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Simple Limbal Epithelial Transplantation

Long-Term Clinical Outcomes in 125 Cases of Unilateral Chronic Ocular Surface Burns
  • Sayan Basu
    Affiliations
    Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India

    Center for Regenerative Ophthalmology, L. V. Prasad Eye Institute, Hyderabad, India

    Prof. Brien Holden Eye Research Center, Champalimaud Translational Centre for Eye Research, L. V. Prasad Eye Institute, Hyderabad, India
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  • Shraddha P. Sureka
    Affiliations
    Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India
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  • Swapna S. Shanbhag
    Affiliations
    Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India
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  • Abhinav R. Kethiri
    Affiliations
    Center for Regenerative Ophthalmology, L. V. Prasad Eye Institute, Hyderabad, India

    Prof. Brien Holden Eye Research Center, Champalimaud Translational Centre for Eye Research, L. V. Prasad Eye Institute, Hyderabad, India
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  • Vivek Singh
    Affiliations
    Center for Regenerative Ophthalmology, L. V. Prasad Eye Institute, Hyderabad, India

    Prof. Brien Holden Eye Research Center, Champalimaud Translational Centre for Eye Research, L. V. Prasad Eye Institute, Hyderabad, India
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  • Virender S. Sangwan
    Correspondence
    Correspondence: Virender S. Sangwan, MBBS, MS, Kallam Anji Reddy Campus, L. V. Prasad Eye Institute, Room 606B, Sixth Floor, Hyderabad 500034, India.
    Affiliations
    Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India

    Center for Regenerative Ophthalmology, L. V. Prasad Eye Institute, Hyderabad, India

    Prof. Brien Holden Eye Research Center, Champalimaud Translational Centre for Eye Research, L. V. Prasad Eye Institute, Hyderabad, India
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Open AccessPublished:February 17, 2016DOI:https://doi.org/10.1016/j.ophtha.2015.12.042

      Purpose

      This study describes the long-term clinical outcomes of autologous simple limbal epithelial transplantation (SLET), a relatively new technique of limbal stem cell transplantation.

      Design

      This was a single-center prospective interventional cases series.

      Participants

      This study included 125 patients, 65 adults and 60 children who developed unilateral limbal stem cell deficiency (LSCD) after suffering with ocular surface burns and underwent SLET between 2010 and 2014.

      Methods

      A 1-clock hour limbal biopsy sample was obtained from the unaffected eye. At the same sitting, the recipient eye was surgically prepared and the donor tissue was divided into small pieces and transplanted using an amniotic membrane scaffold with fibrin glue.

      Main Outcome Measures

      The diagnosis and outcome in every case was validated by 5 independent masked assessors. The primary outcome measure was restoration of a completely epithelized, stable, and avascular corneal surface. The secondary outcome measure was improvement in visual acuity. Complications, risk factors for failure, and immunohistochemistry analysis of corneas that underwent SLET also were described.

      Results

      At a median postoperative follow-up of 1.5 years (range, 1–4 years), 95 of 125 eyes (76%; 95% confidence interval, 68.5%–83.5%) maintained a successful outcome. Kaplan-Meier analysis revealed a comparable survival probability at 1 year of 80% in adults and 72% in children (P = 0.304). Two-line improvement in visual acuity was seen in 75.2%, and 67% of successful cases attained 20/60 or better vision (P < 0.0001). Progressive conjunctivalization occurred in 18.4% of eyes. The clinical factors associated with failure were identified as acid injury, severe symblepharon, SLET combined with keratoplasty, and postoperative loss of transplants (P ≤ 0.0075). Success rates were comparable among faculty and trainees (P = 0.71). Immunohistochemistry revealed successful regeneration of normal corneal epithelium (CK3+/12+) without admixture of conjunctiva cells (Muc5AC/CK19) and replenishment of limbal stem cell (ΔNp63α+/ABCG2+) reserve.

      Conclusions

      Autologous SLET is an effective, reliable and replicable technique for long-lasting corneal regeneration and vision restoration in unilateral chronic ocular surface burns. Simple limbal epithelial transplantation is probably preferable to other techniques of limbal stem cell transplantation, particularly where cell cultivation facilities are unavailable.

      Abbreviations and Acronyms:

