Modified Simple Limbal Epithelial Transplantation Using Cryopreserved Amniotic Membrane for Unilateral Limbal Stem Cell Deficiency GUILLERMO AMESCUA, MARWAN ATALLAH, NEDA NIKPOOR, ANAT GALOR, AND VICTOR L. PEREZ
PURPOSE:
To report the results of simple limbal epithelial transplantation using a double-layered cryopreserved amniotic membrane graft for the management of unilateral limbal stem cell deficiency. DESIGN: Retrospective case series. METHODS: Four consecutive patients with unilateral partial (2 eyes) and total (2 eyes) limbal stem cell deficiency secondary to ocular surface burns (2 eyes), trauma (1 eye) and conjunctival melanoma treatment (1 eye) underwent modified simple limbal epithelial transplantation at Bascom Palmer Eye Institute. Preoperative and postoperative visual acuity and quality of corneal epithelium were evaluated. RESULTS: The patients were followed up for a mean ± standard deviation of 7.5 ± 1.3 months. The donor eye returned to a healthy state in all patients. All patients had significant improvement in visual acuity and resolution of ocular pain in the affected eye. Regular corneal epithelium and a quiet ocular surface were obtained in all patients by a median of 4 weeks. CONCLUSIONS: Compared with the currently used surgical techniques for management of limbal stem cell deficiency, simple limbal epithelial transplantation seems to be a safe, reproducible, and effective alternative. The use of a double layer allows more protection for the explant without impacting outcomes. Also, the use of cryopreserved amniotic membrane allows surgeons to perform this procedure in the Unites States. More cases with longer follow-up will be needed to assess the outcomes further. (Am J Ophthalmol 2014;158: 469–475. Ó 2014 by Elsevier Inc. All rights reserved.)
L
IMBAL STEM CELL DEFICIENCY IS A COMMON PHENO-
typic end point of various ocular pathologic features, the most common of which are severe inflammatory insults to the ocular surface such as chemical or thermal burns.1,2 Other causes include immunologically induced inflammation such as Stevens-Johnson syndrome and Accepted for publication Jun 3, 2014. From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida. Inquiries to Guillermo Amescua, Ocular Surface Service, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 900 NW 17 Street, Suite 340C, Miami, FL 33136; e-mail: [email protected] 0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2014.06.002
Ó
2014 BY
ocular cicatricial pemphigoid, along with mechanical, infectious, iatrogenic, idiopathic, and hereditary causes.2,3 As limbal stem cells fail to generate and maintain basal cells for the corneal epithelium, conjunctival epithelium starts invading the superficial cornea. Patients usually have pain and photophobia secondary to recurrent corneal erosions. Superficial neovascularization and conjunctivalization also emerge and eventually lead to visual impairment. Visual rehabilitation is difficult in these patients because corneal transplantations almost universally fail.4,5 Efforts have been made to reinstate a healthy ocular surface with limbal stem cell transplantation. However, limitations in the available techniques have limited its widespread use. Traditional conjunctival limbal autografts harvested from the nonaffected eye are limited by the degree of limbal stem cell deficiency in the affected eye and the risk of destabilizing the ocular surface in the good eye.6–8 Keratolimbal allografts usually are reserved for bilateral limbal stem cell deficiency, given the need for systemic immunosuppression or otherwise guarded longterm success. Outcomes after autologous ex vivo cultivated limbal epithelial transplantation have been promising with faster epithelialization, less inflammation, and less scarring than traditional autograft.9 This technique, however, is limited by the high cost and the need for a good manufacturing practice facility to process and expand the cells properly. Currently in the United States, there is no Food and Drug Administration approval for using this type of facility to expand limbal stem cells ex vivo from the healthy eye of a patient and then transplant them back to the patient’s affected eye. Recently, Sangwan and associates have introduced simple limbal epithelial transplantation, a novel approach that avoids the drawbacks of the existing techniques.10 In his technique, a small (2 3 2-mm) donor limbal graft from the unaffected eye is harvested and divided into smaller pieces that are then expanded in vivo in the stem cell-deficient eye with the use of a fresh amniotic membrane and fibrin glue. Herein we describe our modification where 2 amniotic membrane layers are used to sandwich and protect the harvested limbal stem cells. Using this technique, we demonstrate the safety and efficacy of using cryopreserved amniotic membrane grafts, a Food and Drug Administration-approved alternative in the United States.
ELSEVIER INC. ALL
RIGHTS RESERVED.
