015-20942. 21. Vazirani J, Basu S, Sangwan V. Successful simple limbal epithelial transplantation (SLET) in lime injury-induced limbal stem cell deficiency with ocular surface granuloma. BMJ Case Rep 2013. doi: 10.1136/bcr-2013-009405. 22. Lal I, Panchal BU, Basu S, Sangwan VS. In-vivo expansion of autologous limbal stem cell using simple limbal epithelial transplantation for treatment of limbal stem cell deficiency. BMJ Case Rep 2013. doi: 10.1136/bcr-2013009247. 23. Bhalekar S, Basu S, Lal I, Sangwan VS. Successful autologous simple limbal epithelial transplantation (SLET) in previously failed paediatric limbal transplantation for ocular surface burns. BMJ Case Rep 2013. doi: 10.1136/bcr2013-009888. 24. Barreiro TP, Santos MS, Vieira AC, et al. Comparative study of conjunctival limbal transplantation not associated with the use of amniotic membrane transplantation for treatment of total limbal deficiency secondary to chemical injury. Cornea 2014; 33:716–720. 25. Pauklin M, Fuchsluger TA, Westekemper H, et al. Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency. Dev Ophthalmol 2010; 45:57–70. 26. Sejpal K, Ali MH, Maddileti S, et al. Cultivated limbal epithelial transplantation in children with ocular surface burns. JAMA Ophthalmol 2013; 131:731– 736. 27. Ahmad S. Concise review: limbal stem cell deficiency, dysfunction, and distress. Stem Cells Transl Med 2012; 1:110–115. 28. Guillermo A, Djalilian A, Jeng B. Simple limbal epithelial transplant: promising results in the right patients. EyeNet Magazine 2016; 20:25–27. 29. Gomes JA, dos Santos MS, Cunha MC, et al. Amniotic membrane transplantation for partial and total limbal stem cell deficiency secondary to chemical burn. Ophthalmology 2003; 110:466–473. 30. Dua HS, Gomes JA, Singh A. Corneal epithelial wound healing. Br J Ophthalmol 1994; 78:401–408. 31. Basu S, Mohamed A, Chaurasia S, et al. Clinical outcomes of penetrating keratoplasty after autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol 2011; 152:917–924.

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Simple limbal epithelial transplantation.

Simple limbal epithelial transplant (SLET) is a technique for addressing limbal stem cell deficiency. Limbal tissue from a donor eye, typically the pa...
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