      BCVA (best-corrected visual acuity), CI (confidence interval), CLET (cultivated limbal epithelial transplantation), hAM (human amniotic membrane), HR (hazard ratio), LSCD (limbal stem cell deficiency), SLET (simple limbal epithelial transplantation)
      A delicately thin layer of stratified but nonkeratinized squamous epithelium covers the corneal surface. This epithelial cover is renewed continuously as younger cells migrate inward from the periphery and older cells are lost from the surface.
      • Shapiro M.S.
      • Friend J.
      • Thoft R.A.
      Corneal re-epithelialization from the conjunctiva.
      The constant source of corneal epithelial cells is believed to be the limbus, which is the annular transitional area between the cornea and the sclera.
      • Shapiro M.S.
      • Friend J.
      • Thoft R.A.
      Corneal re-epithelialization from the conjunctiva.
      Corneal epithelial stem cells have been identified deep within a protected microenvironment or niche at the limbal palisades of Vogt.
      • Schermer A.
      • Galvin S.
      • Sun T.-T.
      Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells.
      • Ksander B.R.
      • Kolovou P.E.
      • Wilson B.J.
      • et al.
      ABCB5 is a limbal stem cell gene required for corneal development and repair.
      When the limbus is intact, corneal epithelial defects heal promptly. But when the limbus is damaged, either because of injury or inflammation, the normal corneal epithelial physiologic features are disrupted. Delay or failure in corneal epithelialization leads to conjunctival encroachment over the cornea, vascularization, and nonhealing epithelial defects.
      • Dua H.S.
      • Azuara-Blanco A.
      Limbal stem cells of the corneal epithelium.
      The consequent clinical condition, termed limbal stem cell deficiency (LSCD), is a rare but severe cause of corneal blindness.
      Fortunately, transplantation of healthy limbal tissue can reverse LSCD and restore a normal corneal surface.
      • Kenyon K.R.
      • Tseng S.C.
      Limbal autograft transplantation for ocular surface disorders.
      • Pellegrini G.
      • Traverso C.E.
      • Franzi A.T.
      • et al.
      Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium.
      In the last 3 decades, both the understanding of limbal biology and the techniques of limbal transplantation have evolved considerably.
      • Baylis O.
      • Figueiredo F.
      • Henein C.
      • et al.
      13 years of cultured limbal epithelial cell therapy: a review of the outcomes.
      • Shortt A.J.
      • Secker G.A.
      • Notara M.D.
      • et al.
      Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results.
      Although conjunctival-limbal or keratolimbal grafting continues to be practiced,
      • Miri A.
      • Al-Deiri B.
      • Dua H.S.
      Long-term outcomes of autolimbal and allolimbal transplants.
      transplantation of ex vivo–cultivated limbal epithelial sheets has become popular in many centers worldwide.
      • Baylis O.
      • Figueiredo F.
      • Henein C.
      • et al.
      13 years of cultured limbal epithelial cell therapy: a review of the outcomes.
      • Shortt A.J.
      • Secker G.A.
      • Notara M.D.
      • et al.
      Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results.
      Regulatory issues and the expenses of maintaining a clinical-grade laboratory limit the use of ex vivo cultivation, whereas conventional limbal grafting requires no special infrastructure, but is technically demanding and carries some risk to the donor eye.
      • Ramachandran C.
      • Basu S.
      • Sangwan V.S.
      • Balasubramanian D.
      Concise review: the coming of age of stem cell treatment for corneal surface damage.
      No head-to-head trials have been conducted, and it is unclear whether one technique is more effective than the other. Therefore, availability of resources or individual preference, rather than scientific evidence, usually determines which technique a particular surgeon or center adopts.
      Having performed more than 1000 ex vivo–cultivated limbal epithelial transplantation (CLET) procedures and having reported long-term outcomes comparable with those of other groups,
      • Ramachandran C.
      • Basu S.
      • Sangwan V.S.
      • Balasubramanian D.
      Concise review: the coming of age of stem cell treatment for corneal surface damage.
      • Sangwan V.S.
      • Basu S.
      • Vemuganti G.K.
      • et al.
      Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.
      • Basu S.
      • Fernandez M.M.
      • Das S.
      • et al.
      Clinical outcomes of xeno-free allogeneic cultivated limbal epithelial transplantation for bilateral limbal stem cell deficiency.
      • Sangwan V.S.
      • Jain R.
      • Basu S.
      • et al.
      Transforming ocular surface stem cell research into successful clinical practice.
      we adopted a novel technique called simple limbal epithelial transplantation (SLET) in 2010.
      • Sangwan V.S.
      • Basu S.
      • MacNeil S.
      • Balasubramanian D.
      Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency.
      Simple limbal epithelial transplantation essentially showed that direct transplantation of a tiny limbal fragment could reverse LSCD without needing ex vivo expansion.
      • Sangwan V.S.
      • Basu S.
      • MacNeil S.
      • Balasubramanian D.
      Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency.
      After that initial report, 2 other groups independently replicated the successful outcomes in varied indications using slight modifications of the original technique.
      • Amescua G.
      • Atallah M.
      • Nikpoor N.
      • et al.
      Modified simple limbal epithelial transplantation using cryopreserved amniotic membrane for unilateral limbal stem cell deficiency.
      • Mittal V.
      • Jain R.
      • Mittal R.
      Ocular surface epithelialization pattern after simple limbal epithelial transplantation: an in vivo observational study.
      However, for wider acceptance of any new technique, the results need to be validated in larger numbers and with longer follow-up. Therefore, in this study, we report the outcomes of autologous SLET in a large cohort of patients with unilateral LSCD after sustaining ocular surface burns.

      Methods

       Study Approval, Design, and Subjects

      The Ethics Committee of the L. V. Prasad Eye Institute, Hyderabad, India, prospectively approved this study. After evaluating the results of the initial pilot trial involving 6 patients,
      • Sangwan V.S.
      • Basu S.
      • MacNeil S.
      • Balasubramanian D.
      Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency.
      the committee approved SLET as an alternative option to ex vivo CLET for the treatment of LSCD. This study was conducted in strict adherence to the tenets of the Declaration of Helsinki. All adults and legal guardians of children who underwent SLET gave informed written consent for all procedures described in this study.
      All 163 consecutive patients who underwent SLET between October 1, 2010, and March 31, 2014, were considered for analysis. Of 163 patients, 125 patients met the following inclusion criteria: (1) a documented history of chemical or thermal burns, and (2) presence of unilateral (defined as no history or clinical signs of ocular surface disease in the other eye) LSCD (defined as total or partial superficial corneal vascularization, punctate fluorescein staining of the corneal surface with or without persistent epithelial defects, conjunctivalization of the corneal surface, and absence of limbal palisades of Vogt). The 39 cases that were excluded from this study included 12 cases with unknown cause of LSCD, 11 cases of bilateral LSCD, 5 cases of primary or recurrent pterygium, 5 cases of LSCD occurring after ocular surface tumor excision, 3 cases of LSCD occurring after radiotherapy for intraocular tumors, and 3 cases of LSCD without visual potential in which SLET was performed for cosmetic correction. Patients with untreated concurrent ocular problems, such as severe dry eye disease (Schirmer's test I measure of less than 10 mm of wetting at 5 minutes), entropion, trichiasis, lagophthalmos, glaucoma, and infection, were not considered for surgery.