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FIGURE 1. Photographs obtained before and after simple limbal epithelial transplantation in patients with limbal stem cell deficiency. (Top row) Photographs from a 74-year-old woman with limbal stem cell deficiency of the right eye secondary to surgical excision with cryotherapy and topical mitomycin C for biopsy proven conjunctival melanoma. The patient had chronic photophobia and foreign body sensation at presentation. (Top left) Fluorescein staining showing the chronic and recurrent central epithelial defect. (Top right) Six months after simple limbal epithelial transplantation using cryopreserved amniotic membrane, the cornea re-epithelialized and cleared after dissolution of the amniotic membrane. The patient reported resolution of symptoms. No episodes of recurrent corneal erosions were seen during the 6 months of follow-up after surgery. (Second row) Photographs from a 57-year-old man with limbal stem cell deficiency resulting from an ammonia injury. Ocular surface reconstruction with amniotic membrane graft failed and the patient sought treatment for tearing, photophobia, and poor vision. (Second row left) Preoperative photograph obtained the day of surgery demonstrating 360 degrees of superficial corneal neovascularization and conjunctivalization with irregular corneal epithelium. (Second row right) Six months after simple limbal epithelial transplantation using cryopreserved amniotic membrane, a noninflamed ocular surface was restored with a regular corneal epithelium. The patient reported resolution of symptoms. (Third row) Photographs from a 48-year-old woman with limbal stem cell deficiency resulting from an acid chemical burn sought treatment for pain, photophobia, and decrease in vision. (Third row left) Examination revealed conjunctivalization and pannus of the left cornea
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METHODS THIS STUDY WAS A RETROSPECTIVE CASE SERIES OF 4
consecutive patients. Through it, we present our experience at Bascom Palmer Eye Institute and the Miami Veterans Affairs Hospital using the simple limbal epithelial transplantation technique to manage unilateral partial or complete limbal stem cell deficiency. The study was conducted in accordance with the tenets of the Declaration of Helsinki, federal and Florida laws, and the Health Insurance Portability and Accountability Act. This study was exempted from approval by the University of Miami and Miami Veterans Affairs Hospital Institutional Review Boards because it was considered nongeneralizable human research by each. The diagnosis of limbal stem cell deficiency was made clinically by confirming in the affected eye an irregular corneal epithelium, superficial corneal vascularization, persistent epithelial defects, and, in the cases of total limbal stem cell deficiency, a complete conjunctivalization of the cornea (Figure 1, Left). Posterior segment evaluation was carried out by ultrasound biomicroscopy in patients where visualization was not possible. Three surgeries were performed by one author (G.A.), and 1 surgery was performed by another author (A.G.). We followed the technique described by Sangwan and associates.10 The original technique describes the use of fresh amniotic membrane. This option is not available in the United States; instead, we used cryopreserved amniotic membrane (AmnioGraft size B; Biotissue, Inc., Miami, Florida, USA). In brief, and similar to what was described in their article, a 2 3 2-mm area was marked across the supranasal limbus of the donor eye. A subconjunctival dissection was carried out 1 mm into the clear cornea to excise the limbal tissue. The donor tissue then was placed in balanced salt solution. In the affected eye, a 360-degree conjunctival peritomy was performed and the fibrovascular pannus was excised. Bleeding was controlled by gentle cauterization, and the corneal and limbal surface was polished with a diamond bur. An amniotic membrane graft was placed stroma side down on the bare ocular surface and was secured with fibrin glue (Tisseel; Baxter, Deerfield, Illinois, USA). The donor limbal tissue was cut into approximately 12 to 15 pieces with Vannas scissors and placed on the amniotic membrane overlying the cornea in a spiral fashion, sparing the visual axis. The tissue then was secured with fibrin glue.10 We then modified the original simple limbal epithelial transplantation technique by placing
the harvested tissue between the 2 layers of amniotic membrane with the intention of replicating a fetal environment for the stem cells. The second amniotic membrane graft was sutured with 10-0 nylon in a purse string fashion around the limbal area, and then a bandage contact lens (18-mm Kontur lens; Kontur Contact Lens, Co., Inc., California, USA) was placed. All patients were administered topical broad-spectrum antibiotics until the contact lens was removed and prednisolone acetate 1% for 4 to 6 weeks. The bandage contact lens was removed 10 to 14 days after surgery, and the nylon suture was removed after the membrane dissolved. Clinical examination was performed at 1 day then 1 week then 1, 3, and 4 months after surgery, then at 2- to 3-month intervals. Clinical examination by biomicroscopy was documented.
RESULTS DEMOGRAPHICS AND CLINICAL DETAILS OF THE 4 PATIENTS
are provided in the Table. Two females and 2 males were included with a mean age 6 standard deviation of 59.8 6 10.9 years. Duration of limbal stem cell deficiency varied between 3 and 45 years, with a median of 15.5 years. Two eyes of 2 patients had partial unilateral limbal stem cell deficiency secondary to chemical injury (1 eye) and trauma (1 eye), and 2 eyes had total unilateral limbal stem cell deficiency secondary to chemical injury (1 eye) and conjunctival melanoma treatment (1 eye). All patients reported symptoms before surgery, including ocular pain, photophobia, and foreign body sensation. Corneal findings included chronic or recurrent epithelial defects (4 eyes), corneal neovascularization and partial corneal conjunctivalization (2 eyes), complete corneal conjunctivalization (2 eyes), stromal scarring (3 eyes), and pyogenic granuloma formation (1 eye). All patients had bilateral wet ocular surface. There were no intraoperative or postoperative complications in any of the 4 cases. After surgery, the donor eye in all patients returned to its healthy state by the first week (Figure 2, Left), whereas in the grafted eye, the amniotic membrane sandwich was seen in all patients (Figure 2, Right). On slit-lamp examination, the amniotic membranes were no longer visible after a median of 4 weeks, and the cornea had a stable epithelium with a quiet ocular surface. Ultra–high-resolution optical coherence tomography performed at 4 months after surgery for
(Third row right) Image obtained 4 months after simple limbal epithelial transplantation using cryopreserved amniotic membrane showing a healthy corneal epithelium. The patient did not report any pain or photophobia. (Bottom row) Photographs from a 55-year-old man with longstanding limbal stem cell deficiency secondary to trauma reporting mainly of chronic eye pain, irritation, and poor vision. (Bottom left) Image showing corneal conjunctivalization with a pyogenic granuloma formation, irregular corneal epithelium, corneal stromal scarring, and neovascularization. (Bottom right) Six months after simple limbal epithelial transplantation using cryopreserved amniotic membrane, the corneal epithelium was regular and the patient reported complete resolution of symptoms.
