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ARTICLE Barcelona-Cincinnati technique for limbal transplantation Jose L. Güell, MD, PhD1,2,3; Merce Morral, MD, PhD2,4; Oscar Gris, MD, PhD2; Daniel Elies, MD2; Felicidad Manero, MD2; Nerea Saenz, MD, PhD5 PURPOSE: To describe a new technique of allogenic limbal transplantation for total 28 limbal stem cell deficiencies (LSCD). METHODS: The “Barcelona-Cincinnati” technique combines a cadaveric keratolimbal allograft and transplantation of ex vivo expanded limbal cells on amniotic membrane (AM). A 1 x 3-mm biopsy of limbal tissue was obtained from a living donor for ex vivo culture on AM for 2-3 weeks. Two 120° corneoscleral rims were obtained from the cadaveric donor. A 360° limbal peritomy was performed on the recipient eye, and the cadaveric crescents were sutured on the superior and inferior limbus. Abnormal fibrovascular pannus and epithelium were removed from the surface of the cornea and peripheral areas. A penetrating keratoplasty was then performed. Finally, AM with ex vivo expanded limbal cells was sutured over the cornea epithelial-side down. Postoperatively, topical steroids and systemic immunosuppression with methylprednisolone and cyclosporine A were prescribed and, in the absence of an immunological reaction, tapered off. RESULTS: Two eyes of two patients with bilateral total LSCD were treated. Six years postoperatively, Case 1 maintained a stable ocular surface. Case 2, however, presented recurrence of LSCD 17 months after surgery. CONCLUSION: Although prospective comparative studies including more patients and a longer follow-up are required, the Barcelona-Cincinnati technique may be an effective alternative for bilateral total LSCD while decreasing the risk for living donors. J Emmetropia 2012; 3: 124-128 INTRODUCTION Treatment of limbal stem cell deficiency (LSCD) is challenging. In partial LSCD involving the visual axis, sequential sector conjunctival epitheliectomy (SSCE) is the treatment of choice.1 If fibrovascular pannus covers the cornea, a sector limbal transplant may also be required. Submitted: 06/22/2012 Revised: 07/24/2012 Accepted: 07/26/2012 1 2 3 4 5 Director of the Cornea and Refractive Surgery Unit, Instituto Microcirugia Ocular, Barcelona, Spain. Instituto de Microcirugia Ocular (IMO), Barcelona, Spain. Associate Professor of Ophthalmology at the “Universitat Autonoma de Barcelona” (UAB), Barcelona, Spain. Department of Cornea and Anterior Segment Diseases, and Refractive Surgery of the Institut Clinic d’Oftalmologia, Hospital Clinic i Provincial, Barcelona, Spain. Hospital 12 de Octubre, Madrid. Spain. Financial Disclosure: None of the authors has any financial or proprietary interest in any material or method mentioned. Corresponding Author: Jose L Güell, MD. Instituto Microcirugia Ocular Barcelona, Spain. E-mail: [email protected] 124 © 2010 SECOIR Sociedad Española de Cirugía Ocular Implanto-Refractiva In total LSCD, transplantation of limbal tissue is the mainstay. In bilateral cases, living donors are preferred over cadaveric donors, as the fresh tissue contains a higher number of stem cells and can be human leukocyte antigen (HLA) matched, reducing the risk of rejection.2 Holland, et al. described the Cincinnati technique, which combines a living-related conjunctival limbal allograft (lr-CLAL) and a keratolimbal allograft (KLAL) for LSCD associated with conjunctival deficiency.3 This technique allowed transplantation of limbal stem cells from two different sources, thereby increasing the density of viable transplanted cells, and healthy conjunctiva, which improves the local microenvironment conditions by decreasing conjunctival inflammation and replacing goblet cells.3 However, the potential risk of iatrogenic LSCD in the living donor must be considered.4-6 To decrease such risk, ex vivo expansion of limbus-derived cells with or without a substrate (fibrin, collagen or amnion), first described by Pellegrini et al. in 1997, is a viable option for total, unilateral or bilateral LSCD.7-11 The surgical techniques for ex vivo expansion of limbal stem cells and KLAL have been described.2,3,7,12-14 