       Outcome Measures of Efficacy

      In recipient eyes, the primary outcome measure was the success of SLET, defined clinically as a completely epithelized, clinically stable, and avascular corneal surface (Fig 1A–J). Failure was defined as the occurrence of progressive conjunctivalization of the cornea encroaching onto the central 8 mm, occurrence of persistent epithelial defects, or both (Fig 1P–T). Occurrence of microbial keratitis and need for repeat surgery were additional criteria for failure. Survival time was calculated in months from the date of SLET to the date of failure or the date of last follow-up, depending on the clinical outcome. The secondary outcome measure of efficacy was the change in best-corrected visual acuity (BCVA) at each postoperative follow-up visit.
      Figure thumbnail gr1
      Figure 1Slit-lamp photographs showing the typical clinical course after simple limbal epithelial transplantation (SLET). A, Total limbal stem cell deficiency in the right eye of a 6-year-old child with lime burns. B, Same eye 1 day after SLET showing fibrin glue and SLET transplants under the contact lens. C, Same eye 1 month after SLET showing intact transplants with an epithelized and avascular corneal surface. D, Same eye 6 months after SLET showing peripheral conjunctivalization from 4 to 5 o'clock and 7 to 10 o'clock. E, Same eye 2 years after SLET showing no progression in conjunctivalization and maintenance of a stable epithelized corneal surface. F, G, Left eye of a 15-year old boy with total limbal stem cell deficiency (LSCD) and persistent epithelial defect (PED) after lime burns. He had previously undergone 3 amniotic membrane grafts without resolution. H, Same eye 3 months after SLET showing a quiet and stable ocular surface with anterior stromal haze in the area of the PED. I, J, At 1 and 3 years after SLET, the surface remained stable and there was significant reduction in the stromal scarring and improvement in vision over time. K, L, Right eye of the same patient showing the area of the 1-clock-hour (cornea shown divided into 12 clock hours by bluedashed lines) biopsy site (yellow dashed lines with blue asterisks). M–O, Same eye (M) 1 year, (N) 2 years, and (O) 3 years after SLET showing a stable surface without donor site LSCD. P, Total LSCD in the left eye of a 5-year-old girl after lime burns. Q, R, Same eye at (Q) 1 week and (R) 1 month after SLET showing the presence of SLET transplants and an epithelized corneal surface. S, Same eye 6 months after SLET showing recurrence of conjunctivalization superiorly, inferonasally, and inferotemporally. T, Same eye 1 year after SLET showing progression of conjunctivalization and failure of SLET.

       Outcome Measures of Safety

      The outcome measures of safety were intraoperative and postoperative complications of both limbal biopsy and SLET in the donor and recipient eye.

       Surgical Technique of Simple Limbal Epithelial Transplantation

      We followed the surgical technique that has been described previously for total LSCD
      • Sangwan V.S.
      • Basu S.
      • MacNeil S.
      • Balasubramanian D.
      Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency.
      with certain modifications for partial LSCD cases (Supplemental Appendix 1 and Supplemental Fig 1, available at www.aaojournal.org). All tissue samples excised during SLET or keratoplasty during or after SLET were processed in a standardized fashion for histopathologic and immunohistochemical analysis, as described in Supplemental Appendix 2 and Supplemental Table 1 (available at www.aaojournal.org).

       Postoperative Care and Follow-up Schedule

      All patients underwent comprehensive ophthalmic examinations of both eyes at every follow-up visit. Patients were seen on days 1, 7, 30 (at 1 month) or day 42 (at 6 weeks), 90, and at 3-month intervals thereafter. For the entire duration of the first year after surgery, patients were contacted by telephone if they missed a scheduled visit, and the next earliest possible appointment was arranged for them. This was done proactively to ensure that all patients completed at least 1 year of follow-up after the procedure. Patients were prescribed ciprofloxacin 0.3% eye drops (Cipla India, Mumbai, India) 4 times daily for 1 week and prednisolone acetate 1% eye drops (Alcon Laboratories Pvt. Ltd, Bangalore, India) 6 times daily tapered weekly over 6 weeks in both eyes. The bandage contact lens (BCL) was removed from the recipient eye on day 7 and carboxymethyl cellulose 0.5% eye drops (Allergan India Pvt. Ltd, Bangalore, India) were added in the recipient eye.

       Data Collection

      Data were collected at every visit in a predesigned format and the completed form was filed in the medical record. These data included patient age and gender, type and date of injury, details of prior ocular procedures, Snellen BCVA, intraocular pressure, presence or absence of lid abnormalities, dry eye disease, symblepharon, degree of limbal involvement, intraoperative surgical details, postoperative complications, duration of follow-up, and status of the ocular surface at each visit (slit-lamp findings including fluorescein staining).