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20/50 Yes (3) 6
20/50 Yes (1) 8
Counting fingers at 1 m Counting fingers at 1 m
20/30 7 20/200
Yes (3)
9 20/400
Yes (2)
Follow-Up (mos)
20/40
Postoperative Best-Corrected Visual Acuity Stabilization of Ocular Surface (wks) Preoperative Best-Corrected Visual Acuity
DISCUSSION
Partial Mechanical trauma (15) Left 55 4
Male
Partial Left Female 48 3
57 2
Male
Right
Chemical (ammonium) injury (45) Chemical injury (16)
Total
Excisional biopsy, cryotherapy, intraoperative and postoperative mitomycin C, cataract extraction with intraocular lens placement Ocular restoration with amniotic membrane graft Ocular restoration with amniotic membrane graft Ocular restoration with amniotic membrane graft Total Melanoma excision þ mitomycin C (3) Right Female 74 1
Age (y)
Sex
Eye
Limbal Stem Cell Deficiency Cause (Time; y)
Limbal Stem Cell Deficiency Degree
Preoperative Treatment
IN THIS STUDY, THE SIMPLE LIMBAL EPITHELIAL TRANS-
Patient No.
TABLE. Demographic Data, Diagnosis, Follow-up, and Primary Outcomes of Patients Undergoing Limbal Epithelial Transplantation Using Cryopreserved Amniotic Membrane for Unilateral Limbal Stem Cell Deficiency
472
Patient 1 showed a normalized corneal epithelium and a hyperreflective subepithelial layer, likely the residual amniotic membrane graft in the recipient eye (Figure 3). A stable surface with no evidence of epithelial defects or conjunctivalization was maintained in all recipient eyes at a mean 6 standard deviation follow-up of 7.5 6 1.3 months (Figure 1). In all 4 patients, residual islets of tissue in the paracentral cornea were observed after surgery. Explants initially were thick and thinned out over a period of 3 months such that they were barely visible. Best-corrected visual acuity improved from worse than 20/200 in all recipient eyes before surgery to 20/50 or better in all 4 patients (Table). After surgery, all patients had complete resolution of pre-existing ocular symptoms including pain, tearing, and photophobia.
plantation technique was used to restore corneal epithelium in 4 patients with unilateral partial or total stem cell deficiency using cryopreserved amniotic membrane graft. Moreover, a double amniotic membrane graft step was used as modification to protect the transplanted limbal stem cells. This procedure was easy to perform and involves only simple surgical techniques that can be replicated by most ophthalmic surgeons. We strongly believe that this surgical technique is a good option for patients with unilateral limbal stem cell deficiency. Ideal candidates for this procedure are patients with unilateral stem cell deficiency and a clinically noninflamed wet ocular surface. The migration of stem cells during in vivo expansion is not well understood. Some groups have suggested that during corneal healing, cells first repopulate the limbus, and then heal centripetally.11,12 As such, it is not clear whether the location of the planted cells is truly an issue. Although we do not yet have experience in patients with stromal opacity and limbal stem cell deficiency, based on studies of other similar transplantation techniques,13 our recommendation is to perform keratoplasty followed by a limbal stem cell transplantation with in vivo expansion as a staged procedure. Alternatively, in case of partial stem cell deficiency, a same-day procedure can be performed. Our modified simple limbal epithelial transplantation technique closely compares with the original technique in terms of outcomes. Both our and the previous report attained 100% graft success rate with restoration of the corneal epithelium and achieved an improvement in visual acuity of at least 2 lines in all patients.10 The simple limbal epithelial transplantation technique has significant advantages over conventional surgical options in that it requires only a small amount of donor tissue as opposed to conjunctival limbal autografts, with no need to use a
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FIGURE 2. Slit-lamp photographs of a donor and a recipient limbal stem cell-deficient eye, both 3 days after simple limbal epithelial transplantation using cryopreserved amniotic membrane. (Left) Donor eye showing resolution of the epithelial defect at the donor site at the limbus and supranasal conjunctiva. (Right) Recipient eye showing the double-layer amniotic membrane with stem cell islets.
FIGURE 3. Ultra–high-resolution optical coherence tomography from a previously stem cell-deficient cornea performed 4 months after a modified simple limbal epithelial transplantation using cryopreserved amniotic membrane, where limbal tissue from the healthy eye was expanded in vivo. The scan shows a normalized corneal epithelium, and a hyperreflective subepithelial layer, likely the residual amniotic membrane grafted versus the Bowman membrane. Mild anterior stromal scarring can be noted.
good manufacturing practice facility for ex vivo expansion as required in cultivated limbal epithelial transplantation, nor the use of systemic immunosuppression to prevent rejection, as required in keratolimbal allografts. Furthermore, outcomes of simple limbal epithelial transplantation are comparable with or better than outcomes of other techniques. With respect to conjunctival limbal autografts, a review of the literature revealed that vision was improved in 90% of cases (n ¼ 39), with 94% (n ¼ 49) restoration of the ocular surface with large grafts (>120 degrees). Visual improvement decreased to 60% (n ¼ 22) and frequency of ocular surface restoration decreased to 91% (n ¼ 22) when smaller grafts were attempted to avoid jeopardizing the donor eye.14 With respect to cultivated limbal epithelial transplantation, in 14 studies evaluating the outcomes of ex vivo cultivated grafts, 66% of patients with burns, malignancy, or trauma (n ¼ 313) had improvement in visual acuity and 79% (n ¼ 541) had a successful autologous graft.15–28 The cost of the procedure, however, was reported to reach 8 times those of conjunctival limbal autografts and simple limbal epithelial transplantation,10 provided a specialized facility is available. VOL. 158, NO. 3
With respect to keratolimbal allografts, a review of the literature revealed that short-term outcomes are comparable with the above values. Success rates drop, however, with longer follow-up to less than 60% at 2 years and 50% at 3 years.14 This is largely because of allograft rejection. Furthermore, Krakauer and associates reported a 68% rate of adverse effects related to immunosuppression.29 Exciting research is being conducted on stem cells that will no doubt improve available techniques. This includes research on new carrier methods for the stem cells,30 new sources of stem cells (bone marrow),31 and new delivery systems.32,33 In the meantime, of the available stem cell transplant procedures, and based on our and previous reports, simple limbal epithelial transplantation is an excellent option because it can expand limbal stem cells in vivo without the need for a stem cell expansion facility. We believe that our modification to the procedure offers a protective environment to the limbal stem cells in case, for example, the patient loses the bandage contact lens early in the postoperative course. Also, the use of cryopreserved amniotic membranes, an alternative approved by the United States Food and Drug Administration, yields similar results to the originally
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used fresh membranes. More research with longer follow-up will be needed to assess the outcomes further of this new, exciting surgical procedure. To conclude, this study demonstrated that compared with the currently used surgical techniques for management of limbal stem cell deficiency, simple limbal epithelial transplantation seems to be a safe, reproducible, and
effective alternative. The use of a so-called sandwich technique provided protection to the graft and the stem cell niche without negatively impacting the clinical outcome. Furthermore, the use of Food and Drug Administrationapproved cryopreserved tissue allows surgeons to perform simple limbal epithelial transplantation in the United States.
ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST and none were reported. Supported by Center Core Grant P30EY014801 from the National Institutes of Health, Bethesda, Maryland; and unrestricted grant from Research to Prevent Blindness, Inc, New York, New York; and Grant W81XWH-09-1-0675 from the Department of Defense, Washington, DC. Dr Perez is the Walter G. Ross Distinguished Chair in Ophthalmic Research. Involved in Design and conduct of study (G.A., A.G., M.A.); Collection (N.N., M.A., G.A.), management (G.A., A.G.), analysis (M.A., G.A., A.G.), and interpretation (M.A., G.A., N.N., A.G.) of data; and Preparation (G.A., M.A., A.G.) and review or approval (N.N., V.L.P.) of manuscript. The authors thanks Virender S. Sangwan and Sayan Basu, Cornea and Anterior Segment Service, L. V. Prasad Eye Institute, Hyderabad, Andhra Pradesh, India, for sharing their knowledge of this novel surgical technique.
REFERENCES 1. Shortt AJ, Secker GA, Notara MD, et al. Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol 2007;52(5): 483–502. 2. Nishida K. Tissue engineering of the cornea. Cornea 2003; 22(7 Suppl):S28–S34. 3. Limbal stem cell transplantation: an evidence-based analysis. Ont Health Technol Assess Ser 2008;8(7):1–58. 4. Reinhard T, Spelsberg H, Henke L, et al. Long-term results of allogeneic penetrating limbo-keratoplasty in total limbal stem cell deficiency. Ophthalmology 2004;111(4):775–782. 5. Krachmer JH, Mannis MJ, Holland EJ. Cornea. 3rd ed. Maryland Heights, Missouri: Elsevier Inc.; 2011:1499. 6. Miri A, Said DG, Dua HS. Donor site complications in autolimbal and living-related allolimbal transplantation. Ophthalmology 2011;118(7):1265–1271. 7. Basti S, Mathur U. Unusual intermediate-term outcome in three cases of limbal autograft transplantation. Ophthalmology 1999;106(5):958–963. 8. Chen JJ, Tseng SC. Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. Invest Ophthalmol Vis Sci 1991;32(8):2219–2233. 9. Ang LPK, Sotozono C, Koizumi N, Suzuki T, Inatomi T, Kinoshita S. A comparison between cultivated and conventional limbal stem cell transplantation for Stevens-Johnson syndrome. Am J Ophthalmol 2007;143(1):178–180. 10. 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(7):931–934. 11. Dua HS, Forrester JV. The corneoscleral limbus in human corneal epithelial wound healing. Am J Ophthalmol 1990; 110(6):646–656. 12. Yeung AM-H, Schlo¨tzer-Schrehardt U, Kulkarni B, Tint NL, Hopkinson A, Dua HS. Limbal epithelial crypt: a model for corneal epithelial maintenance and novel limbal regional variations. Arch Ophthalmol 2008;126(5): 665–669. 13. Solomon A, Ellies P, Anderson DF, et al. Long-term outcome of keratolimbal allograft with or without penetrating kerato-
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plasty for total limbal stem cell deficiency. Ophthalmology 2002;109(6):1159–1166. Liang L, Sheha H, Li J, Tseng SCG. Limbal stem cell transplantation: new progresses and challenges. Eye (Lond) 2009; 23(10):1946–1953. 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(9057):990–993. Schwab IR. Cultured corneal epithelia for ocular surface disease. Trans Am Ophthalmol Soc 1999;97:891–986. Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea 2000;19(4):421–426. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343(2):86–93. Rama P, Bonini S, Lambiase A, et al. Autologous fibrincultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 2001;72(9):1478–1485. Grueterich M, Espana EM, Touhami A, Ti S-E, Tseng SCG. Phenotypic study of a case with successful transplantation of ex vivo expanded human limbal epithelium for unilateral total limbal stem cell deficiency. Ophthalmology 2002; 109(8):1547–1552. Sangwan VS, Vemuganti GK, Iftekhar G, Bansal AK, Rao GN. Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury: a case report of unique application. Cornea 2003;22(5):478–481. Sangwan VS, Vemuganti GK, Singh S, Balasubramanian D. Successful reconstruction of damaged ocular outer surface in humans using limbal and conjunctival stem cell culture methods. Biosci Rep 2003;23(4):169–174. Sangwan VS, Murthy SI, Vemuganti GK, Bansal AK, Gangopadhyay N, Rao GN. Cultivated corneal epithelial transplantation for severe ocular surface disease in vernal keratoconjunctivitis. Cornea 2005;24(4):426–430. Sangwan VS, Matalia HP, Vemuganti GK, et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J Ophthalmol 2006;54(1):29–34.
