REVIEW
Systematic Review and Meta-analysis on Transplantation of Ex Vivo Cultivated Limbal Epithelial Stem Cell on Amniotic Membrane in Limbal Stem Cell Deficiency Yuan Zhao, MD,* and Lei Ma, MD†
Purpose: Ex vivo cultured limbal epithelial transplantation (CLET) with amniotic membrane (AM) as the substrate is a relatively new type of surgical therapy in treating limbal stem cell deficiency (LSCD). We summarize available evidence for determining the efficiency of this technique by a systematic review and metaanalysis.
Methods: Searching the following electronic databases, MEDLINE, EMBASE, and the Cochrane Library, we analyzed the selected articles in 5 main aspects: donor screening, culture methods, evidence of cultivated stem cells, subjective symptoms, and adverse events through systematic review. Specifically, meta-analysis was used in evaluating improvements in ocular surface and visual acuity.
Results: A total of 18 articles involving 572 eyes of 562 patients were available. The rate of success and 2-line improvement in bestcorrected visual acuity (BCVA) was 67% [95% confidence interval (CI), 0.59–0.75; I2 = 60%] and 62% (95% CI, 0.57–0.66; I2 = 37.7%), respectively; and no difference was found both in success rate [odds ratio (OR), 1.35; 95% CI, 0.63–2.89; I2 = 46%] and visual acuity outcome (OR, 1.53; 95% CI, 0.67–3.45; I2 = 42.1%) between autograft and allograft.
Conclusions: CLET is efficacious in patients with LSCD, and no difference both in success rate and visual acuity outcome between autograft and allograft was found. Overall safety profile was good, with most side effects being transient and amenable to subsequent treatments. The long-term results of autograft and allograft will inform future treatment algorithms and techniques with random control trials and better-designed analysis. Received for publication October 16, 2014; revision received January 2, 2015; accepted January 19, 2015. Published online ahead of print March 19, 2015. From the *Department of Clinical Medicine, Shanxi Medical University, Taiyuan, People’s Republic of China; and †Department of Ophthalmology, Second Teaching Hospital of Shanxi Medical University, Taiyuan, People’s Republic of China. Supported by National Natural Science Foundation of China (Grant 30973248); Shanxi Provincial Foundation for Returned Scholars (Grant 200860, 200856). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.corneajrnl.com). Reprints: Lei Ma, MD, Department of Ophthalmology, Second Teaching Hospital of Shanxi Medical University, 382# Wuyi Rd, Taiyuan, Shanxi 030001, People’s Republic of China (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
592
| www.corneajrnl.com
Key Words: limbal stem cell deficiency, amniotic membrane, ex vivo cultured limbal epithelial transplantation, meta-analysis, systematic review (Cornea 2015;34:592–600)
S
tem cells of adult tissues, which are relatively undifferentiated and self renewing, serve as a reservoir for cell replacement to maintain homeostasis of the tissue mass.1,2 In the cornea, equivalent populations of limbal epithelial stem cells (LESCs) generate and renew the corneal epithelium throughout life and are thus essential for maintaining corneal transparency and vision.3 Various diseases, such as chemical or thermal injury, UV and ionizing radiation, Stevens–Johnson syndrome, advanced ocular cicatricial pemphigoid, contact lens overwear, multiple surgeries, antimetabolites, extensive microbial infection, or aniridia can lead to limbal stem cell deficiency (LSCD).4–6 These conditions can make patients feel pain and photophobia, and even result in chronic inflammation, conjunctival epithelial ingrowth (conjunctivalization) invading the cornea, corneal vascularization, poor epithelial integrity manifested as an irregular surface, recurrent epithelial erosion or persistent epithelial ulceration, and destruction of the basement membrane, leading to fibrous ingrowth.7 However, conjunctivalization is central to the diagnosis of LSCD.8 Moreover, clinical signs observed are epithelial haze, superficial subepithelial vascularization, persistent or recurrent epithelial defects, epithelial and stromal inflammation, late fluorescein staining, and loss of the limbal palisades of Vogt.8–10 Impression cytology, to identify goblet cells, or monoclonal antibodies to cytokeratin 3 and cytokeratin19 to confirm a conjunctival phenotype, can usually be used to make additional confirmation.9,11 LSCD may be classified as partial or total with limbal stem cells partially or totally depleted, resulting in varying degrees of stem cell deficiency with abnormalities and conjunctivalization of the corneal surface.9,12 Although the conservative options available for managing LSCD, whether partial or total, are intensive lubrication, nonpreserved artificial tear drops, bandage contact lenses, and autologous serum eye drops, only the latter is supported by evidence in the literature.13,14 It must be noted that corneal transplantation, which involves replacing the central cornea (excluding the limbus), cannot be used as a treatment option for LSCD due to the lack of host limbal stem cells to replace the epithelium overlying the corneal graft.15,16 In partial LSCD, it has been demonstrated that repeated debridement of migrating Cornea Volume 34, Number 5, May 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
conjunctival epithelium in the acute phase following injury, known as sequential sector conjunctival epitheliectomy (SSCE), can reduce or prevent conjunctival ingrowth,17 and it may be combined with amniotic membrane transplantation (AMT).18 When LSCD is total, a population of autologous or allogeneic LESCs must be transplanted if a stable corneal epithelial phenotype is to be regained.8 A variety of techniques have been developed to transplant limbal stem cells, such as keratolimbal lamellar allograft (KLAL),19 which was the first to be introduced and the most widely used, and also conjunctival-limbal autografts (CLAU) and living related conjunctival-limbal allografts (lr-CLAL).6 All these surgical options may be combined with penetrating keratoplasty (PK) or deep lamellar keratoplasty (DLK), with or without cataract surgery. A novel method of transplanting limbal stem cells is through ex vivo cultured limbal epithelial transplantation (CLET), which has theoretical advantages over conventional treatments. The limbal epithelial cells are harvested from the contralateral healthy eye (or from a normal area of a partially stem cell deficient eye), living related donors, or from fresh tissue of deceased donors (in cases of bilateral total limbal stem cell deficiency) by a minimal (eg, 2 · 2 mm2) limbal biopsy, which minimizes the risk of precipitating stem cell failure in the donor eye and provides the option of taking a further biopsy, if required. CLET may also have a reduced risk of allograft rejection because antigenpresenting macrophages do not survive the process of ex vivo culture.20 Cryopreserving excess ex vivo cultured cells for a prolonged period21 could also provide a source of additional cells for transplantation, should the need arise. Various materials (amniotic membrane, fibrin, contact lens, etc.) may be used as a cell substrate in the ex vivo culture. But human amniotic membrane (HAM), which promotes epithelialization, inhibits fibrosis, has anti-inflammatory and antiangiogenic properties, and possesses antimicrobial and antiviral properties and a high hydraulic conductivity,22,23 with low or no immunogenicity,24 has become the widely used material25–42 acting as a surrogate environmental stem cell niche.43 The use of ex vivo cultured LESCs to treat corneal LSCD in humans was first described by Pellegrini et al44 in 1997. Subsequently, plenty of case series and reviews have reported the efficiency and evidence of this treatment.12,25–43,45–47 However, with the lack of sufficient cases or valid measurements, they failed to provide a convincing assessment of this surgery. Moreover, there is controversy regarding the clinical outcome between different sources of limbal stem cells, such as autograft and allograft. Therefore, our study aims to view all relevant published articles to determine the overall success rate and visual acuity outcome of CLET through a systematic review and meta-analysis and to analyze whether the outcome is different between autograft and allograft, expecting that it may provide some suggestions for this procedure.
MATERIALS AND METHODS Search Strategy The search was performed in July 2014, which was restricted to English language reports without published time Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Systematic Review and Meta-analysis
limits. Using the combined key words “limbal stem cell deficiency” and “amniotic membrane,” we searched the following electronic databases: MEDLINE, EMBASE, and the Cochrane Library. Trying to increase the recall rate, we did not use “cultured limbal epithelial transplantation” as key words. In addition, reference lists were scanned to identify any additional reports.
Inclusion Criteria Qualified studies included in the meta-analysis must meet the following criteria. Participants: All age groups with different kinds of LSCD. Interventions: Ex vivo cultured limbal epithelial cells with AM as a substrate for transplantation, including those who received conservative treatments or other (except this intervention) surgeries previously. Preoperative evaluation of patients and postoperative outcome measures should both be obtained in each article. Accordingly, variables such as age, gender, different ex vivo cultivated methods, source of limbal stem cells, cause, extent of LSCD, duration between injury and surgery, and various previous treatments all are potential sources of heterogeneity.
Article Selection Titles and abstracts were reviewed to exclude any obviously irrelevant reports and full-text articles were ordered for studies potentially suitable for inclusion. Case reports, review articles, animal or laboratory studies, conference abstracts, and letters were excluded.