       Validation of Diagnosis and Outcome by Independent Masked Assessors

      Five fellowship-trained cornea specialists (3 ocular surface disease specialists and 2 refractive surgeons) volunteered as assessors to validate the investigator's assessment of the diagnosis of LSCD and the outcome of treatment in every case based on 5 objective criteria: visual acuity, grade of symblepharon, grade of conjunctivalization, grade of corneal vascularization, and clock hours of limbal involvement. The grading system was adapted from Sotozono et al
      • Sotozono C.
      • Ang L.P.
      • Koizumi N.
      • et al.
      New grading system for the evaluation of chronic ocular manifestations in patients with Stevens-Johnson syndrome.
      (Supplemental Fig 2, available at www.aaojournal.org). They were masked to the identity of the patient and the nature of the procedure performed. In cases where there was disagreement between the assessors or investigators, the majority opinion of the independent assessors was taken as the final diagnosis or outcome of treatment. The details of the assessment procedure are provided in Supplemental Appendix 3 (available at www.aaojournal.org).

       Statistical Analysis

      MedCalc software version 11.4.3.0 (MedCalc Software, Mariakerke, Belgium) was used for data analysis. Continuous parametric data were reported as mean (±standard deviation) and nonparametric data were reported as median with range. Agreement between different assessors was analyzed using Light's κ statistic.
      • Hallgren K.A.
      Computing inter-rater reliability for observational data: an overview and tutorial.
      Success rates were reported as percentages with 95% confidence intervals (CI) or Kaplan-Meier survival probability rates. The Fisher exact test was used to compare categorical data. A multivariate Cox proportional hazards model with stepwise elimination using Akaike information criterion was designed (after checking the assumptions and interactions of the model) to test the association between graft failure and clinical variables. Patients younger than 16 years were considered children because 16 years is the legal age for valid informed consent in India. A 2-tailed P value less than 0.05 was considered statistically significant.

      Results

      The study included 125 patients, 65 adults and 60 children, with unilateral LSCD occurring after ocular burns. Total LSCD was seen in the affected eye in 55 adults and 52 children, and the remaining 18 eyes had partial LSCD ranging from 6 to 9 clock hours of limbal involvement. Table 1 summarizes the baseline features of the cohort and compares each characteristic between adults and children. Males were significantly more common among adults than children (P = 0.0234). This is explained by industrial trauma being more common among adults, compared with domestic accidents being the major cause among children. In urban and semiurban India, the industrial workforce comprises mainly adult men, who therefore are more likely to become injured than women. Accidental edible lime–related trauma was the most common cause of LSCD in children, whereas in adults other causes, including industrial alkali and thermal burns and vitriolage, were more common (P = 0.0001). The proportion of eyes having advanced symblepharon (51.6% vs. 27.7%; P = 0.029) and those needing keratoplasty along with SLET (8.3% vs. 3%; P = 0.256) was greater in children than in adults, respectively, indicating the greater seriousness of injury in children. The minimum follow-up after SLET in both groups was 1 year, ranging from 1 to 3.5 years in children and 1 to 4 years in adults (P = 0.7). Twenty-two patients were followed up for 2 years, 6 patients were followed up for 3 years, and 2 patients were followed up for 4 years.
      Table 1Baseline Characteristics of Patients Undergoing Simple Limbal Epithelial Transplantation for Limbal Stem Cell Deficiency Occurring after Ocular Burns
      CharacteristicChildrenAdultsP Value
      Sex
       Female27 (45)16 (24.6)0.0234
       Male33 (55)49 (75.4)
      Laterality
       Right31 (51.7)32 (49.2)0.855
       Left29 (48.3)33 (50.8)
      Cause of ocular burn
       Alkali50 (83.3)37 (56.9)0.006
       Acid6 (10)8 (12.3)
       Blast0 (0)8 (12.3)
       Cement3 (5)7 (10.8)
       Unknown chemical1 (1.7)5 (7.7)
      Prior ocular surgery
       AMG38 (63.3)33 (50.8)0.254
       LSCT5 (8.3)3 (4.6)
       Others6 (10)8 (12.3)
       None11 (18.4)21 (32.3)
      BCVA at presentation
       20/200 or worse (blindness)56 (93.3)60 (92.3)0.873
       20/70 to 20/160 (low vision)3 (5)3 (4.6)
       20/60 or better1 (1.7)2 (3.1)
      Symblepharon
       Limited to conjunctiva10 (16.7)10 (15.4)0.029
       Extending to limbus9 (15)17 (26.1)
       Extending to cornea31 (51.6)18 (27.7)
       Absent10 (16.7)20 (30.8)
      Combined SLET and keratoplasty
       Yes5 (8.3)2 (3)0.256
       No55 (91.7)63 (97)
      Extent of LSCD (clock hours)
       12 (total)52 (86.7)55 (84.6)0.744
       6–11 (partial)8 (13.3)10 (15.4)
       <60 (0)0 (0)
      Duration between ocular burn and SLET
       3–5 mos13 (21.7)9 (13.8)0.063
       6 mos–1 yr29 (48.3)21 (32.4)
       >1 yr18 (30)35 (53.8)
      Follow-up after SLET (mos)
       12–1743 (71.7)53 (81.5)0.47
       18–234 (6.7)4 (6.2)
       24–3511 (18.3)6 (9.2)
       36–482 (3.3)2 (3.1)
      AMG = amniotic membrane grafting; BCVA = best-corrected visual acuity; LSCD = limbal stem cell deficiency; SLET = simple limbal epithelial transplantation.
      Data are no. (%) unless otherwise indicated. Boldface values indicate statistically significant P values.

       Agreement between Investigators and Independent Masked Assessors

      The overall agreement between individual assessors and the investigators both for diagnosis (Light's κ, 0.93; 95% CI, 0.86–0.99) and treatment outcome (Light's κ, 0.89; 95% CI, 0.83–0.96) were excellent. There was 100% agreement between the initial impression of the investigators and the final consensus opinion of the assessors.