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25. Nakamura T, Inatomi T, Sotozono C, Koizumi N, Kinoshita S. Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease. Acta Ophthalmol Scand 2004;82(4):468–471. 26. Nakamura T, Inatomi T, Sotozono C, et al. Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease. Ophthalmology 2006;113(10):1765–1772. 27. Basu S, Ali H, Sangwan VS. Clinical outcomes of repeat autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol 2012;153(4): 643–650. 650.e1–2. 28. Marchini G, Pedrotti E, Pedrotti M, et al. Long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns. Clin Experiment Ophthalmol 2012;40(3): 255–267.
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29. Krakauer M, Welder JD, Pandya HK, Nassiri N, Djalilian AR. Adverse effects of systemic immunosuppression in keratolimbal allograft. J Ophthalmol 2012. article ID 576712, 5 pages. 30. Feng Y, Borrelli M, Reichl S, Schrader S, Geerling G. Review of alternative carrier Materials for ocular surface reconstruction. Curr Eye Res 2014;39(6):541–552. 31. Rohaina CM, Then KY, Ng AMH, et al. Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane. Transl Res 2014;163(3):200–210. 32. Brown KD, Low S, Mariappan I, et al. Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency. Tissue Eng Part A 2014;20(3-4):646–655. 33. Bhalekar S, Sangwan VS, Basu S. Growth of corneal epithelial cells over in situ therapeutic contact lens after simple limbal epithelial transplantation (SLET). BMJ Case Rep 2013. http://dx.doi.org/10.1136/bcr-2013-009113.
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Biosketch Guillermo Amescua, MD received his medical degree from the Ignacio A. Santos-Tech of Monterrey School of Medicine. He completed an ophthalmology residency at the University of Pittsburgh School of Medicine. He then completed two fellowship trainings in Cornea, and Uveitis at the Bascom Palmer Eye Institute where he currently is an assistant professor. His areas of clinical practice are ocular surface and cataracts. His research interests are in infectious keratitis, limbal stem cell deficiency and keratoprosthesis.
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Biosketch Victor L. Perez, MD received his medical doctorate from the University of Puerto Rico. He performed his ophthalmology residency training, and cornea and uveitis fellowships at Harvard Medical School Department of Ophthalmolgy. He completed two research fellowships in Ocular Immunology at Schepens Eye Research Institute and Brigham & Women’s Hospital. He is the director of the Ocular Surface Center and is the Walter G. Ross Distinguished Chair in Ophthalmic Research Programs at the Bascom Palmer Eye Institute.
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PURPOSE:
To report the results of simple limbal epithelial transplantation using a double-layered cryopreserved amniotic membrane graft for the management of unilateral limbal stem cell deficiency. DESIGN: Retrospective case series. METHODS: Four consecutive patients with unilateral partial (2 eyes) and total (2 eyes) limbal stem cell deficiency secondary to ocular surface burns (2 eyes), trauma (1 eye) and conjunctival melanoma treatment (1 eye) underwent modified simple limbal epithelial transplantation at Bascom Palmer Eye Institute. Preoperative and postoperative visual acuity and quality of corneal epithelium were evaluated. RESULTS: The patients were followed up for a mean ± standard deviation of 7.5 ± 1.3 months. The donor eye returned to a healthy state in all patients. All patients had significant improvement in visual acuity and resolution of ocular pain in the affected eye. Regular corneal epithelium and a quiet ocular surface were obtained in all patients by a median of 4 weeks. CONCLUSIONS: Compared with the currently used surgical techniques for management of limbal stem cell deficiency, simple limbal epithelial transplantation seems to be a safe, reproducible, and effective alternative. The use of a double layer allows more protection for the explant without impacting outcomes. Also, the use of cryopreserved amniotic membrane allows surgeons to perform this procedure in the Unites States. More cases with longer follow-up will be needed to assess the outcomes further. (Am J Ophthalmol 2014;158: 469–475. Ó 2014 by Elsevier Inc. All rights reserved.)
L
IMBAL STEM CELL DEFICIENCY IS A COMMON PHENO-
typic end point of various ocular pathologic features, the most common of which are severe inflammatory insults to the ocular surface such as chemical or thermal burns.1,2 Other causes include immunologically induced inflammation such as Stevens-Johnson syndrome and Accepted for publication Jun 3, 2014. From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida. Inquiries to Guillermo Amescua, Ocular Surface Service, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 900 NW 17 Street, Suite 340C, Miami, FL 33136; e-mail: [email protected] 0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2014.06.002
Ó
2014 BY
ocular cicatricial pemphigoid, along with mechanical, infectious, iatrogenic, idiopathic, and hereditary causes.2,3 As limbal stem cells fail to generate and maintain basal cells for the corneal epithelium, conjunctival epithelium starts invading the superficial cornea. Patients usually have pain and photophobia secondary to recurrent corneal erosions. Superficial neovascularization and conjunctivalization also emerge and eventually lead to visual impairment. Visual rehabilitation is difficult in these patients because corneal transplantations almost universally fail.4,5 Efforts have been made to reinstate a healthy ocular surface with limbal stem cell transplantation. However, limitations in the available techniques have limited its widespread use. Traditional conjunctival limbal autografts harvested from the nonaffected eye are limited by the degree of limbal stem cell deficiency in the affected eye and the risk of destabilizing the ocular surface in the good eye.6–8 Keratolimbal allografts usually are reserved for bilateral limbal stem cell deficiency, given the need for systemic immunosuppression or otherwise guarded longterm success. Outcomes after autologous ex vivo cultivated limbal epithelial transplantation have been promising with faster epithelialization, less inflammation, and less scarring than traditional autograft.9 This technique, however, is limited by the high cost and the need for a good manufacturing practice facility to process and expand the cells properly. Currently in the United States, there is no Food and Drug Administration approval for using this type of facility to expand limbal stem cells ex vivo from the healthy eye of a patient and then transplant them back to the patient’s affected eye. Recently, Sangwan and associates have introduced simple limbal epithelial transplantation, a novel approach that avoids the drawbacks of the existing techniques.10 In his technique, a small (2 3 2-mm) donor limbal graft from the unaffected eye is harvested and divided into smaller pieces that are then expanded in vivo in the stem cell-deficient eye with the use of a fresh amniotic membrane and fibrin glue. Herein we describe our modification where 2 amniotic membrane layers are used to sandwich and protect the harvested limbal stem cells. Using this technique, we demonstrate the safety and efficacy of using cryopreserved amniotic membrane grafts, a Food and Drug Administration-approved alternative in the United States.