Quality Assessment The quality of all included articles was assessed using a validated checklist consisted of 14 questions (see Table, Supplemental Digital Content 1, http://links.lww. com/ICO/A265)12 according to a relevant study by Cauchi et al.12 This checklist was developed by the Review Body for Interventional Procedures, which is an independent review body that carries out systematic reviews for the National Institute for Health and Clinical Excellence Interventional Procedures Program and was adapted from several sources. Following are details used to evaluate the included studies. The prognostic factors were described as age, gender, etiology, lid abnormalities, treatments before CLET, intervals between injury and this surgery, type of LSCD, source of limbal stem cells, and extent of dry eye. Studies including most of these items (at least 8 of the 9) were considered as “YES.” The objective outcome measures may be defined as explicit description of clinical with or without impression cytology findings of stability of the ocular surface and visual acuity. All important outcomes such as subjective symptoms and objective outcomes should be included. Scientific scales and scores of these outcomes, which add possibility for statistic analysis, would be better. Additionally, 6 months was set as a reasonable follow-up period.48 www.corneajrnl.com |
593
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Zhao and Ma
Data Extraction Data such as study design, participant numbers, diagnoses, follow-up, cause and type of LSCD, source of limbal stem cells, culture method, clinical outcome, and adverse events were analyzed. The main clinical outcome was success rate of this treatment, which was defined as the reestablishment of a stable and transparent corneal epithelium, resolution of corneal conjunctivalization and persistent epithelial defect, and regression of corneal vascularization. (And if the definition is different or unclear in some articles, we would work out the new success rate according to the postoperative descriptions reported in these articles). Secondary outcome was an improvement in vision of at least 2 Snellen lines of bestcorrected visual acuity (BCVA). Available raw data of visual acuity were analyzed. In detail, visual acuity (VA) “hand motion” was calculated as 3.0 logarithm of the minimum angle of resolution (logMAR), “counting fingers at 1/2-ft distance” as 2.6 logMAR, “counting fingers at 1-ft distance” as 2.3 logMAR, and “counting fingers at 2-ft distance” as 2.0 logMAR.31 Thus, VA from hand motion (HM) to counting fingers (CF) was considered as a 2-line improvement.
FIGURE 1. Flowchart showing process of article selection.
All 18 studies were noncomparative case series (Table 1). However, lack of high-quality objective data in the form
of randomized controlled trials (RCT) is an inherent problem with surgical procedures as they were less suited to this study design than medical interventions. In accordance with the inclusion criteria, only partially available data were extracted from the study by Schwab.42 Basically, a “yes” to a quality assessment question represented a positive measure of quality. Seventeen of 18 studies (94.4%) had a representative sample, 9 of 18 (50%) outlined a recruitment period, 16 of 18 (88.9%) had objective outcome measures, and 15 of 18 (83.3%) had a clear description of the 3 main findings. Six of 18 studies (33.3%) had inclusion/exclusion criteria clearly described. Twelve of 18 studies (66.7%) had a minimum follow-up period of more than 6 months. Only 2 of 18 studies (11.1%) reported that an experienced surgeon performed the procedures. Three of 18 studies (11.7%) considered all relevant outcomes and identified all prognostic factors, and 4 of 18 (22.2%) collected data prospectively. Two of 18 studies had participants entering the study at a similar stage of disease progression and gave the information on dropouts, and no study specifically stated blinding of outcome assessors (Table 2 and Fig. 2). Totally, 572 eyes of 562 patients were studied with the size of the population ranging from 4 to 200, and approximately 74.6% were men. The overall mean age was 25 years.43 The follow-up period ranged from 1 to 118 months, the minimum being 6 months. The etiology of LSCD in these studies showed that the most common cause was chemical burn (74.1%). In 7 articles, 82 eyes (33.7%) were clinically diagnosed as partial LSCD, which were with or without the results of impressing cytology and cytokeratin profiling. Of the 572 eyes, 486 (85.0%) received autografts, and 86 (15.0%) received allografts from corneas of deceased donors or living related donors. However, 5 studies noted the duration between injury and surgery.25,26,28,29,39
594
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Statistical Analysis R-3.0.3 software was used for meta-analysis. For the overall success rate and visual outcome, we calculated the combined ratio using Freeman–Tukey double arcsine transformation methods with metaprop package, and effect size and 95% confidence intervals (CI) were calculated. For comparing outcomes of autograft and allograft, we adopted odds ratios (OR) and 95% CI using metabin package. If significant heterogeneity was observed (P , 0.05), a random-effects model was used for pooling the data; otherwise, a fixed-effects model was used (P . 0.05). We conducted regression analysis to assess publication bias (P . 0.05 was considered no publication bias), and when the sample was small, funnel plot was used for assessment.
RESULTS Study Identification A total of 389 articles of potential interest were identified by the original literature search (Fig. 1), including 140 duplications (1 article existed with 3 different data). Reviewing the abstracts, 208 articles were excluded for either not concerned with CLET or not meeting the inclusion criteria. Furthermore, the remaining 40 full-text articles were read in detail. A further 22 exclusions were made for the following reasons: conference papers, 5; reviews, 4; case reports, 6; without postoperative outcome, 2; and 5 with unqualified intervention. Eventually, a total of 18 papers were available.
Study Characteristics
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Systematic Review and Meta-analysis
TABLE 1. Characteristics of Included Studies
25
Sejpal et al Subramaniam et al26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al38 Sharma et al30,39,49 Tsai et al40 Koizumi et al41 Schwab et al37,42
Cause
Extent of LSCD
Donor
Country
Design
No. Eyes/ No. Patients
Follow-up, Mean 6 SD (range), m
CB
Others
Partial
Total
Autograft
Allograft
India India Norway India Iran India Thailand United Kingdom Belgium United Kingdom Germany Japan United States Japan India Taiwan Japan United States
RCS RCS RCS RCS CS CS PCS PCS CS PCS CS RCS CS RCS CS CS RCS PCS
107/107 40/39 9/9 200/200 8/8 50/50 19/18 8/8 18/18 10/10 44/38 9/9 7/7 13/13 4/4 6/6 13/11 7/7
41.2 6 26 (12–118) 33.4 6 29.2 (1–87) 18.4 (11–28) 36 6 19.2 (12–91.2) 34.0 6 13.5 (6–48) 11.0 6 8.0 (1.5–25) 26.1 6 13.5 (6–47) 19 (13–20) 22 (4–43) NA 28.5 6 14.9 (9–73) 14.6 6 4.4 (6–20) 11.57 (6–19) NA 19.5 6 7.4 (9–26) 15 6 2 (12–18) 11.2 6 1.3 (9–13) 4.43 (2–8)
NA 34 4 179 5 41 13 6 7 4 NA 1 6 2 3 3 3 1
NA 6 5 21 3 9 6 2 11 6 NA 8 1 11 1 3 10 6/7
15 35 5 0 0 NA 8 0 3 NA 12 0 NA 0 0 4 0 NA
92 5 4 200 8 NA 11 8 15 NA 32 9 NA 13 4 2 13 NA
107 40 9 200 8 34 12 8 15 3 30 2 3 0 4 6 0 5
0 0 0 0 0 16 7 0 3 7 14 7 4 13 0 0 13 2
Reference
CB, chemical burn; CS, case series; NA, not available; PCS, prospective case series; RCS, retrospective case series.
Donor Screening
stated human amniotic membrane screening. Taken together, these findings highlight the absence of clearly reporting tissue screening although it might have been performed.
Four of 16 studies32,34,35,38 (the remaining 2 had no autografts used) were screened for autologous donors. Screening of live donors was reported in 5 of 8 studies,34,35,37,38,42 which used living allogeneic limbal cells. That corneas from deceased donors were used as a source of limbal stem cells for culture30,31,33,34,36,41 was not explicitly stated whether these were screened. Additionally, 4 of 18 studies29–31,42 explicitly
Ex Vivo Culture Methods As for the ex vivo culture, we specifically focus on the following 4 aspects: culture system, the type of AM, and
TABLE 2. Quality Items Per Study Reference 25
Sejpal et al Subramaniam et al26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al38 Sharma et al30,39,49 Tsai et al40 Koizumi et al41 Schwab et al37,42
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Y Y Y Y Y Y Y Y Y U Y Y Y Y Y Y Y Y
Y Y N Y Y N Y Y N N U U N N N N N N
N U N U U N N Y N U N Y N U N N U N
U U U U U U U U U U Y Y U U U U U U
N Y N Y N N N N N N N N N N Y N N N
N N N N U U Y Y U Y U N U N U U N Y
Y Y Y Y Y N N N N N Y Y N N N N Y Y
N N N N U U U U U U U N U N U U N U
U U U U U U U U U U Y U U U U Y U U
Y Y N U Y Y Y Y Y Y Y Y Y Y Y Y Y Y
N N N N N N N Y Y N Y N N N N N N N
Y N Y Y Y N Y Y N N Y Y Y N Y Y Y N
N N N N N N N N Y N N N N N N N N Y
Y Y N U Y Y Y Y Y Y Y Y Y Y Y Y N Y
N, no; U, unclear; Y, yes.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.corneajrnl.com |
595
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Zhao and Ma
FIGURE 2. Bar graph showing the proportion of studies per quality item.
whether using mouse 3T3 fibroblast or airlifting or not. Consequently, of the 17 studies involved, 14 used explant systems, 2 used suspension systems, and 1 used both. Denuded AM with the absence of the amniotic epithelial cell was commonly used in 12 studies, although the remaining 5 studies either used intact AM or stated unclearly. 3T3 fibroblasts were usually used to contribute to cultivated stem cells ex vivo. However, of the 17 relative studies, only 2 reported the use of 3T3 fibroblasts, 1 with unclear documentation and another with it in allograft culture, but not in autograft. Moreover, airlifting was also not used in 13 articles, with 2 unclearly reported (Table 3).