       Efficacy of Simple Limbal Epithelial Transplantation

      At the final follow-up visit after SLET, 95 of 125 eyes (76%; 95% CI, 68.51–83.49%) maintained a successfully regenerated stable corneal surface without progressive conjunctivalization, development of persistent epithelial defect, infection, or need for repeat SLET (Fig 2). Among patients with total LSCD, successful outcomes were observed in 44 of 55 adult eyes (80%; 95% CI, 69.43–90.5%) and in 37 of 52 pediatric eyes (71.2%; 95% CI, 58.8–83.44%; P = 0.69). Among patients with partial LSCD, successful outcomes were observed in 8 of 10 adult eyes (80%; 95% CI, 55.21–104.79%) and in 6 of 8 pediatric eyes (75%; 95% CI, 44.99–105.01%; P = 0.79). Kaplan-Meier analysis revealed a survival probability of 80% at 1 year and beyond in adults and of 72% at 1 year and 66% subsequently in children (Fig 3; P = 0.304). The success rates of SLET performed by the senior surgeon with more than 10 years of experience in ocular surface procedures (V.S.S.) was comparable with that of a less experienced surgeon with 5 years of experience (S.B.) and also with that of cornea fellows in training (P = 0.71; Fig 4A).
      Figure thumbnail gr2
      Figure 2Slit-lamp photographs showing 2-year clinical outcomes of simple limbal epithelial transplantation (SLET). A–J, Patients with partial limbal stem cell deficiency (LSCD) after ocular burns: (A–F) preoperative photographs and (F–J) their corresponding 2-year postoperative photographs showing a completely epithelized and stable corneal surface. K–U, Eyes with total LSCD: (K–O) preoperative clinical photographs and (P–T) corresponding 2-year postoperative photographs after SLET showing excellent anatomic outcomes.
      Figure thumbnail gr3
      Figure 3Graphs showing anatomic and visual outcomes of simple limbal epithelial transplantation (SLET). A, Successful regeneration of the corneal surface described as maintenance of a completely epithelized, avascular, and stable corneal surface without progressive conjunctivalization was observed in 80% eyes of adults and 71.2% eyes of children (P = 0.69) with total limbal stem cell deficiency (LSCD). Among patients with partial LSCD, successful outcomes were observed in 80% eyes of adults and 76% eyes of children (P = 0.79). B, Kaplan-Meier analysis showing a survival probability of 80% at 1 year and beyond in adults and 72% at 1 year and 66% subsequently in children (P = 0.304). C, D, Best-corrected visual acuity (BCVA) improved significantly after SLET both in (C) children and (D) adults (P < 0.0001).
      Figure thumbnail gr4
      Figure 4Kaplan-Meier survival curves comparing the success rates of simple limbal epithelial transplantation (SLET) in different subgroups. A, The survival rate was comparable between the senior ocular surface surgeon with more than 10 years of experience (V.S.S.), a comparatively less experienced surgeon with 5 years of experience (S.B.), and cornea fellows in training. The survival rate was affected adversely when (B) symblepharon extending onto the cornea was present before surgery, (C) SLET was combined with keratoplasty during surgery, and (D) the SLET transplants were lost after surgery.
      There was significant improvement in BCVA after SLET compared with baseline (Fig 3). Among the 95 cases with successful outcome, 64 (67%), or more than two thirds of eyes, recovered a BCVA of 20/60 or better. Among the remaining 31 cases, dense amblyopia (n = 12; 38.7%), stromal scarring (n = 14; 45.2%), and cataract (n = 5; 16%) were noted to be the causes of suboptimal visual improvement. Penetrating keratoplasty and cataract surgery were performed in 10 and 5 eyes, respectively, for further visual gain. A 2-line improvement in BCVA compared with baseline was seen in 94 of 125 eyes (75.2%) at the final follow-up visit.

       Risk Factors of Failure

      A Cox proportional hazards model predicted the following preoperative, intraoperative, and postoperative risk factors for SLET failure: history of acid burns (hazard ratio [HR], 3.6; 95% CI, 1.2–10.4; P = 0.0075), presence of symblepharon extending onto the cornea (HR, 7.8; 95% CI, 3.2–18.9; P < 0.0001; Fig 4B), keratoplasty combined with SLET (HR, 11.6; 95% CI, 4.2–32.2; P = 0.0001; Fig 4C), and postoperative loss of SLET transplants (HR, 22.8; 95% CI, 8.1–64.2; P < 0.0001; Fig 4D).

       Safety of Limbal Biopsy in the Donor Eye

      None (95% CI, 0–3.6%) of the donor or fellow eyes demonstrated any donor site LSCD or other complications. Typically, the donor site epithelial defect had completely healed by 1 week (Fig 1K–M). The most common observation in the donor eye was subconjunctival hemorrhage in 35 of 125 eyes (28%), which resolved by 1 month (Fig 1K–M).