ELSEVIER INC. ALL
RIGHTS RESERVED.
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FIGURE 1. Photographs obtained before and after simple limbal epithelial transplantation in patients with limbal stem cell deficiency. (Top row) Photographs from a 74-year-old woman with limbal stem cell deficiency of the right eye secondary to surgical excision with cryotherapy and topical mitomycin C for biopsy proven conjunctival melanoma. The patient had chronic photophobia and foreign body sensation at presentation. (Top left) Fluorescein staining showing the chronic and recurrent central epithelial defect. (Top right) Six months after simple limbal epithelial transplantation using cryopreserved amniotic membrane, the cornea re-epithelialized and cleared after dissolution of the amniotic membrane. The patient reported resolution of symptoms. No episodes of recurrent corneal erosions were seen during the 6 months of follow-up after surgery. (Second row) Photographs from a 57-year-old man with limbal stem cell deficiency resulting from an ammonia injury. Ocular surface reconstruction with amniotic membrane graft failed and the patient sought treatment for tearing, photophobia, and poor vision. (Second row left) Preoperative photograph obtained the day of surgery demonstrating 360 degrees of superficial corneal neovascularization and conjunctivalization with irregular corneal epithelium. (Second row right) Six months after simple limbal epithelial transplantation using cryopreserved amniotic membrane, a noninflamed ocular surface was restored with a regular corneal epithelium. The patient reported resolution of symptoms. (Third row) Photographs from a 48-year-old woman with limbal stem cell deficiency resulting from an acid chemical burn sought treatment for pain, photophobia, and decrease in vision. (Third row left) Examination revealed conjunctivalization and pannus of the left cornea
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METHODS THIS STUDY WAS A RETROSPECTIVE CASE SERIES OF 4
consecutive patients. Through it, we present our experience at Bascom Palmer Eye Institute and the Miami Veterans Affairs Hospital using the simple limbal epithelial transplantation technique to manage unilateral partial or complete limbal stem cell deficiency. The study was conducted in accordance with the tenets of the Declaration of Helsinki, federal and Florida laws, and the Health Insurance Portability and Accountability Act. This study was exempted from approval by the University of Miami and Miami Veterans Affairs Hospital Institutional Review Boards because it was considered nongeneralizable human research by each. The diagnosis of limbal stem cell deficiency was made clinically by confirming in the affected eye an irregular corneal epithelium, superficial corneal vascularization, persistent epithelial defects, and, in the cases of total limbal stem cell deficiency, a complete conjunctivalization of the cornea (Figure 1, Left). Posterior segment evaluation was carried out by ultrasound biomicroscopy in patients where visualization was not possible. Three surgeries were performed by one author (G.A.), and 1 surgery was performed by another author (A.G.). We followed the technique described by Sangwan and associates.10 The original technique describes the use of fresh amniotic membrane. This option is not available in the United States; instead, we used cryopreserved amniotic membrane (AmnioGraft size B; Biotissue, Inc., Miami, Florida, USA). In brief, and similar to what was described in their article, a 2 3 2-mm area was marked across the supranasal limbus of the donor eye. A subconjunctival dissection was carried out 1 mm into the clear cornea to excise the limbal tissue. The donor tissue then was placed in balanced salt solution. In the affected eye, a 360-degree conjunctival peritomy was performed and the fibrovascular pannus was excised. Bleeding was controlled by gentle cauterization, and the corneal and limbal surface was polished with a diamond bur. An amniotic membrane graft was placed stroma side down on the bare ocular surface and was secured with fibrin glue (Tisseel; Baxter, Deerfield, Illinois, USA). The donor limbal tissue was cut into approximately 12 to 15 pieces with Vannas scissors and placed on the amniotic membrane overlying the cornea in a spiral fashion, sparing the visual axis. The tissue then was secured with fibrin glue.10 We then modified the original simple limbal epithelial transplantation technique by placing
the harvested tissue between the 2 layers of amniotic membrane with the intention of replicating a fetal environment for the stem cells. The second amniotic membrane graft was sutured with 10-0 nylon in a purse string fashion around the limbal area, and then a bandage contact lens (18-mm Kontur lens; Kontur Contact Lens, Co., Inc., California, USA) was placed. All patients were administered topical broad-spectrum antibiotics until the contact lens was removed and prednisolone acetate 1% for 4 to 6 weeks. The bandage contact lens was removed 10 to 14 days after surgery, and the nylon suture was removed after the membrane dissolved. Clinical examination was performed at 1 day then 1 week then 1, 3, and 4 months after surgery, then at 2- to 3-month intervals. Clinical examination by biomicroscopy was documented.