Evidence of the Presence of Stem Cells in Cultures To provide evidence for the presence of stem cells in the transplanted cell population, various methods from
different aspects were involved. A monolayer of cells being achieved for transplantation after 10 to 15 days were reported in 2 studies,26,28 whereas 4 other studies30–32,37 reported a multilayered epithelium. A well-conserved basal layer formed by cuboidal cells with positive hematoxylin–eosin staining expression was described,32,36 and was further confirmed by the formation of a primitive epithelium with a prominent basal layer of cuboidal cells possessing a high nucleus–cytoplasm ratio observed in transmission electron microscopy (TEM).32 The expression of specific markers, such as stem cell–positive markers (p63, ABCG2, and K19) and the differentiation markers (K3/K12), was detected in the same culture.26,30–33,36 Expression of more K19 and less K3/K12 in the basal layer of the cultured cells was reported in several studies.30,32,36 The colony-forming efficiency of representative cultures, which was used to analyze the presence of stem cells, resulted in a proportion varying from 2% to 9%.36 Corneal buttons from eyes that underwent
TABLE 3. Culture Method Reference Sejpal et al25 Subramaniam et al26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al38 Sharma et al30,39,49 Tsai et al40 Koizumi et al41
System
AM
3T3 Fibroblast
Airlifting
Time (days)
Explant Explant Explant Explant Explant Explant Explant Explant Explant Suspension Explant Explant and suspension Suspension Explant Explant Explant Explant
dAM dAM UC dAM dAM dAM dAM iAM dAM iAM iAM dAM dAM dAM dAM UC dAM
(2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) Auto (2), Allo (+) (+) (2) UC (2) (+)
(2) (2) (2) (2) UC (2) (+) UC (2) (2) (2) (2) (2) (2) (2) (2) (+)
10–14 10–15 14–21 10–14 10–14 7–15 17–28 10–14 14 14–21 14 14 21–28 12–20 14 14–21 32
dAM, denuded AM; iAM, intact AM; UC, unclear.
596
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Systematic Review and Meta-analysis
subsequent PK were found to have multiple layers of stratified epithelium, similar to normal corneal phenotype with positive staining of p63, CK3, and CK12.31,39
Improvement in Ocular Surface Eighteen articles25–42 reported a success rate (Table 4) with 381 successful procedures in a total of 572 eyes. The pooled success rate was 67% (95% CI, 0.59–0.75; I2 = 60%, P = 0.0006; Fig. 3A) using the random-effects model. Seven studies,30,31,33–37 which included 157 eyes, reported the success rate of autograft (99 eyes) and allograft (58 eyes) simultaneously. Consequently, in terms of the success rate, there may be no significant difference between autograft and allograft (OR, 1.35; 95% CI, 0.63–2.89; I2 = 46%, P . 0.05; Fig. 3B).
Visual Acuity Seventeen articles,25,27–42 which included 532 eyes, reported a 2-line improvement in BCVA in 335 eyes. The pooled rate was 62% (95% CI, 0.57–0.66; I2 = 37.7%, P . 0.05; Fig. 3C). Seven studies30,31,33–37 reported visual acuity outcome of autograft and allograft, with 78 and 49 eyes, respectively. As a result, there may be no difference between the 2 groups with regard to 2-line improvement in BCVA (OR, 1.53; 95% CI, 0.67–3.45; I2 = 42.1%, P . 0.05; Fig. 3D).
Egger test, there was slight publication bias. Funnel plots for success rate and a 2-line improvement of BCVA of autograft and allograft were generated (data not shown).
Subjective Symptoms Four studies27,32,33,35 reported that the postoperative pain and discomfort of the eye were reduced, whereas only 2 of them used a numerical pain score to record the preoperative and postoperative results for data analysis.
Complications and Adverse Events As for the condition of donor eyes, which was mentioned in 4 studies,25,28,30,32 no complications were reported. Following CLET, in 7 studies25,28,31,34,37,38,41 it was reported that 18 eyes were suffering from microbial keratitis, but through fortified topical antibiotics or emergency keratoplasty 11 eyes were fully cured. Furthermore, perforation or corneal thinning requiring tissue adhesive application occurred in 14 eyes,25,28,33,35,38 whereas 58 eyes had hemorrhage under the AM,25,28,35 but this diminished spontaneously. Other complications were epithelial rejection, endothelial rejection, corneal allograft rejection, granuloma, and panophthalmitis.25,29,37,41 Five studies30,32,36,39,40 stated no complications.
DISCUSSION Publication Bias After analyzing the overall success rate (t = 1.25, P = 0.228) and outcome of visual acuity (t = 1.75, P = 0.10) using
The optimum method of graft culture is yet to be determined. Some authors43,50 supported that intact HAM favored the preservation and expansion of limbal stem cells,
TABLE 4. Clinical Results Success Rate, n/N (%) Reference
All 25
Sejpal et al Subramaniam et al‡26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al‡38 Sharma et al30,39,49 Tsai et al40 Koizumi et al‡41 Schwab et al37,42
53/107 18/40 5/9 142/200 5/8 37/50 14/19 8/8 12/18 7/10 37/44 9/9 4/7 5/13 4/4 6/6 10/13 5/7
(49.5) (45) (55.6) (71.0) (62.5) (74.0) (73.7) (100.0) (66.7) (70.0) (84.1) (100.0) (57.1) (38.5) (100.0) (100.0) (76.9) (71.4)
BCVA of At least 2-line Improvement, n/N (%)
Autograft
Allograft
All
Autograft
Allograft
53/107 (49.5) 18/40 (45) 5/9 (55.6) 142/200 (71.0) 5/8 (62.5) 28/34 (82.4) 8/12 (66.7) 8/8 (100.0) 10/15 (66.7) 1/3 (33.3) 27/30 (90.0) 2/2 (100.0) 1/3 (33.3) — 4/4 (100.0) 6/6 (100.0) — 4/5 (80.0)
— — — — — 9/16 (56.3) 6/7 (85.7) — 2/3 (66.7) 6/7 (85.7) 10/14 (71.4) 7/7 (100.0) 3/4 (75.0) 5/13 (38.5) — — 10/13 (76.9) 1/2 (50.0)
58/107 (54.2) NA 2/9 (22.2) 121/200 (60.5) 7/8 (87.5) 34/50 (68.0) 13/19 (68.4) 7/8 (87.5) 10/18 (55.6) 5/10 (50.0) 32/44 (72.7)† 9/9 (100.0) 7/7 (100.0) 10/13 (76.9) 4/4 (100.0) 3/6 (50.0) 10/13 (76.9) 3/7 (42.9)
58/107 (54.2) NA 2/9 (22.2) 121/200 (60.5) 7/8 (87.5) 12/13 (92.3)* 8/12 (66.7) 7/8 (87.5) 8/15 (53.3) 1/3 (66.7) 21/30 (70.0)† 2/2 (200.0) 3/3 (100.0) — 4/4 (100.0) 3/6 (50.0) — 2/5 (40.0)
— — — — — 2/7 (28.6)* 5/7 (71.4) — 2/3 (66.7) 4/7 (57.1) 9/14 (64.3)† 7/7 (100.0) 4/4 (100.0) 10/13 (76.9) — — 12/13 (92.3) 1/2 (50.0)
*From partial data given by the article. †Data from the article with contradiction. ‡Success rate was not directly given in these articles, and it was inferred by our definition of success. BCVA, best-corrected visual acuity.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.corneajrnl.com |
597
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Zhao and Ma
Cornea Volume 34, Number 5, May 2015
FIGURE 3. Forest plot for the outcomes. A, Forest plot for pooled success rate of CLET with AM. B, Forest plot for success rate of autograft and allograft. C, Forest plot for a 2-line improvement of BCVA by CLET with AM. D, Forest plot for a 2-line improvement in BCVA of autograft and allograft. Shweta Sharma et al39 and Ray Jui-Fang Tsai et al40 in A and C.
whereas denuded HAM promoted a corneal epithelial phenotype. Rendal-Vázquez ME et al51 reported that a normal limbal epithelium, 1 to 2 cell layers thick, of which the basal layer of cells had high expression of the putative LESC markers p63 and ABCG2, was well formed on intact AM. However, Baharvand et al52 showed that denuded HAM provided a superior niche for LESC proliferation and phenotype maintenance in vitro. As to the culture system, a study by Li et al53 showed that, during explant outgrowth, a proportion of epithelial progenitor cells invade the limbal stroma and, by undergoing epithelial–mesenchymal transition, reduce the quantity of progenitor cells on the explant surface. But that the limbal explant culture epithelial outgrowth covered the AM much earlier than the suspension culture was also reported.32 Because the use of animalderived components in ex vivo expansion of epithelial stem cells poses potential risks of pathogen transmission, Sharma SM et al49 who made a comparison between human limbal epithelium cocultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and no feeder cells (NF) found that epithelial cells grown on a DF feeder layer maintained a stem cell–like phenotype, comparable with cells grown on a 3T3 feeder layer. Reports of eliminating the use of feeder cells completely, in terms of xeno-free culture,28 have been widespread, and long-term observation may be necessary to draw a conclusion. Airlifting was used in some studies to cause
stratification of the epithelial cells. However, Li et al54 reported that stratification promoted by airlifting was coupled with squamous metaplasia in human limbal explants. The expression of all markers, stem cell markers and differentiated markers, in the culture suggests that cultured limbal epithelial cells contained a heterogeneous population of corneal progenitor cells and matured corneal epithelial cells.30 To improve the success rate, it is of great importance to ensure the proportion of stem cells in the culture. To date, there is no definitive marker of limbal stem cells, and the most promising candidates are p63 and ABCG2. Rama et al55 suggested an association between their clinical results and the percentage of cells in the culture that stained positive for p63. It was found that those LESCs cultivated with more than 3% p63 bright cells were associated with a success rate of 78% in clinical transplantation, whereas less than 3% p63 bright cells were only successful in 11%. Evidently, functional assays preserving the colony-forming ability of transplanted cells is also important, as the capacity of transplanted cells continuing to regenerate the ocular surface after prolonged periods is an indirect indicator of the long-term proliferative potential of transplanted cells.36 In the present study, consistent with the reports by Shortt et al46 and Baylis et al56 no difference both in success rate and visual acuity outcome between autograft and allograft was found, although we used meta-analysis and strict inclusion criteria to try to improve the reliability of the outcomes. The
598
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
result may provide some suggestion for surgeons to choose the source of limbus. There is no doubt that for unilateral disease, the fellow healthy eye or a normal region of an eye that was partially stem cell deficient is the best source of tissue. However, when the disease is bilateral, the options are deceased or living related donors, although with subsequent immunosuppression required, which may also lead to a promising outcome at least in a limited postoperative period. Definitely, whether the long-term effect of these 2 sources is of no difference or not needs further research.