       Safety of Simple Limbal Epithelial Transplantation in the Recipient Eye

      Complications of SLET are summarized in Table 2. Recurrence of progressive conjunctivalization was the most common complication in 23 of 125 recipient eyes (18.4%). Progressive symblepharon was seen in 21 of 125 eyes (16.8%). Hemorrhage under the human amniotic membrane (hAM) graft was observed in 10 of 125 eyes (8%); 9 cases resolved spontaneously, and in 1 case, the blood had to be drained by puncturing the hAM with a 26-gauge needle. Partial loss of SLET transplants was noted in 7 of 125 eyes (5.6%). In all of these cases, the patient had lost the contact lens within the first week and showed recurrence of LSCD over varying lengths of time. Sterile or microbial keratitis developed in 8 of 125 eyes (6.4%). Corneal melting with perforation was seen in 2 eyes (1.6%).
      Table 2Postoperative Complications of Simple Limbal Epithelial Transplantation for Limbal Stem Cell Deficiency Occurring after Ocular Burns
      ComplicationChildrenAdultsP Value
      Donor eye
       Subconjunctival hemorrhage17 (28.3)18 (27.7)0.93
       Pyogenic granuloma2 (3.3)0 (0)0.44
       LSCD0 (0)0 (0)1
      Recipient eye
       Conjunctivalization14 (23.3)9 (13.8)0.17
       Symblepharon12 (20)9 (13.8)0.47
       Hemorrhage under hAM4 (6.7)6 (9.2)0.84
       Loss of transplants4 (6.7)3 (4.6)0.91
       Detached hAM3 (5)1 (1.5)0.55
       Keratitis5 (8.3)3 (4.6)0.63
       Corneal perforation2 (3.3)0 (0)0.44
       Lignocaine allergy0 (0)1 (1.5)0.97
      hAM = human amniotic membrane graft; LSCD = limbal stem cell deficiency.
      Data are no. (%) unless otherwise indicated.

       Histopathologic and Immunofluorescence Analysis

      Histopathologic and immunofluorescence analysis showed the following results. In all cases, the excised pannus showed the presence of variably thick, stratified squamous epithelium with occasional goblet cells and underlying fibrocollagenous matrix laden with plasma–lymphocytic infiltrate (Fig 5A). This confirmed the clinical diagnosis of LSCD. In 10 cases, areas of absent epithelium with underlying necrosis suggestive of persistent epithelial defect were seen. In 7 cases, basophilic deposits suggestive of calcification were seen in the subepithelial tissue. Corneas that underwent SLET showed the presence of a smooth stratified squamous epithelium without goblet cells identical to normal corneal tissue over a thick basement membrane with positive periodic acid–Schiff staining suggestive of persistent hAM (Fig 5A). Immunohistochemistry examination confirmed the presence of corneal markers CK3 and CK12 in corneas that underwent SLET, which were absent in the excised pannus. Similarly, conjunctival markers MuC5AC and CK19 were present in the excised pannus and absent in corneas that underwent SLET (Fig 5A). Immunohistochemistry examination of corneas that underwent SLET also showed positive results for epithelial progenitor cell marker p63 (Fig 5B) and focal basal cell positive results for putative limbal epithelial stem cell markers ΔNp63α and ABCG2 (Fig 5C). In summary, immunohistochemistry analysis showed regeneration of a normal corneal epithelium with focal retention of limbal epithelial stem cells in the basal epithelial layers after SLET.
      Figure thumbnail gr5
      Figure 5Photomicrographs showing immunohistochemistry analysis results using fluorescence-labeled antibodies. A, Left column represents sections of the ocular surface pannus excised from eyes with clinically diagnosed limbal stem cell deficiency (LSCD) occurring after ocular burns. Middle column represents sections of corneas excised during penetrating keratoplasty from eyes of patients who had previously undergone successful simple limbal epithelial transplantation (SLET). Right column represents sections of a normal cadaveric cornea as controls. Immunohistochemistry analysis confirmed the diagnosis of LSCD in the excised pannus, which showed negative results for corneal cytokeratin markers (CK3/12) and positive results for conjunctival markers (CK19, Mu5Ac). Corneal sections obtained after SLET showed stratified squamous epithelium without goblet cells with cytokeratin expression identical to that of normal control corneas (CK3+/12+, CK19/Mu5Ac) over a thick basement membrane with positive periodic acid–Schiff results suggestive of persistent human amniotic membrane (blue asterisks). B, Top row represents sections of corneas that underwent SLET and the bottom row represents sections of normal cadaveric human corneas. Positive nuclear expression of epithelial progenitor cell marker p63 is seen in both corneas that underwent SLET and normal corneas. C, Left column represents sections of corneas that underwent SLET and the right column represents sections of normal human cadaveric limbus. Positive expression (white arrows) of putative limbal epithelial stem cell markers ΔNp63α and ABCG2 is seen focally in the basal epithelial layers in corneas that underwent SLET, similar to that seen in the normal limbus. This indicates the persistence of limbal epithelial stem cells on the cornea after SLET. White asterisks indicate artifacts.