RESULTS DEMOGRAPHICS AND CLINICAL DETAILS OF THE 4 PATIENTS
are provided in the Table. Two females and 2 males were included with a mean age 6 standard deviation of 59.8 6 10.9 years. Duration of limbal stem cell deficiency varied between 3 and 45 years, with a median of 15.5 years. Two eyes of 2 patients had partial unilateral limbal stem cell deficiency secondary to chemical injury (1 eye) and trauma (1 eye), and 2 eyes had total unilateral limbal stem cell deficiency secondary to chemical injury (1 eye) and conjunctival melanoma treatment (1 eye). All patients reported symptoms before surgery, including ocular pain, photophobia, and foreign body sensation. Corneal findings included chronic or recurrent epithelial defects (4 eyes), corneal neovascularization and partial corneal conjunctivalization (2 eyes), complete corneal conjunctivalization (2 eyes), stromal scarring (3 eyes), and pyogenic granuloma formation (1 eye). All patients had bilateral wet ocular surface. There were no intraoperative or postoperative complications in any of the 4 cases. After surgery, the donor eye in all patients returned to its healthy state by the first week (Figure 2, Left), whereas in the grafted eye, the amniotic membrane sandwich was seen in all patients (Figure 2, Right). On slit-lamp examination, the amniotic membranes were no longer visible after a median of 4 weeks, and the cornea had a stable epithelium with a quiet ocular surface. Ultra–high-resolution optical coherence tomography performed at 4 months after surgery for
(Third row right) Image obtained 4 months after simple limbal epithelial transplantation using cryopreserved amniotic membrane showing a healthy corneal epithelium. The patient did not report any pain or photophobia. (Bottom row) Photographs from a 55-year-old man with longstanding limbal stem cell deficiency secondary to trauma reporting mainly of chronic eye pain, irritation, and poor vision. (Bottom left) Image showing corneal conjunctivalization with a pyogenic granuloma formation, irregular corneal epithelium, corneal stromal scarring, and neovascularization. (Bottom right) Six months after simple limbal epithelial transplantation using cryopreserved amniotic membrane, the corneal epithelium was regular and the patient reported complete resolution of symptoms.
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20/50 Yes (3) 6
20/50 Yes (1) 8
Counting fingers at 1 m Counting fingers at 1 m
20/30 7 20/200
Yes (3)
9 20/400
Yes (2)
Follow-Up (mos)
20/40
Postoperative Best-Corrected Visual Acuity Stabilization of Ocular Surface (wks) Preoperative Best-Corrected Visual Acuity
DISCUSSION
Partial Mechanical trauma (15) Left 55 4
Male
Partial Left Female 48 3
57 2
Male
Right
Chemical (ammonium) injury (45) Chemical injury (16)
Total
Excisional biopsy, cryotherapy, intraoperative and postoperative mitomycin C, cataract extraction with intraocular lens placement Ocular restoration with amniotic membrane graft Ocular restoration with amniotic membrane graft Ocular restoration with amniotic membrane graft Total Melanoma excision þ mitomycin C (3) Right Female 74 1
Age (y)
Sex
Eye
Limbal Stem Cell Deficiency Cause (Time; y)
Limbal Stem Cell Deficiency Degree
Preoperative Treatment
IN THIS STUDY, THE SIMPLE LIMBAL EPITHELIAL TRANS-
Patient No.
TABLE. Demographic Data, Diagnosis, Follow-up, and Primary Outcomes of Patients Undergoing Limbal Epithelial Transplantation Using Cryopreserved Amniotic Membrane for Unilateral Limbal Stem Cell Deficiency
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Patient 1 showed a normalized corneal epithelium and a hyperreflective subepithelial layer, likely the residual amniotic membrane graft in the recipient eye (Figure 3). A stable surface with no evidence of epithelial defects or conjunctivalization was maintained in all recipient eyes at a mean 6 standard deviation follow-up of 7.5 6 1.3 months (Figure 1). In all 4 patients, residual islets of tissue in the paracentral cornea were observed after surgery. Explants initially were thick and thinned out over a period of 3 months such that they were barely visible. Best-corrected visual acuity improved from worse than 20/200 in all recipient eyes before surgery to 20/50 or better in all 4 patients (Table). After surgery, all patients had complete resolution of pre-existing ocular symptoms including pain, tearing, and photophobia.
plantation technique was used to restore corneal epithelium in 4 patients with unilateral partial or total stem cell deficiency using cryopreserved amniotic membrane graft. Moreover, a double amniotic membrane graft step was used as modification to protect the transplanted limbal stem cells. This procedure was easy to perform and involves only simple surgical techniques that can be replicated by most ophthalmic surgeons. We strongly believe that this surgical technique is a good option for patients with unilateral limbal stem cell deficiency. Ideal candidates for this procedure are patients with unilateral stem cell deficiency and a clinically noninflamed wet ocular surface. The migration of stem cells during in vivo expansion is not well understood. Some groups have suggested that during corneal healing, cells first repopulate the limbus, and then heal centripetally.11,12 As such, it is not clear whether the location of the planted cells is truly an issue. Although we do not yet have experience in patients with stromal opacity and limbal stem cell deficiency, based on studies of other similar transplantation techniques,13 our recommendation is to perform keratoplasty followed by a limbal stem cell transplantation with in vivo expansion as a staged procedure. Alternatively, in case of partial stem cell deficiency, a same-day procedure can be performed. Our modified simple limbal epithelial transplantation technique closely compares with the original technique in terms of outcomes. Both our and the previous report attained 100% graft success rate with restoration of the corneal epithelium and achieved an improvement in visual acuity of at least 2 lines in all patients.10 The simple limbal epithelial transplantation technique has significant advantages over conventional surgical options in that it requires only a small amount of donor tissue as opposed to conjunctival limbal autografts, with no need to use a
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FIGURE 2. Slit-lamp photographs of a donor and a recipient limbal stem cell-deficient eye, both 3 days after simple limbal epithelial transplantation using cryopreserved amniotic membrane. (Left) Donor eye showing resolution of the epithelial defect at the donor site at the limbus and supranasal conjunctiva. (Right) Recipient eye showing the double-layer amniotic membrane with stem cell islets.