Limitations of Current Study The included studies in our search recruited participants of all ages and both genders, covered different regions, and accommodated different types of LSCD. Stage of disease in each patient, duration between injury and surgery, culture methods, interventions both in the primary procedure and in subsequent interventions, and outcome measures used to define successful treatment varied in different studies. Most studies were not prospective, and it was unclear whether recruitment was consecutive in more than 80% of studies. Specifically, not all studies reported a minimum follow-up of more than 6 months. Additionally, most work, such as article selection, data extraction, and assessment of studies, was carried out by a single person. And there probably exists publication bias according to the funnel plot analysis when comparing autograft and allograft. All these contribute to heterogeneity. In the future, standardized methods of assessing success of both objective and subjective outcomes may be required. The long-term results of autograft and allograft will inform future treatment algorithms and techniques with random control trials and better-designed analysis.
ACKNOWLEDGMENTS The authors thank S.Q. Li of Statistics Department, Shanxi Medical University, for providing statistical advice, and the following persons for their assistance during the search: Q. Chen, Dr R. Y. Zhu, Dr L. Hu, and Z. Zhang. REFERENCES 1. Leblond CP. The life history of cells in renewing systems. Am J Anat. 1981;160:114–158. 2. Potten CS, Morris RJ. Epithelial stem cells in vivo. J Cell Sci Suppl. 1988;10:45–62. 3. Pellegrini G, Golisano O, Paterna P, et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol. 1999;145:769–782. 4. Dua HS, Azuara-Blanco A. Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency. Br J Ophthalmol. 2000;84: 273–278. 5. Espana EM, Di Pascuale MA, He H, et al. Characterization of corneal pannus removed from patients with total limbal stem cell deficiency. Invest Ophthalmol Vis Sci. 2004;45:2961–2966. 6. Santos MS, Gomes JA, Hofling-Lima AL, et al. Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency. Am J Ophthalmol. 2005;140: 223–230. 7. Tseng SC. Concept and application of limbal stem cells. Eye (Lond). 1989;3:141–157.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Systematic Review and Meta-analysis
8. Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol. 2000;44:415–425. 9. Nishida K, Kinoshita S, Ohashi Y, et al. Ocular surface abnormalities in aniridia. Am J Ophthalmol. 1995;120:368–375. 10. Tseng SCG, Espana EM, Di Pascuale MA. Stem cell transplantation for ocular surface diseases. In: Tasman W, Jaeger EA, eds. Duanes Clinical Ophthalmology on CD-rom. Baltimore, MD: Lippincott Williams & Wilkins; 2005. 11. Calonge M, Diebold Y, Saez V, et al. Impression cytology of the ocular surface: a review. Exp Eye Res. 2004;78:457–472. 12. Cauchi PA, Ang GS, Azuara-Blanco A, et al. A systematic literature review of surgical interventions for limbal stem cell deficiency in humans. Am J Ophthalmol. 2008;146:251–259. 13. Geerling G, Maclennan S, Hartwig D. Autologous serum eye drops for ocular surface disorders. Br J Ophthalmol. 2004;88:1467–1474. 14. Young AL, Cheng AC, Ng HK, et al. The use of autologous serum tears in persistent corneal epithelial defects. Eye (Lond). 2004;18:609–614. 15. Ahmad S, Kolli S, Lako M, et al. Stem cell therapies for ocular surface disease. Drug Discov Today. 2010;15:306–313. 16. Ahmad S, Osei-Bempong C, Dana R, et al. The culture and transplantation of human limbal stem cells. J Cell Physiol. 2010;225:15–19. 17. Dua HS. The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol. 1998;82:1407–1411. 18. Tseng SCG, Prabhasawat P, Barton K, et al. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch Ophthalmol. 1998;116:431–441. 19. Espana EM, Di Pascuale M, Grueterich M, et al. Keratolimbal allograft in corneal reconstruction. Eye (Lond). 2004;18:406–417. 20. Shortt AJ, Tuft SJ, Daniels JT. Corneal stem cells in the eye clinic. Br Med Bull. 2011;100:209–225. 21. Yeh HJ, Yao CL, Chen HI, et al. Cryopreservation of human limbal stem cells ex vivo expanded on amniotic membrane. Cornea. 2008;27:327–333. 22. Dua HS, Gomes JA, King AJ, et al. The amniotic membrane in ophthalmology. Surv Ophthalmol. 2004;49:51–77. 23. Gomes JA, Romano A, Santos MS, et al. Amniotic membrane use in ophthalmology. Curr Opin Ophthalmol. 2005;16:233–240. 24. Akle CA, Adinolfi M, Welsh KI, et al. Immunogenicity of human amniotic epithelial cells after transplantation into volunteers. Lancet. 1981;2:1003–1005. 25. Sejpal K, Ali MH, Maddileti S, et al. Cultivated limbal epithelial transplantation in children with ocular surface burns. JAMA Ophthalmol. 2013;131:731–736. 26. Subramaniam SV, Sejpal K, Fatima A, et al. Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface diaorders: long-term survival analysis. Indian J Ophthalmol. 2013;61: 202–207. 27. Pathak M, Cholidis S, Haug K, et al. Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series. Acta Ophthalmol. 2013;91:769–775. 28. Sangwan VS, Basu S, Vemuganti GK, et al. Clinical outcome of xenofree autologous cultivated limbal epithelial transplantation: a 10-year study. Br J Ophthalmol. 2011;95:1525–1529. 29. Baradaran-Rafii A, Ebrahimi M, Kanavi MR, et al. Midterm outcomes of autologous cultivated limbal stem cell transplantation with or without penetrating keratoplasty. Cornea. 2010;29:502–509. 30. Sharma S, Tandon R, Mohanty S, et al. Culture of corneal limbal epithelial stem cell: experience from benchtop to bedside in a tertiary care hospital in India. Cornea. 2011;30:1223–1232. 31. Prabhasawat P, Ekpo P, Uiprasertkul M, et al. Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction. Clin Ophthalmol. 2012;6:1483–1492. 32. Kolli S, Ahmad S, Lako M, et al. Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency. Stem Cells. 2010;28:597–610. 33. Zakaria N, Possemiers T, Dhubhghaill SN, et al. Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation. J Transl Med. 2014;3:58. 34. Shortt AJ, Secker GA, Rajan MS, et al. Ex vivo expansion and transplantation of limbal epithelial stem cells. Ophthalmology. 2008; 115:1989–1997.
www.corneajrnl.com |
599
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Zhao and Ma
Cornea Volume 34, Number 5, May 2015
35. Pauklin M, Fuchsluger TA, Westekemper H, et al. Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency. Dev Ophthalmol. 2010;45:57–70. 36. 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;113:1765–1772. 37. Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea. 2000;19:421–426. 38. Shimazaki J, Aiba M, Goto E, et al. Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders. Ophthalmology. 2002;109:1285–1290. 39. Sharma S, Tandon R, Mohanty S, et al. Phenotypic evaluation of severely damaged ocular surface after reconstruction by cultured limbal epithelial cell transplantation. Ophthalmic Res. 2013;50:59–64. 40. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2000; 343:86–93. 41. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology. 2001; 108:1569–1574. 42. Schwab IR. Cultured corneal epithelial for ocular surface disease. Trans Am Ophthalmol Soc. 1999;97:891–986. 43. Grueterich M, Espana EM, Tseng SC. Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche. Surv Ophthalmol. 2003;48:631–646. 44. Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349:990–993. 45. Ramaesh K, Dhillon B. Ex vivo expansion of corneal limbal epithelial/stem cells for corneal surface reconstruction. Eur J Ophthalmol. 2003;13: 515–524.