      Discussion

      This study showed that, as indicated by initial reports,
      • Sangwan V.S.
      • Basu S.
      • MacNeil S.
      • Balasubramanian D.
      Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency.
      • Amescua G.
      • Atallah M.
      • Nikpoor N.
      • et al.
      Modified simple limbal epithelial transplantation using cryopreserved amniotic membrane for unilateral limbal stem cell deficiency.
      • Mittal V.
      • Jain R.
      • Mittal R.
      Ocular surface epithelialization pattern after simple limbal epithelial transplantation: an in vivo observational study.
      • Vazirani J.
      • Basu S.
      • Sangwan V.
      Successful simple limbal epithelial transplantation (SLET) in lime injury-induced limbal stem cell deficiency with ocular surface granuloma.
      • Bhalekar S.
      • Basu S.
      • Lal I.
      • Sangwan V.S.
      Successful autologous simple limbal epithelial transplantation (SLET) in previously failed paediatric limbal transplantation for ocular surface burns.
      SLET was successful in the long-term regeneration of the corneal surface in a large cohort of patients with unilateral LSCD occurring after ocular burns. Simple limbal epithelial transplantation was equally effective in children and adults as well as in total and partial LSCD. In addition to surface restoration, most patients undergoing SLET reported a significant improvement in visual acuity. We also showed that the corneal surface after SLET was identical to that of the native cornea, comprising uniform nonkeratinized stratified squamous epithelium without goblet cells or vascularization. Because SLET requires only minimal donor tissue and does not require any clinical-grade laboratory support, these results make SLET an attractive alternative to conventional limbal grafting or ex vivo CLET for the treatment of LSCD.
      Table 3 provides an overall comparison of the results of this study with that of other large series (more than 20 cases) of autologous CLET and conjunctival–limbal autografting for the treatment of unilateral LSCD.
      • Kenyon K.R.
      • Tseng S.C.
      Limbal autograft transplantation for ocular surface disorders.
      • Sangwan V.S.
      • Basu S.
      • Vemuganti G.K.
      • et al.
      Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.
      • Rama P.
      • Matuska S.
      • Paganoni G.
      • et al.
      Limbal stem-cell therapy and long-term corneal regeneration.
      • Pauklin M.
      • Fuchsluger T.A.
      • Westekemper H.
      • et al.
      Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency.
      • Barreiro T.P.
      • Santos M.S.
      • Vieira A.C.
      • 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.
      Because the inclusion criteria and definition of success vary across studies, superficial comparisons sometimes can lead to deeply flawed conclusions. Therefore, it may be worthwhile to limit the comparison with our own experience with autologous CLET. We have reported an overall success rate of 71.4% in total LSCD
      • Sangwan V.S.
      • Basu S.
      • Vemuganti G.K.
      • et al.
      Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.
      and 75% in partial LSCD,
      • Vazirani J.
      • Basu S.
      • Kenia H.
      • et al.
      Unilateral partial limbal stem cell deficiency: contralateral versus ipsilateral autologous cultivated limbal epithelial transplantation.
      rates that are comparable with the outcomes of SLET in this study. In children, however, SLET seemed to have a much better success rate than CLET (71% vs. 37%).
      • Sejpal K.
      • Ali M.H.
      • Maddileti S.
      • et al.
      Cultivated limbal epithelial transplantation in children with ocular surface burns.
      Although one would intuitively expect better results in pediatric patients owing to the greater regenerative potential of younger donor tissue, this effect may have been dampened by the greater severity of injury in children.
      Table 3Outcomes of Different Techniques of Autologous Limbal Stem Cell Transplantation for Treatment of Unilateral Limbal Stem Cell Deficiency
      AuthorsYearTechniqueNo. of EyesAnatomic Success Rate (%)2-Line Visual Acuity Gain (%)Follow-up Range (yrs)
      Kenyon and Tseng
      • Kenyon K.R.
      • Tseng S.C.
      Limbal autograft transplantation for ocular surface disorders.
      1998CLAu2677650.2–3.75
      Rama et al
      • Rama P.
      • Matuska S.
      • Paganoni G.
      • et al.
      Limbal stem-cell therapy and long-term corneal regeneration.
      2010CLET10768541–10
      Pauklin et al
      • Pauklin M.
      • Fuchsluger T.A.
      • Westekemper H.
      • et al.
      Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency.
      2010CLET3077730.8–6
      Sangwan et al
      • Sangwan V.S.
      • Basu S.
      • Vemuganti G.K.
      • et al.
      Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.
      2011CLET20071.460.31–7.6
      Barreiro TP et al
      • Barreiro T.P.
      • Santos M.S.
      • Vieira A.C.
      • 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.
      2014CLAu2387800.9–3
      Basu et al2015SLET12575.2751–4
      CLAu = conjunctival–limbal autografting; CLET = cultivated limbal epithelial transplantation; SLET = simple limbal epithelial transplantation.
      The findings of this study question the paradigm regarding the minimum amount of limbal tissue that is necessary to regenerate the entire corneal surface. It seems that the conventional assumption of needing 3 to 6 clock hours of donor tissue based on animal studies may not necessarily hold true in humans.
      • Kenyon K.R.
      • Tseng S.C.
      Limbal autograft transplantation for ocular surface disorders.
      Even the pioneers of limbal transplantation themselves have questioned this and have tried using lesser amounts of limbal tissue, with successful results.
      • Kheirkhah A.
      • Raju V.K.
      • Tseng S.C.
      Minimal conjunctival limbal autograft for total limbal stem cell deficiency.
      Simple limbal epithelial transplantation also challenges the usefulness of ex vivo cultivation, considering the additional costs involved. These results suggest that, at least in terms of clinical efficacy, it does not seem to matter whether a 1-clock-hour limbal biopsy sample is cultured ex vivo on a Petri dish with laboratory reagents or in vivo on the corneal surface itself. The visual outcomes after SLET are particularly impressive. It is known that limbal biopsies yield both epithelial and mesenchymal stem cells and that the mesenchymal cells can modulate corneal wound healing and can ameliorate scarring.
      • Basu S.
      • Hertsenberg A.J.
      • Funderburgh M.L.
      • et al.
      Human limbal biopsy-derived stromal stem cells prevent corneal scarring.
      It may be possible that keeping the epithelial-mesenchymal microenvironment intact in SLET has a beneficial effect on the corneal stroma and results in less scarring, and hence better visual outcomes.