We propose the “Barcelona-Cincinnati technique”, which ISSN: 2171-4703 BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION combines a cadaveric KLAL and ex-vivo expansion of limbal stem cells on amniotic membrane (AM) from a living donor, with the aim of increasing the number of viable cells while decreasing the risk in the living donor. METHODS All patients were fully informed of the details and possible risks of the specific procedure. Written informed consent to perform the surgical procedure was obtained from all patients before surgery in accordance with the Declaration of Helsinki, and the study was approved by the ethics committee of our institution, Instituto de Microcirugia Ocular, and the Universitat Autonoma de Barcelona. All eyes were operated by the same surgeon (JLG). The “Barcelona-Cincinnati technique” combines ex vivo expansion of limbal stem cells on amniotic membrane (AM) from a living donor, and a cadaveric KLAL. In these two cases, penetrating keratoplasty (PK) was also performed as a single-stage procedure. 125 Preparation of cadaveric donor tissue and the recipient eye, and placement of donor tissue For KLAL, the posterior half of each hemisection of the corneoscleral rim was removed on the cadaveric donor. A 360° limbal peritomy was performed on the recipient eye. Abnormal fibrovascular pannus and epithelium were removed from the cornea and peripheral areas. The cadaveric donor crescents were placed with the donor corneal edge overlying the recipient superior and inferior limbus with interrupted 10-0 Nylon sutures at the corneal margin, and interrupted 9-0 Vycril sutures at the conjunctival margin.2,14 Penetrating keratoplasty PK was then performed as a single-stage procedure using the same donor as for the KLAL. An 8.5-mm Hessburg-Barron punch was used to obtain the corneal graft, and an 8-mm trephine was performed on the recipient´s eye. The corneal graft was then sutured with 16 interrupted 10-0 Nylon sutures. The epithelium of the donor was not removed. Ex vivo expansion of limbal stem cells Briefly, a 1 × 3-mm biopsy of limbal tissue was obtained for ex vivo culture. The superior limbus is preferred as it is thought to contain a higher density of stem cells. The obtained tissue was placed with Ham’s F12 medium containing 50 µg/ml gentamicin and 1.25 µg/ml amphotericin B until it was processed. Limbal tissue was exposed for 5 min to Dispase II (1.2 U/ml in Mg2+ and Ca2+ free Hank’s balanced salt solution HBSS) at 37 °C under humidified 5% CO2. The explants were then cultured in DMEM medium, which is a 1:1 mixture of DMEM and Ham’s F12 medium containing 5 ng/ml epithelial growth factor (EGF), 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml sodium selenite, 0.5 mg/ml hydrocortisone, 30 ng/ml cholera toxin A, 0.5% dimethylsulfoxide (DMSO), 50 mg/ ml gentamicin, 1.25 mg/ml amphotericin B and 5% autologous serum, at 37ºC under 5% CO2 and 95% humidity. The medium was renewed every 2-3 days. For allogeneic related transplantation, donor serum was used and for allogeneic non-related transplantation, ABO tested blood bank serum was employed. The limbal epithelial cell explants were plated onto the center of the basement membrane side of the AM denuded of epithelium. The extent of the limbal epithelium outgrowth, which exhibits a compact uniform cell layer, was monitored with a phase contrast microscope until it covered an area of 2-3 cm in diameter (2-3 weeks later). Bacteriological testing, including mycoplasma and Gram´s test, was performed to assess microorganism contamination.12 Implantation of ex vivo expanded limbal stem cells After the KLAL and PK were completed, the AM containing ex vivo expanded limbal cells was sutured epithelial-side down with a running 10-0 Nylon suture so that the cultivated cells were in direct contact with the ocular surface. The biopsy specimen was left located in the limbal area (Figure 1). Finally, subconjunctival methylprednisolone was injected. Mechanical protection was achieved by placing a bandage contact lens over the graft at the end of surgery, leaving it in situ until complete reabsorption of the AM. Postoperative