46. 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;52:483–502. 47. Shortt AJ, Secker GA, Lomas RJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelia cells. Biomaterials. 2009;30:1056–1065. 48. Sangwan VS, Matalia HP, Vemuganti GK, et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J Ophthalmol. 2006;54:29–34. 49. SM Sharma, Fuchsluger T, Ahmad S, et al. Comparative analysis of human-derived feeder layers with 3T3 fibroblasts for the ex vivo expansion of human limbal and oral epithelium. Stem Cell Rev. 2012;8:696–705. 50. Li W, He H, Kuo CL, et al. Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane. Invest Ophthalmol Vis Sci. 2006;47:2381–2389. 51. Rendal-Vázquez ME, San-Luis-Verdes A, Yebra-Pimentel-Vilar MT, et al. Culture of limbal stem cells on human amniotic membrane. Cell Tissue Bank. 2012;13:513–519. 52. Baharvand H, Heidari M, Ebrahimi M, et al. Proteomic analysis of epithelium-denuded human amniotic membrane as a limbal stem cell niche. Mol Vis. 2007;13:1711–1721. 53. Li W, Hayashida Y, He H, et al. The fate of limbal epithelial progenitor cells during explant culture on intact amniotic membrane. Invest Ophthalmol Vis Sci. 2007;48:605–613. 54. Li W, Hayashida Y, Chen YT, et al. Air exposure induced squamous metaplasia of human limbal epithelium. Invest Ophthalmol Vis Sci. 2008; 49:154–162. 55. Rama P, Matuska S, Paganoni G, et al. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363:147–155. 56. O1 Baylis, Figueiredo F, Henein C, et al. 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. J Cell Biochem. 2011; 112:993–1002.
600
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Systematic Review and Meta-analysis on Transplantation of Ex Vivo Cultivated Limbal Epithelial Stem Cell on Amniotic Membrane in Limbal Stem Cell Deficiency Yuan Zhao, MD,* and Lei Ma, MD†
Purpose: Ex vivo cultured limbal epithelial transplantation (CLET) with amniotic membrane (AM) as the substrate is a relatively new type of surgical therapy in treating limbal stem cell deficiency (LSCD). We summarize available evidence for determining the efficiency of this technique by a systematic review and metaanalysis.
Methods: Searching the following electronic databases, MEDLINE, EMBASE, and the Cochrane Library, we analyzed the selected articles in 5 main aspects: donor screening, culture methods, evidence of cultivated stem cells, subjective symptoms, and adverse events through systematic review. Specifically, meta-analysis was used in evaluating improvements in ocular surface and visual acuity.
Results: A total of 18 articles involving 572 eyes of 562 patients were available. The rate of success and 2-line improvement in bestcorrected visual acuity (BCVA) was 67% [95% confidence interval (CI), 0.59–0.75; I2 = 60%] and 62% (95% CI, 0.57–0.66; I2 = 37.7%), respectively; and no difference was found both in success rate [odds ratio (OR), 1.35; 95% CI, 0.63–2.89; I2 = 46%] and visual acuity outcome (OR, 1.53; 95% CI, 0.67–3.45; I2 = 42.1%) between autograft and allograft.
Conclusions: CLET is efficacious in patients with LSCD, and no difference both in success rate and visual acuity outcome between autograft and allograft was found. Overall safety profile was good, with most side effects being transient and amenable to subsequent treatments. The long-term results of autograft and allograft will inform future treatment algorithms and techniques with random control trials and better-designed analysis. Received for publication October 16, 2014; revision received January 2, 2015; accepted January 19, 2015. Published online ahead of print March 19, 2015. From the *Department of Clinical Medicine, Shanxi Medical University, Taiyuan, People’s Republic of China; and †Department of Ophthalmology, Second Teaching Hospital of Shanxi Medical University, Taiyuan, People’s Republic of China. Supported by National Natural Science Foundation of China (Grant 30973248); Shanxi Provincial Foundation for Returned Scholars (Grant 200860, 200856). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflicts of interest to disclose. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.corneajrnl.com). Reprints: Lei Ma, MD, Department of Ophthalmology, Second Teaching Hospital of Shanxi Medical University, 382# Wuyi Rd, Taiyuan, Shanxi 030001, People’s Republic of China (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
592
| www.corneajrnl.com
Key Words: limbal stem cell deficiency, amniotic membrane, ex vivo cultured limbal epithelial transplantation, meta-analysis, systematic review (Cornea 2015;34:592–600)
S
tem cells of adult tissues, which are relatively undifferentiated and self renewing, serve as a reservoir for cell replacement to maintain homeostasis of the tissue mass.1,2 In the cornea, equivalent populations of limbal epithelial stem cells (LESCs) generate and renew the corneal epithelium throughout life and are thus essential for maintaining corneal transparency and vision.3 Various diseases, such as chemical or thermal injury, UV and ionizing radiation, Stevens–Johnson syndrome, advanced ocular cicatricial pemphigoid, contact lens overwear, multiple surgeries, antimetabolites, extensive microbial infection, or aniridia can lead to limbal stem cell deficiency (LSCD).4–6 These conditions can make patients feel pain and photophobia, and even result in chronic inflammation, conjunctival epithelial ingrowth (conjunctivalization) invading the cornea, corneal vascularization, poor epithelial integrity manifested as an irregular surface, recurrent epithelial erosion or persistent epithelial ulceration, and destruction of the basement membrane, leading to fibrous ingrowth.7 However, conjunctivalization is central to the diagnosis of LSCD.8 Moreover, clinical signs observed are epithelial haze, superficial subepithelial vascularization, persistent or recurrent epithelial defects, epithelial and stromal inflammation, late fluorescein staining, and loss of the limbal palisades of Vogt.8–10 Impression cytology, to identify goblet cells, or monoclonal antibodies to cytokeratin 3 and cytokeratin19 to confirm a conjunctival phenotype, can usually be used to make additional confirmation.9,11 LSCD may be classified as partial or total with limbal stem cells partially or totally depleted, resulting in varying degrees of stem cell deficiency with abnormalities and conjunctivalization of the corneal surface.9,12 Although the conservative options available for managing LSCD, whether partial or total, are intensive lubrication, nonpreserved artificial tear drops, bandage contact lenses, and autologous serum eye drops, only the latter is supported by evidence in the literature.13,14 It must be noted that corneal transplantation, which involves replacing the central cornea (excluding the limbus), cannot be used as a treatment option for LSCD due to the lack of host limbal stem cells to replace the epithelium overlying the corneal graft.15,16 In partial LSCD, it has been demonstrated that repeated debridement of migrating Cornea Volume 34, Number 5, May 2015
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
conjunctival epithelium in the acute phase following injury, known as sequential sector conjunctival epitheliectomy (SSCE), can reduce or prevent conjunctival ingrowth,17 and it may be combined with amniotic membrane transplantation (AMT).18 When LSCD is total, a population of autologous or allogeneic LESCs must be transplanted if a stable corneal epithelial phenotype is to be regained.8 A variety of techniques have been developed to transplant limbal stem cells, such as keratolimbal lamellar allograft (KLAL),19 which was the first to be introduced and the most widely used, and also conjunctival-limbal autografts (CLAU) and living related conjunctival-limbal allografts (lr-CLAL).6 All these surgical options may be combined with penetrating keratoplasty (PK) or deep lamellar keratoplasty (DLK), with or without cataract surgery. A novel method of transplanting limbal stem cells is through ex vivo cultured limbal epithelial transplantation (CLET), which has theoretical advantages over conventional treatments. The limbal epithelial cells are harvested from the contralateral healthy eye (or from a normal area of a partially stem cell deficient eye), living related donors, or from fresh tissue of deceased donors (in cases of bilateral total limbal stem cell deficiency) by a minimal (eg, 2 · 2 mm2) limbal biopsy, which minimizes the risk of precipitating stem cell failure in the donor eye and provides the option of taking a further biopsy, if required. CLET may also have a reduced risk of allograft rejection because antigenpresenting macrophages do not survive the process of ex vivo culture.20 Cryopreserving excess ex vivo cultured cells for a prolonged period21 could also provide a source of additional cells for transplantation, should the need arise. Various materials (amniotic membrane, fibrin, contact lens, etc.) may be used as a cell substrate in the ex vivo culture. But human amniotic membrane (HAM), which promotes epithelialization, inhibits fibrosis, has anti-inflammatory and antiangiogenic properties, and possesses antimicrobial and antiviral properties and a high hydraulic conductivity,22,23 with low or no immunogenicity,24 has become the widely used material25–42 acting as a surrogate environmental stem cell niche.43 The use of ex vivo cultured LESCs to treat corneal LSCD in humans was first described by Pellegrini et al44 in 1997. Subsequently, plenty of case series and reviews have reported the efficiency and evidence of this treatment.12,25–43,45–47 However, with the lack of sufficient cases or valid measurements, they failed to provide a convincing assessment of this surgery. Moreover, there is controversy regarding the clinical outcome between different sources of limbal stem cells, such as autograft and allograft. Therefore, our study aims to view all relevant published articles to determine the overall success rate and visual acuity outcome of CLET through a systematic review and meta-analysis and to analyze whether the outcome is different between autograft and allograft, expecting that it may provide some suggestions for this procedure.
MATERIALS AND METHODS Search Strategy The search was performed in July 2014, which was restricted to English language reports without published time Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Systematic Review and Meta-analysis
limits. Using the combined key words “limbal stem cell deficiency” and “amniotic membrane,” we searched the following electronic databases: MEDLINE, EMBASE, and the Cochrane Library. Trying to increase the recall rate, we did not use “cultured limbal epithelial transplantation” as key words. In addition, reference lists were scanned to identify any additional reports.
Inclusion Criteria Qualified studies included in the meta-analysis must meet the following criteria. Participants: All age groups with different kinds of LSCD. Interventions: Ex vivo cultured limbal epithelial cells with AM as a substrate for transplantation, including those who received conservative treatments or other (except this intervention) surgeries previously. Preoperative evaluation of patients and postoperative outcome measures should both be obtained in each article. Accordingly, variables such as age, gender, different ex vivo cultivated methods, source of limbal stem cells, cause, extent of LSCD, duration between injury and surgery, and various previous treatments all are potential sources of heterogeneity.