      Mittal et al
      • Mittal V.
      • Jain R.
      • Mittal R.
      Ocular surface epithelialization pattern after simple limbal epithelial transplantation: an in vivo observational study.
      recently described the epithelization pattern after SLET in a clinical setting. They showed that epithelization starts from the limbal transplants over the hAM on the second postoperative day and that ocular surface epithelialization is completed within 2 weeks, which is similar to our experience. Interestingly, they also noted variations in the epithelization rate depending on the size of the transplants and the age of the donor.
      • Mittal V.
      • Jain R.
      • Mittal R.
      Ocular surface epithelialization pattern after simple limbal epithelial transplantation: an in vivo observational study.
      Using ultra high-resolution optical coherence tomography, Amescua et al
      • Amescua G.
      • Atallah M.
      • Nikpoor N.
      • et al.
      Modified simple limbal epithelial transplantation using cryopreserved amniotic membrane for unilateral limbal stem cell deficiency.
      showed persistence of the hAM while epithelial cells stratified over it, which correlates well with the histopathologic findings of this study. It is important to note that hAM plays a critical role in promoting and preserving the stemness of the limbal epithelial stem cells,
      • Mariappan I.
      • Kacham S.
      • Purushotham J.
      • et al.
      Spatial distribution of niche and stem cells in ex vivo human limbal cultures.
      • Chen S.Y.
      • Han B.
      • Zhu Y.T.
      • et al.
      HC-HA/PTX3 purified from amniotic membrane promotes BMP signaling in limbal niche cells to maintain quiescence of limbal epithelial progenitor/stem cells.
      and the membrane's persistence after SLET may contribute to the prolonged success of the procedure. Both epithelial progenitor cells (p63+) and limbal epithelial stem cells (ΔNp63α+/ABCG2+) were observed in the basal layers of the regenerated epithelium, next to the retained hAM, many months after SLET.
      This study also sheds new light on the possible factors responsible for recurrence of LSCD and failure of SLET. The presence of symblepharon extending up to the cornea before surgery could indicate some form of conjunctival deficiency, and outcomes may improve further if the symblepharon is addressed before or at the time of SLET. Inadvertent corneal perforation at the time of dissection should be avoided because it necessitates penetrating keratoplasty, and this in turn adversely impacts the outcome of SLET. We previously showed a similar detrimental impact of combining penetrating keratoplasty with CLET.
      • Basu S.
      • Mohamed A.
      • Chaurasia S.
      • et al.
      Clinical outcomes of penetrating keratoplasty after autologous cultivated limbal epithelial transplantation for ocular surface burns.
      Therefore, it may be advisable to identify those eyes with extremely thin corneas by performing optical coherence tomography or ultrasound biomicroscopy before surgery. We speculate that such cases may fare better if a lamellar corneal graft is planned along with SLET. Early loss of SLET transplants is another factor that may result in recurrence of LSCD. This finding confirms the observation made by Konomi et al
      • Konomi K.
      • Satake Y.
      • Shimmura S.
      • et al.
      Long-term results of amniotic membrane transplantation for partial limbal deficiency.
      that hAM alone, without limbal transplantation, may not be sufficient for corneal surface regeneration, even in cases with partial LSCD. To prevent this rare complication, a temporary tarsorrhaphy or an additional layer of hAM
      • Amescua G.
      • Atallah M.
      • Nikpoor N.
      • et al.
      Modified simple limbal epithelial transplantation using cryopreserved amniotic membrane for unilateral limbal stem cell deficiency.
      may be considered in cases where the contact lens is unstable or is likely to be lost early (e.g., in very young children).
      The major strengths of this study are that it was planned prospectively, it had a large sample, and the diagnosis we made and outcomes we assessed in every case were validated by 5 independent masked assessors. Although LSCD was defined clinically in this study, the surface pannus excised from all eyes was subjected to histopathologic examination to confirm the diagnosis. Other more objective means, such as confocal microscopy or impression cytologic analysis, could have been used. However, considering that almost half of the cohort constituted children, it would have meant extra procedures under anesthesia at each follow-up visit, which was not feasible. We compared our results with historical controls instead of conducting a comparative clinical trial. However, for a noninferiority trial of SLET compared with CLET or conjunctival–limbal autografting, assuming a 2-tailed α error of 5%, power of 80%, and a 75% success rate for CLET or conjunctival–limbal autografting per the published literature, a sample size of 800 eyes in each group is needed. Because LSCD occurring after ocular burns is a rare condition, achieving this number, even if a multicenter trial is attempted, is impractical. In our institute, approximately 100 new cases of chronic ocular burns are seen every year, and therefore, it would take us 16 years just to enroll enough patients for such a study.
      Transplantation of ex vivo–cultivated limbal epithelial sheets is currently in vogue and is accepted as standard of care, particularly in Europe and Japan. Although we have published the largest clinical series of this technique with excellent outcomes over the past decade in more than 1000 cases, we realized that this technique was prohibitively expensive and therefore was out of reach of most cornea-blind individuals, most of whom live in the developing world. Because of regulatory restrictions, it is not practiced in many countries, including the United States. To circumvent these limitations, we developed the technique of SLET, drawing from our experience with ex vivo cultivation. This study showed that the results of SLET are extremely promising and that SLET potentially could make ex vivo cultivation redundant and limbal transplantation accessible to hundreds of thousands of people with corneal blindness worldwide.

      Acknowledgments

      The authors thank the following individuals for serving as independent masked assessors who validated the diagnosis and treatment outcomes of patients included in this study: James Chodosh, MD, MPH, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts; Guillermo Amescua, MD, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida; Jayesh Vazirani, MS, Cornea and Refractive Service, Maheshwari Eye Care Hospital, Rajkot, Gujarat, India; and Sunita Chaurasia, MS, and Jagadesh Reddy, MS, Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India.

      Supplementary Data

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