regimen The immediate postoperative topical regimen was: tobramycin and dexamethasone (Tobradex® Alcon, El Masnou, Barcelona, Spain) drops five times per day, timolol 0.5% (Cusimolol 0.5%®, Alcon) twice a day (BID), and chloramphenicol and dexamethasone ointment (Oftalmolosa Cusi De Icol®, Alcon) at bedtime. Additionally, systemic immunosuppression was achieved with methylprednisolone (Urbason®, Sanofi Aventis, Barcelona, Spain; 0.5 mg/kg/day) and cyclosporine A (Sandimmun®, Novartis SA, Barcelona, Spain; 5 mg/kg/12 hours, maintaining blood levels of 100-400 ng/ml). Patients were monitored for potential side effects, especially nephrotoxicity. Blood tests and blood pressure were checked on a weekly basis for the first month, every 2-3 weeks for the second and JOURNAL OF EMMETROPIA - VOL 3, JULY-SEPTEMBER 126 BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION Figure 1. The “Barcelona-Cincinnati technique”. (A) The cadaveric donor crescents are sutured superiorly and inferiorly. Interrupted 10-0 Nylon sutures are used to secure the graft to the recipient´s cornea, and interrupted 9-0 Vycril sutures are used to secure the graft to the recipient´s conjunctiva and epiesclera. (B) Ex vivo expanded limbal stem cells on amniotic membrane (AM). The AM is sutured epithelial-side down, with the expanded cells contacting the ocular surface. (Arrows: (1) segmental cadaveric donor keratolimbal section; (2) AM with ex vivo expanded limbal stem cells; (3) Original biopsy from a living-related donor). third month, and every two months thereafter. In the absence of an immunological reaction, corticosteroids were discontinued 1 to 2 months after surgery. Low-dose cyclosporine (25-50 mg/day) was maintained for at least 12 months, and then discontinued if possible. RESULTS Two cases were treated. Case 1 presented with total LSCD secondary to congenital aniridia. A limbal biopsy was taken from the patient’s partially HLA-I-compatible sister, and was expanded ex vivo on AM. Three weeks later, penetrating keratoplasty, cadaveric KLAL, and implantation of ex vivo expanded limbal stem cells were performed. The postoperative regimen included: topical combined tobramycin and dexamethasone drops five times per day, timolol 0.5% BID, and chloramphenicol and dexamethasone ointment at bedtime for a month. Additionally, systemic methylprednisolone and cyclosporine A were prescribed for a month. Three weeks postoperatively, a clear corneal graft and localized inflammation on the donor tissue were observed. Topical and systemic steroids were slowly tapered off, but low-dose cyclosporine (25-50 mg/day) was maintained for 12 months and then discontinued. Six years postoperatively, visual acuity (VA) had improved to 20/200, limited by terminal glaucoma, and LSCD had completely resolved (Figure 2). Case 2 presented with bilateral total LSCD secondary to acute chemical injury. A limbal biopsy was taken from the patient’s HLA-I-compatible mother. Three weeks later, penetrating keratoplasty, cadaveric KLAL, and implantation of ex vivo expanded cells were performed. The same postoperative immunosuppressive regime was followed, and three weeks postoperatively visual acuity Figure 2. (A) Clinical photograph of a total limbal stem cell deficiency in the right eye of a patient with congenital anirida. The fellow eye was eviscerated. 360° corneal neovascularization and total corneal clouding can be observed. (B) The conjunctival epithelium appears irregular, with areas of fluorescein pooling and fine neovessels. No epithelial defect is seen. Visual acuity was hand motion. (C) Penetrating keratoplasty, keratolimbal allograft (KLAL) from a cadaveric donor and implantation of ex vivo expanded limbal stem cells were performed. Clinical photograph showing the 3-week postoperative appearance. A clear corneal graft and localized inflammation on the donor tissue can be observed. (D-F) Clinical photograph 1 (D) and 6 years (E-F) after surgery. A clear corneal graft with regular epithelium and no inflammation on the KLAL area are seen. Visual acuity was 20/200, limited by terminal glaucoma. JOURNAL OF EMMETROPIA - VOL 3, JULY-SEPTEMBER BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION 127 Figure 3. (A) Clinical photograph of a total limbal stem cell deficiency secondary to acute chemical ocular burn. The fellow eye was also affected. 