Article Selection Titles and abstracts were reviewed to exclude any obviously irrelevant reports and full-text articles were ordered for studies potentially suitable for inclusion. Case reports, review articles, animal or laboratory studies, conference abstracts, and letters were excluded.
Quality Assessment The quality of all included articles was assessed using a validated checklist consisted of 14 questions (see Table, Supplemental Digital Content 1, http://links.lww. com/ICO/A265)12 according to a relevant study by Cauchi et al.12 This checklist was developed by the Review Body for Interventional Procedures, which is an independent review body that carries out systematic reviews for the National Institute for Health and Clinical Excellence Interventional Procedures Program and was adapted from several sources. Following are details used to evaluate the included studies. The prognostic factors were described as age, gender, etiology, lid abnormalities, treatments before CLET, intervals between injury and this surgery, type of LSCD, source of limbal stem cells, and extent of dry eye. Studies including most of these items (at least 8 of the 9) were considered as “YES.” The objective outcome measures may be defined as explicit description of clinical with or without impression cytology findings of stability of the ocular surface and visual acuity. All important outcomes such as subjective symptoms and objective outcomes should be included. Scientific scales and scores of these outcomes, which add possibility for statistic analysis, would be better. Additionally, 6 months was set as a reasonable follow-up period.48 www.corneajrnl.com |
593
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Zhao and Ma
Data Extraction Data such as study design, participant numbers, diagnoses, follow-up, cause and type of LSCD, source of limbal stem cells, culture method, clinical outcome, and adverse events were analyzed. The main clinical outcome was success rate of this treatment, which was defined as the reestablishment of a stable and transparent corneal epithelium, resolution of corneal conjunctivalization and persistent epithelial defect, and regression of corneal vascularization. (And if the definition is different or unclear in some articles, we would work out the new success rate according to the postoperative descriptions reported in these articles). Secondary outcome was an improvement in vision of at least 2 Snellen lines of bestcorrected visual acuity (BCVA). Available raw data of visual acuity were analyzed. In detail, visual acuity (VA) “hand motion” was calculated as 3.0 logarithm of the minimum angle of resolution (logMAR), “counting fingers at 1/2-ft distance” as 2.6 logMAR, “counting fingers at 1-ft distance” as 2.3 logMAR, and “counting fingers at 2-ft distance” as 2.0 logMAR.31 Thus, VA from hand motion (HM) to counting fingers (CF) was considered as a 2-line improvement.
FIGURE 1. Flowchart showing process of article selection.
All 18 studies were noncomparative case series (Table 1). However, lack of high-quality objective data in the form
of randomized controlled trials (RCT) is an inherent problem with surgical procedures as they were less suited to this study design than medical interventions. In accordance with the inclusion criteria, only partially available data were extracted from the study by Schwab.42 Basically, a “yes” to a quality assessment question represented a positive measure of quality. Seventeen of 18 studies (94.4%) had a representative sample, 9 of 18 (50%) outlined a recruitment period, 16 of 18 (88.9%) had objective outcome measures, and 15 of 18 (83.3%) had a clear description of the 3 main findings. Six of 18 studies (33.3%) had inclusion/exclusion criteria clearly described. Twelve of 18 studies (66.7%) had a minimum follow-up period of more than 6 months. Only 2 of 18 studies (11.1%) reported that an experienced surgeon performed the procedures. Three of 18 studies (11.7%) considered all relevant outcomes and identified all prognostic factors, and 4 of 18 (22.2%) collected data prospectively. Two of 18 studies had participants entering the study at a similar stage of disease progression and gave the information on dropouts, and no study specifically stated blinding of outcome assessors (Table 2 and Fig. 2). Totally, 572 eyes of 562 patients were studied with the size of the population ranging from 4 to 200, and approximately 74.6% were men. The overall mean age was 25 years.43 The follow-up period ranged from 1 to 118 months, the minimum being 6 months. The etiology of LSCD in these studies showed that the most common cause was chemical burn (74.1%). In 7 articles, 82 eyes (33.7%) were clinically diagnosed as partial LSCD, which were with or without the results of impressing cytology and cytokeratin profiling. Of the 572 eyes, 486 (85.0%) received autografts, and 86 (15.0%) received allografts from corneas of deceased donors or living related donors. However, 5 studies noted the duration between injury and surgery.25,26,28,29,39
594
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Statistical Analysis R-3.0.3 software was used for meta-analysis. For the overall success rate and visual outcome, we calculated the combined ratio using Freeman–Tukey double arcsine transformation methods with metaprop package, and effect size and 95% confidence intervals (CI) were calculated. For comparing outcomes of autograft and allograft, we adopted odds ratios (OR) and 95% CI using metabin package. If significant heterogeneity was observed (P , 0.05), a random-effects model was used for pooling the data; otherwise, a fixed-effects model was used (P . 0.05). We conducted regression analysis to assess publication bias (P . 0.05 was considered no publication bias), and when the sample was small, funnel plot was used for assessment.
RESULTS Study Identification A total of 389 articles of potential interest were identified by the original literature search (Fig. 1), including 140 duplications (1 article existed with 3 different data). Reviewing the abstracts, 208 articles were excluded for either not concerned with CLET or not meeting the inclusion criteria. Furthermore, the remaining 40 full-text articles were read in detail. A further 22 exclusions were made for the following reasons: conference papers, 5; reviews, 4; case reports, 6; without postoperative outcome, 2; and 5 with unqualified intervention. Eventually, a total of 18 papers were available.
Study Characteristics
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Systematic Review and Meta-analysis
TABLE 1. Characteristics of Included Studies
25
Sejpal et al Subramaniam et al26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al38 Sharma et al30,39,49 Tsai et al40 Koizumi et al41 Schwab et al37,42
Cause
Extent of LSCD
Donor
Country
Design
No. Eyes/ No. Patients
Follow-up, Mean 6 SD (range), m
CB
Others
Partial
Total
Autograft
Allograft
India India Norway India Iran India Thailand United Kingdom Belgium United Kingdom Germany Japan United States Japan India Taiwan Japan United States
RCS RCS RCS RCS CS CS PCS PCS CS PCS CS RCS CS RCS CS CS RCS PCS
107/107 40/39 9/9 200/200 8/8 50/50 19/18 8/8 18/18 10/10 44/38 9/9 7/7 13/13 4/4 6/6 13/11 7/7
41.2 6 26 (12–118) 33.4 6 29.2 (1–87) 18.4 (11–28) 36 6 19.2 (12–91.2) 34.0 6 13.5 (6–48) 11.0 6 8.0 (1.5–25) 26.1 6 13.5 (6–47) 19 (13–20) 22 (4–43) NA 28.5 6 14.9 (9–73) 14.6 6 4.4 (6–20) 11.57 (6–19) NA 19.5 6 7.4 (9–26) 15 6 2 (12–18) 11.2 6 1.3 (9–13) 4.43 (2–8)
NA 34 4 179 5 41 13 6 7 4 NA 1 6 2 3 3 3 1
NA 6 5 21 3 9 6 2 11 6 NA 8 1 11 1 3 10 6/7
15 35 5 0 0 NA 8 0 3 NA 12 0 NA 0 0 4 0 NA
92 5 4 200 8 NA 11 8 15 NA 32 9 NA 13 4 2 13 NA
107 40 9 200 8 34 12 8 15 3 30 2 3 0 4 6 0 5
0 0 0 0 0 16 7 0 3 7 14 7 4 13 0 0 13 2
Reference
CB, chemical burn; CS, case series; NA, not available; PCS, prospective case series; RCS, retrospective case series.
Donor Screening
stated human amniotic membrane screening. Taken together, these findings highlight the absence of clearly reporting tissue screening although it might have been performed.
Four of 16 studies32,34,35,38 (the remaining 2 had no autografts used) were screened for autologous donors. Screening of live donors was reported in 5 of 8 studies,34,35,37,38,42 which used living allogeneic limbal cells. That corneas from deceased donors were used as a source of limbal stem cells for culture30,31,33,34,36,41 was not explicitly stated whether these were screened. Additionally, 4 of 18 studies29–31,42 explicitly
Ex Vivo Culture Methods As for the ex vivo culture, we specifically focus on the following 4 aspects: culture system, the type of AM, and
TABLE 2. Quality Items Per Study Reference 25
Sejpal et al Subramaniam et al26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al38 Sharma et al30,39,49 Tsai et al40 Koizumi et al41 Schwab et al37,42
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Y Y Y Y Y Y Y Y Y U Y Y Y Y Y Y Y Y
Y Y N Y Y N Y Y N N U U N N N N N N
N U N U U N N Y N U N Y N U N N U N
U U U U U U U U U U Y Y U U U U U U
N Y N Y N N N N N N N N N N Y N N N
N N N N U U Y Y U Y U N U N U U N Y
Y Y Y Y Y N N N N N Y Y N N N N Y Y
N N N N U U U U U U U N U N U U N U
U U U U U U U U U U Y U U U U Y U U
Y Y N U Y Y Y Y Y Y Y Y Y Y Y Y Y Y
N N N N N N N Y Y N Y N N N N N N N
Y N Y Y Y N Y Y N N Y Y Y N Y Y Y N
N N N N N N N N Y N N N N N N N N Y
Y Y N U Y Y Y Y Y Y Y Y Y Y Y Y N Y
N, no; U, unclear; Y, yes.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.corneajrnl.com |
595
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Zhao and Ma
FIGURE 2. Bar graph showing the proportion of studies per quality item.
whether using mouse 3T3 fibroblast or airlifting or not. Consequently, of the 17 studies involved, 14 used explant systems, 2 used suspension systems, and 1 used both. Denuded AM with the absence of the amniotic epithelial cell was commonly used in 12 studies, although the remaining 5 studies either used intact AM or stated unclearly. 3T3 fibroblasts were usually used to contribute to cultivated stem cells ex vivo. However, of the 17 relative studies, only 2 reported the use of 3T3 fibroblasts, 1 with unclear documentation and another with it in allograft culture, but not in autograft. Moreover, airlifting was also not used in 13 articles, with 2 unclearly reported (Table 3).