FIGURE 3. Ultra–high-resolution optical coherence tomography from a previously stem cell-deficient cornea performed 4 months after a modified simple limbal epithelial transplantation using cryopreserved amniotic membrane, where limbal tissue from the healthy eye was expanded in vivo. The scan shows a normalized corneal epithelium, and a hyperreflective subepithelial layer, likely the residual amniotic membrane grafted versus the Bowman membrane. Mild anterior stromal scarring can be noted.
good manufacturing practice facility for ex vivo expansion as required in cultivated limbal epithelial transplantation, nor the use of systemic immunosuppression to prevent rejection, as required in keratolimbal allografts. Furthermore, outcomes of simple limbal epithelial transplantation are comparable with or better than outcomes of other techniques. With respect to conjunctival limbal autografts, a review of the literature revealed that vision was improved in 90% of cases (n ¼ 39), with 94% (n ¼ 49) restoration of the ocular surface with large grafts (>120 degrees). Visual improvement decreased to 60% (n ¼ 22) and frequency of ocular surface restoration decreased to 91% (n ¼ 22) when smaller grafts were attempted to avoid jeopardizing the donor eye.14 With respect to cultivated limbal epithelial transplantation, in 14 studies evaluating the outcomes of ex vivo cultivated grafts, 66% of patients with burns, malignancy, or trauma (n ¼ 313) had improvement in visual acuity and 79% (n ¼ 541) had a successful autologous graft.15–28 The cost of the procedure, however, was reported to reach 8 times those of conjunctival limbal autografts and simple limbal epithelial transplantation,10 provided a specialized facility is available. VOL. 158, NO. 3
With respect to keratolimbal allografts, a review of the literature revealed that short-term outcomes are comparable with the above values. Success rates drop, however, with longer follow-up to less than 60% at 2 years and 50% at 3 years.14 This is largely because of allograft rejection. Furthermore, Krakauer and associates reported a 68% rate of adverse effects related to immunosuppression.29 Exciting research is being conducted on stem cells that will no doubt improve available techniques. This includes research on new carrier methods for the stem cells,30 new sources of stem cells (bone marrow),31 and new delivery systems.32,33 In the meantime, of the available stem cell transplant procedures, and based on our and previous reports, simple limbal epithelial transplantation is an excellent option because it can expand limbal stem cells in vivo without the need for a stem cell expansion facility. We believe that our modification to the procedure offers a protective environment to the limbal stem cells in case, for example, the patient loses the bandage contact lens early in the postoperative course. Also, the use of cryopreserved amniotic membranes, an alternative approved by the United States Food and Drug Administration, yields similar results to the originally
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used fresh membranes. More research with longer follow-up will be needed to assess the outcomes further of this new, exciting surgical procedure. To conclude, this study demonstrated that compared with the currently used surgical techniques for management of limbal stem cell deficiency, simple limbal epithelial transplantation seems to be a safe, reproducible, and
effective alternative. The use of a so-called sandwich technique provided protection to the graft and the stem cell niche without negatively impacting the clinical outcome. Furthermore, the use of Food and Drug Administrationapproved cryopreserved tissue allows surgeons to perform simple limbal epithelial transplantation in the United States.
ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST and none were reported. Supported by Center Core Grant P30EY014801 from the National Institutes of Health, Bethesda, Maryland; and unrestricted grant from Research to Prevent Blindness, Inc, New York, New York; and Grant W81XWH-09-1-0675 from the Department of Defense, Washington, DC. Dr Perez is the Walter G. Ross Distinguished Chair in Ophthalmic Research. Involved in Design and conduct of study (G.A., A.G., M.A.); Collection (N.N., M.A., G.A.), management (G.A., A.G.), analysis (M.A., G.A., A.G.), and interpretation (M.A., G.A., N.N., A.G.) of data; and Preparation (G.A., M.A., A.G.) and review or approval (N.N., V.L.P.) of manuscript. The authors thanks Virender S. Sangwan and Sayan Basu, Cornea and Anterior Segment Service, L. V. Prasad Eye Institute, Hyderabad, Andhra Pradesh, India, for sharing their knowledge of this novel surgical technique.
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29. Krakauer M, Welder JD, Pandya HK, Nassiri N, Djalilian AR. Adverse effects of systemic immunosuppression in keratolimbal allograft. J Ophthalmol 2012. article ID 576712, 5 pages. 30. Feng Y, Borrelli M, Reichl S, Schrader S, Geerling G. Review of alternative carrier Materials for ocular surface reconstruction. Curr Eye Res 2014;39(6):541–552. 31. Rohaina CM, Then KY, Ng AMH, et al. Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane. Transl Res 2014;163(3):200–210. 32. Brown KD, Low S, Mariappan I, et al. Plasma polymer-coated contact lenses for the culture and transfer of corneal epithelial cells in the treatment of limbal stem cell deficiency. Tissue Eng Part A 2014;20(3-4):646–655. 33. Bhalekar S, Sangwan VS, Basu S. Growth of corneal epithelial cells over in situ therapeutic contact lens after simple limbal epithelial transplantation (SLET). BMJ Case Rep 2013. http://dx.doi.org/10.1136/bcr-2013-009113.
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Biosketch Guillermo Amescua, MD received his medical degree from the Ignacio A. Santos-Tech of Monterrey School of Medicine. He completed an ophthalmology residency at the University of Pittsburgh School of Medicine. He then completed two fellowship trainings in Cornea, and Uveitis at the Bascom Palmer Eye Institute where he currently is an assistant professor. His areas of clinical practice are ocular surface and cataracts. His research interests are in infectious keratitis, limbal stem cell deficiency and keratoprosthesis.
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Biosketch Victor L. Perez, MD received his medical doctorate from the University of Puerto Rico. He performed his ophthalmology residency training, and cornea and uveitis fellowships at Harvard Medical School Department of Ophthalmolgy. He completed two research fellowships in Ocular Immunology at Schepens Eye Research Institute and Brigham & Women’s Hospital. He is the director of the Ocular Surface Center and is the Walter G. Ross Distinguished Chair in Ophthalmic Research Programs at the Bascom Palmer Eye Institute.
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