360° corneal neovascularization and total corneal clouding can be observed. A central epithelial defect is seen. Visual acuity was counting fingers at 6 feet. (B-C) Penetrating keratoplasty, keratolimbal allograft (KLAL) from a cadaveric donor, and implantation of ex vivo expanded limbal stem cells were performed. Clinical photograph showing the one-month postoperative appearance. A clear corneal graft and localized inflammation on the donor tissue can be observed. The corneal epithelium is smooth. Visual acuity was 20/200. (D-E) Clinical photograph 17 months after surgery. A subtotal epithelial defect is seen, suggesting graft failure and recurrence of the limbal stem cell deficiency. No inflammation on the KLAL area is seen. Visual acuity decreased to counting fingers at 6 feet. (VA) improved from counting fingers to 20/200 with a clear corneal graft. Seventeen months postoperatively, however, VA had decreased to counting fingers, and a subtotal epithelial defect suggested recurrence of the LSCD and failure of the limbal graft. The patient was advised to undergo repeat keratolimbal transplantation, but declined (Figure 3). DISCUSSION Disorders of the ocular surface resulting in both limbal stem cell and conjunctival deficiency are extremely severe and challenging. Total LSCD will require transplantation of a population of autologous or allogeneic limbal epithelial stem cells if a stable corneal epithelial phenotype is to be regained.2 The main limitations of limbal stem cell transplantation are: (1) The need for a high density of viable transplanted cells; (2) Maximal cell compatibility (HLA I-II, and ABO matching)15-17; (3) The need for local and systemic immunosuppression; and (4) Local microenvironment conditions as physiological as possible. Autologous limbal transplants are preferred in unilateral cases to avoid the risk of immune rejection and the side effects of systemic immunosuppression.18 Living donors are also preferred over cadaveric donors, as the tissue is fresh and presents a higher density of limbal epithelial stem cells. However, removal of limbus from a living donor or the contralateral healthy eye is not without risk, and partial stem cell deficiency following limbal biopsy for conjunctival limbal autografts (CLAU) or allografts in previously normal eyes has been described.4-6 Ex vivo expansion of limbus-derived cells constitutes an excellent alternative. Biopsies from livingrelated donors have been shown to be more reliable in terms of successful establishment of cultures than those obtained from cadaveric corneas.19 A progressive loss of donor cells in the recipient eye over time has been described, and is considered the main cause of KLAL failure, with a dramatic decrease in graft survival rate over a 2-year period.20,21 The aim of the “Barcelona-Cincinnati” technique is to provide a greater amount of viable limbal stem cells by using two different sources: KLAL and ex vivo expanded cells. Compared to CLAU and lr-CLAL, the small-sized biopsy minimizes the risk of iatrogenic stem cell failure in the donor eye, and enables another biopsy to be taken if the first fails. However, as no healthy conjunctiva is transplanted, improvement of local microenvironment conditions by reducing conjunctival inflammation and replacing goblet cells is limited. One of the main concerns when two different donors are combined is the theoretical increased risk of immune rejection related to the additional immune stimulation. Compared to CLAU, ex vivo cultured limbal epithelial cell grafts, especially if a cadaveric donor graft is not required, present a reduced risk of rejection due to the absence of antigen-presenting Langerhan´s cells.13 As a direct relationship between ocular surface stem cell transplantation failure and immune rejection has been reported, it is critical that patients are closely monitored for rejection and for compliance with immunosuppressive