Evidence of the Presence of Stem Cells in Cultures To provide evidence for the presence of stem cells in the transplanted cell population, various methods from
different aspects were involved. A monolayer of cells being achieved for transplantation after 10 to 15 days were reported in 2 studies,26,28 whereas 4 other studies30–32,37 reported a multilayered epithelium. A well-conserved basal layer formed by cuboidal cells with positive hematoxylin–eosin staining expression was described,32,36 and was further confirmed by the formation of a primitive epithelium with a prominent basal layer of cuboidal cells possessing a high nucleus–cytoplasm ratio observed in transmission electron microscopy (TEM).32 The expression of specific markers, such as stem cell–positive markers (p63, ABCG2, and K19) and the differentiation markers (K3/K12), was detected in the same culture.26,30–33,36 Expression of more K19 and less K3/K12 in the basal layer of the cultured cells was reported in several studies.30,32,36 The colony-forming efficiency of representative cultures, which was used to analyze the presence of stem cells, resulted in a proportion varying from 2% to 9%.36 Corneal buttons from eyes that underwent
TABLE 3. Culture Method Reference Sejpal et al25 Subramaniam et al26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al38 Sharma et al30,39,49 Tsai et al40 Koizumi et al41
System
AM
3T3 Fibroblast
Airlifting
Time (days)
Explant Explant Explant Explant Explant Explant Explant Explant Explant Suspension Explant Explant and suspension Suspension Explant Explant Explant Explant
dAM dAM UC dAM dAM dAM dAM iAM dAM iAM iAM dAM dAM dAM dAM UC dAM
(2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) Auto (2), Allo (+) (+) (2) UC (2) (+)
(2) (2) (2) (2) UC (2) (+) UC (2) (2) (2) (2) (2) (2) (2) (2) (+)
10–14 10–15 14–21 10–14 10–14 7–15 17–28 10–14 14 14–21 14 14 21–28 12–20 14 14–21 32
dAM, denuded AM; iAM, intact AM; UC, unclear.
596
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
Systematic Review and Meta-analysis
subsequent PK were found to have multiple layers of stratified epithelium, similar to normal corneal phenotype with positive staining of p63, CK3, and CK12.31,39
Improvement in Ocular Surface Eighteen articles25–42 reported a success rate (Table 4) with 381 successful procedures in a total of 572 eyes. The pooled success rate was 67% (95% CI, 0.59–0.75; I2 = 60%, P = 0.0006; Fig. 3A) using the random-effects model. Seven studies,30,31,33–37 which included 157 eyes, reported the success rate of autograft (99 eyes) and allograft (58 eyes) simultaneously. Consequently, in terms of the success rate, there may be no significant difference between autograft and allograft (OR, 1.35; 95% CI, 0.63–2.89; I2 = 46%, P . 0.05; Fig. 3B).
Visual Acuity Seventeen articles,25,27–42 which included 532 eyes, reported a 2-line improvement in BCVA in 335 eyes. The pooled rate was 62% (95% CI, 0.57–0.66; I2 = 37.7%, P . 0.05; Fig. 3C). Seven studies30,31,33–37 reported visual acuity outcome of autograft and allograft, with 78 and 49 eyes, respectively. As a result, there may be no difference between the 2 groups with regard to 2-line improvement in BCVA (OR, 1.53; 95% CI, 0.67–3.45; I2 = 42.1%, P . 0.05; Fig. 3D).
Egger test, there was slight publication bias. Funnel plots for success rate and a 2-line improvement of BCVA of autograft and allograft were generated (data not shown).
Subjective Symptoms Four studies27,32,33,35 reported that the postoperative pain and discomfort of the eye were reduced, whereas only 2 of them used a numerical pain score to record the preoperative and postoperative results for data analysis.
Complications and Adverse Events As for the condition of donor eyes, which was mentioned in 4 studies,25,28,30,32 no complications were reported. Following CLET, in 7 studies25,28,31,34,37,38,41 it was reported that 18 eyes were suffering from microbial keratitis, but through fortified topical antibiotics or emergency keratoplasty 11 eyes were fully cured. Furthermore, perforation or corneal thinning requiring tissue adhesive application occurred in 14 eyes,25,28,33,35,38 whereas 58 eyes had hemorrhage under the AM,25,28,35 but this diminished spontaneously. Other complications were epithelial rejection, endothelial rejection, corneal allograft rejection, granuloma, and panophthalmitis.25,29,37,41 Five studies30,32,36,39,40 stated no complications.
DISCUSSION Publication Bias After analyzing the overall success rate (t = 1.25, P = 0.228) and outcome of visual acuity (t = 1.75, P = 0.10) using
The optimum method of graft culture is yet to be determined. Some authors43,50 supported that intact HAM favored the preservation and expansion of limbal stem cells,
TABLE 4. Clinical Results Success Rate, n/N (%) Reference
All 25
Sejpal et al Subramaniam et al‡26 Pathak et al27 Sangwan et al28,48 Baradaran-Rafii et al29 Sharma et al30,39,49 Prabhasawat et al31 Kolli et al32 Zakaria et al33 Shortt et al20,34,46,47 Pauklin et al35 Nakamura et al36 Schwab et al37,42 Shimazaki et al‡38 Sharma et al30,39,49 Tsai et al40 Koizumi et al‡41 Schwab et al37,42
53/107 18/40 5/9 142/200 5/8 37/50 14/19 8/8 12/18 7/10 37/44 9/9 4/7 5/13 4/4 6/6 10/13 5/7
(49.5) (45) (55.6) (71.0) (62.5) (74.0) (73.7) (100.0) (66.7) (70.0) (84.1) (100.0) (57.1) (38.5) (100.0) (100.0) (76.9) (71.4)
BCVA of At least 2-line Improvement, n/N (%)
Autograft
Allograft
All
Autograft
Allograft
53/107 (49.5) 18/40 (45) 5/9 (55.6) 142/200 (71.0) 5/8 (62.5) 28/34 (82.4) 8/12 (66.7) 8/8 (100.0) 10/15 (66.7) 1/3 (33.3) 27/30 (90.0) 2/2 (100.0) 1/3 (33.3) — 4/4 (100.0) 6/6 (100.0) — 4/5 (80.0)
— — — — — 9/16 (56.3) 6/7 (85.7) — 2/3 (66.7) 6/7 (85.7) 10/14 (71.4) 7/7 (100.0) 3/4 (75.0) 5/13 (38.5) — — 10/13 (76.9) 1/2 (50.0)
58/107 (54.2) NA 2/9 (22.2) 121/200 (60.5) 7/8 (87.5) 34/50 (68.0) 13/19 (68.4) 7/8 (87.5) 10/18 (55.6) 5/10 (50.0) 32/44 (72.7)† 9/9 (100.0) 7/7 (100.0) 10/13 (76.9) 4/4 (100.0) 3/6 (50.0) 10/13 (76.9) 3/7 (42.9)
58/107 (54.2) NA 2/9 (22.2) 121/200 (60.5) 7/8 (87.5) 12/13 (92.3)* 8/12 (66.7) 7/8 (87.5) 8/15 (53.3) 1/3 (66.7) 21/30 (70.0)† 2/2 (200.0) 3/3 (100.0) — 4/4 (100.0) 3/6 (50.0) — 2/5 (40.0)
— — — — — 2/7 (28.6)* 5/7 (71.4) — 2/3 (66.7) 4/7 (57.1) 9/14 (64.3)† 7/7 (100.0) 4/4 (100.0) 10/13 (76.9) — — 12/13 (92.3) 1/2 (50.0)
*From partial data given by the article. †Data from the article with contradiction. ‡Success rate was not directly given in these articles, and it was inferred by our definition of success. BCVA, best-corrected visual acuity.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
www.corneajrnl.com |
597
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Zhao and Ma
Cornea Volume 34, Number 5, May 2015
FIGURE 3. Forest plot for the outcomes. A, Forest plot for pooled success rate of CLET with AM. B, Forest plot for success rate of autograft and allograft. C, Forest plot for a 2-line improvement of BCVA by CLET with AM. D, Forest plot for a 2-line improvement in BCVA of autograft and allograft. Shweta Sharma et al39 and Ray Jui-Fang Tsai et al40 in A and C.