treatment.22 Although we used two different donors (cadaveric for the KLAL and PK, and living-related for ex vivo expansion), we did not introduce any changes in our standard postoperative immunosuppressive regimen. The question of whether case 2 would have succeeded if immunosuppression had been raised remains. Ex vivo expansion from limbal biopsies provides a mixed cell population, which includes limbal stem cells, corneal and conjunctival epithelial cells. Although techniques to check the quality of the cell culture before it is transplanted have evolved considerably in recent years, they were not available at the time we treated these patients. Therefore, the actual proportion of putative stem cells was unclear.7,11,23 One of the main challenges when interpreting the results is to establish whether using a combined approach (e.g. KLAL and ex vivo expansion) is better than using one of the techniques alone. The relative benefit of each cell source can be determined by polymerase chain reaction (PCR) genotyping for DNA microsatellites on the corneal epithelial cells obtained by impression JOURNAL OF EMMETROPIA - VOL 3, JULY-SEPTEMBER 128 BARCELONA-CINCINNATI TECHNIQUE FOR LIMBAL TRANSPLANTATION cytology postoperatively, as long as a blood sample from the cadaveric donor was obtained.9,24 A limitation of our study is that we did not perform such analysis. Several studies have suggested that a two-stage approach of autologous cultivated limbal epithelial transplantation followed by PK successfully restores ocular surface stability and vision, and that a single-stage approach is associated with poorer clinical outcomes and should be avoided.24,25 However, in our experience, we have found similar results with both approaches, although we prefer the one-stage technique as it has two clear advantages: (1) the patient undergoes only one surgical procedure; and (2) the same donor is used for PK and KLAL, which reduces the antigenic load (Personal communication, Güell JL, Gris O. Keratolimbal Allograft Combined with Penetrating Keratoplasty in Severe Chemical injuries. The Cornea Society/Eye Bank Association of America. AAO. America Academy of Ophthalmology. Las Vegas 11-14 November, 2006). This finding has also been reported in other studies.26 In conclusion, the Barcelona-Cincinnati technique represents an encouraging preliminary experience but the limited follow-up, the small number of eyes treated and the lack of a control group, does not allow any conclusion to be drawn. Continued stem cell, pharmacological and surgical research is essential to improve our capacity to treat these major disorders. REFERENCES 1. Dua H. Sequential sector conjunctival epitheliectomy. In: Holland EJ, Mannis M, eds. Ocular Surface Disease, Medical and Surgical Management. New York: Springer, 2002; 168–74. 2. Dua HS, Miri A, Said DG. Contemporary limbal stem cell transplantation - a review. Clin Experiment Ophthalmol. 2010;38:104-17. 3. Biber JM, Skeens HM, Neff KD, Holland EJ. The Cincinnati procedure: technique and outcomes of combined living-related conjunctival limbal allografts and keratolimbal allograft in severe ocular surface failure. Cornea 2011;30:756-71. 4. Miri A, Said DG, Dua HS. Donor site complications in autolimbal and living-related allolimbal transplantation. Ophthalmology. 2011;118:1265-71. 5. Jenkins C, Tuft S, Liu C, et al. Limbal transplantation in the management of chronic contact-lens-associated epitheliopathy. Eye 1993;7:629-633. 6. Tan DT, Ficker LA, Buckley RJ. Limbal transplantation. Ophthalmology. 1996;103:29-36. 7. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997;349:990-3. 8. Meller D, Pires RT, Tseng SC. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol 2002;86:463–71. 9. Daya SM, Watson A, Sharpe JR et al. Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction. Ophthalmology 2005;112:470–7. 10. Koizumi N, Inatomi T, Suzuki T, Sotozono Ch, Kinoshita S. 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Ophthalmology 2002;109:2011-7. JOURNAL OF EMMETROPIA - VOL 3, JULY-SEPTEMBER First author: Jose Luis Güell, MD Instituto de Microcirugia Ocular (IMO) Barcelona, Spain