whereas denuded HAM promoted a corneal epithelial phenotype. Rendal-Vázquez ME et al51 reported that a normal limbal epithelium, 1 to 2 cell layers thick, of which the basal layer of cells had high expression of the putative LESC markers p63 and ABCG2, was well formed on intact AM. However, Baharvand et al52 showed that denuded HAM provided a superior niche for LESC proliferation and phenotype maintenance in vitro. As to the culture system, a study by Li et al53 showed that, during explant outgrowth, a proportion of epithelial progenitor cells invade the limbal stroma and, by undergoing epithelial–mesenchymal transition, reduce the quantity of progenitor cells on the explant surface. But that the limbal explant culture epithelial outgrowth covered the AM much earlier than the suspension culture was also reported.32 Because the use of animalderived components in ex vivo expansion of epithelial stem cells poses potential risks of pathogen transmission, Sharma SM et al49 who made a comparison between human limbal epithelium cocultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and no feeder cells (NF) found that epithelial cells grown on a DF feeder layer maintained a stem cell–like phenotype, comparable with cells grown on a 3T3 feeder layer. Reports of eliminating the use of feeder cells completely, in terms of xeno-free culture,28 have been widespread, and long-term observation may be necessary to draw a conclusion. Airlifting was used in some studies to cause
stratification of the epithelial cells. However, Li et al54 reported that stratification promoted by airlifting was coupled with squamous metaplasia in human limbal explants. The expression of all markers, stem cell markers and differentiated markers, in the culture suggests that cultured limbal epithelial cells contained a heterogeneous population of corneal progenitor cells and matured corneal epithelial cells.30 To improve the success rate, it is of great importance to ensure the proportion of stem cells in the culture. To date, there is no definitive marker of limbal stem cells, and the most promising candidates are p63 and ABCG2. Rama et al55 suggested an association between their clinical results and the percentage of cells in the culture that stained positive for p63. It was found that those LESCs cultivated with more than 3% p63 bright cells were associated with a success rate of 78% in clinical transplantation, whereas less than 3% p63 bright cells were only successful in 11%. Evidently, functional assays preserving the colony-forming ability of transplanted cells is also important, as the capacity of transplanted cells continuing to regenerate the ocular surface after prolonged periods is an indirect indicator of the long-term proliferative potential of transplanted cells.36 In the present study, consistent with the reports by Shortt et al46 and Baylis et al56 no difference both in success rate and visual acuity outcome between autograft and allograft was found, although we used meta-analysis and strict inclusion criteria to try to improve the reliability of the outcomes. The
598
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 5, May 2015
result may provide some suggestion for surgeons to choose the source of limbus. There is no doubt that for unilateral disease, the fellow healthy eye or a normal region of an eye that was partially stem cell deficient is the best source of tissue. However, when the disease is bilateral, the options are deceased or living related donors, although with subsequent immunosuppression required, which may also lead to a promising outcome at least in a limited postoperative period. Definitely, whether the long-term effect of these 2 sources is of no difference or not needs further research.
Limitations of Current Study The included studies in our search recruited participants of all ages and both genders, covered different regions, and accommodated different types of LSCD. Stage of disease in each patient, duration between injury and surgery, culture methods, interventions both in the primary procedure and in subsequent interventions, and outcome measures used to define successful treatment varied in different studies. Most studies were not prospective, and it was unclear whether recruitment was consecutive in more than 80% of studies. Specifically, not all studies reported a minimum follow-up of more than 6 months. Additionally, most work, such as article selection, data extraction, and assessment of studies, was carried out by a single person. And there probably exists publication bias according to the funnel plot analysis when comparing autograft and allograft. All these contribute to heterogeneity. In the future, standardized methods of assessing success of both objective and subjective outcomes may be required. The long-term results of autograft and allograft will inform future treatment algorithms and techniques with random control trials and better-designed analysis.
ACKNOWLEDGMENTS The authors thank S.Q. Li of Statistics Department, Shanxi Medical University, for providing statistical advice, and the following persons for their assistance during the search: Q. Chen, Dr R. Y. Zhu, Dr L. Hu, and Z. Zhang. REFERENCES 1. Leblond CP. The life history of cells in renewing systems. Am J Anat. 1981;160:114–158. 2. Potten CS, Morris RJ. Epithelial stem cells in vivo. J Cell Sci Suppl. 1988;10:45–62. 3. Pellegrini G, Golisano O, Paterna P, et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol. 1999;145:769–782. 4. Dua HS, Azuara-Blanco A. Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency. Br J Ophthalmol. 2000;84: 273–278. 5. Espana EM, Di Pascuale MA, He H, et al. Characterization of corneal pannus removed from patients with total limbal stem cell deficiency. Invest Ophthalmol Vis Sci. 2004;45:2961–2966. 6. Santos MS, Gomes JA, Hofling-Lima AL, et al. Survival analysis of conjunctival limbal grafts and amniotic membrane transplantation in eyes with total limbal stem cell deficiency. Am J Ophthalmol. 2005;140: 223–230. 7. Tseng SC. Concept and application of limbal stem cells. Eye (Lond). 1989;3:141–157.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Systematic Review and Meta-analysis
8. Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol. 2000;44:415–425. 9. Nishida K, Kinoshita S, Ohashi Y, et al. Ocular surface abnormalities in aniridia. Am J Ophthalmol. 1995;120:368–375. 10. Tseng SCG, Espana EM, Di Pascuale MA. Stem cell transplantation for ocular surface diseases. In: Tasman W, Jaeger EA, eds. Duanes Clinical Ophthalmology on CD-rom. Baltimore, MD: Lippincott Williams & Wilkins; 2005. 11. Calonge M, Diebold Y, Saez V, et al. Impression cytology of the ocular surface: a review. Exp Eye Res. 2004;78:457–472. 12. Cauchi PA, Ang GS, Azuara-Blanco A, et al. A systematic literature review of surgical interventions for limbal stem cell deficiency in humans. Am J Ophthalmol. 2008;146:251–259. 13. Geerling G, Maclennan S, Hartwig D. Autologous serum eye drops for ocular surface disorders. Br J Ophthalmol. 2004;88:1467–1474. 14. Young AL, Cheng AC, Ng HK, et al. The use of autologous serum tears in persistent corneal epithelial defects. Eye (Lond). 2004;18:609–614. 15. Ahmad S, Kolli S, Lako M, et al. Stem cell therapies for ocular surface disease. Drug Discov Today. 2010;15:306–313. 16. Ahmad S, Osei-Bempong C, Dana R, et al. The culture and transplantation of human limbal stem cells. J Cell Physiol. 2010;225:15–19. 17. Dua HS. The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol. 1998;82:1407–1411. 18. Tseng SCG, Prabhasawat P, Barton K, et al. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch Ophthalmol. 1998;116:431–441. 19. Espana EM, Di Pascuale M, Grueterich M, et al. Keratolimbal allograft in corneal reconstruction. Eye (Lond). 2004;18:406–417. 20. Shortt AJ, Tuft SJ, Daniels JT. Corneal stem cells in the eye clinic. Br Med Bull. 2011;100:209–225. 21. Yeh HJ, Yao CL, Chen HI, et al. Cryopreservation of human limbal stem cells ex vivo expanded on amniotic membrane. Cornea. 2008;27:327–333. 22. Dua HS, Gomes JA, King AJ, et al. The amniotic membrane in ophthalmology. Surv Ophthalmol. 2004;49:51–77. 23. Gomes JA, Romano A, Santos MS, et al. Amniotic membrane use in ophthalmology. Curr Opin Ophthalmol. 2005;16:233–240. 24. Akle CA, Adinolfi M, Welsh KI, et al. Immunogenicity of human amniotic epithelial cells after transplantation into volunteers. Lancet. 1981;2:1003–1005. 25. Sejpal K, Ali MH, Maddileti S, et al. Cultivated limbal epithelial transplantation in children with ocular surface burns. JAMA Ophthalmol. 2013;131:731–736. 26. Subramaniam SV, Sejpal K, Fatima A, et al. Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface diaorders: long-term survival analysis. Indian J Ophthalmol. 2013;61: 202–207. 27. Pathak M, Cholidis S, Haug K, et al. Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series. Acta Ophthalmol. 2013;91:769–775. 28. Sangwan VS, Basu S, Vemuganti GK, et al. Clinical outcome of xenofree autologous cultivated limbal epithelial transplantation: a 10-year study. Br J Ophthalmol. 2011;95:1525–1529. 29. Baradaran-Rafii A, Ebrahimi M, Kanavi MR, et al. Midterm outcomes of autologous cultivated limbal stem cell transplantation with or without penetrating keratoplasty. Cornea. 2010;29:502–509. 30. Sharma S, Tandon R, Mohanty S, et al. Culture of corneal limbal epithelial stem cell: experience from benchtop to bedside in a tertiary care hospital in India. Cornea. 2011;30:1223–1232. 31. Prabhasawat P, Ekpo P, Uiprasertkul M, et al. Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction. Clin Ophthalmol. 2012;6:1483–1492. 32. Kolli S, Ahmad S, Lako M, et al. Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency. Stem Cells. 2010;28:597–610. 33. Zakaria N, Possemiers T, Dhubhghaill SN, et al. Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation. J Transl Med. 2014;3:58. 34. Shortt AJ, Secker GA, Rajan MS, et al. Ex vivo expansion and transplantation of limbal epithelial stem cells. Ophthalmology. 2008; 115:1989–1997.
www.corneajrnl.com |
599
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Zhao and Ma
Cornea Volume 34, Number 5, May 2015
35. Pauklin M, Fuchsluger TA, Westekemper H, et al. Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency. Dev Ophthalmol. 2010;45:57–70. 36. 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;113:1765–1772. 37. Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea. 2000;19:421–426. 38. Shimazaki J, Aiba M, Goto E, et al. Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders. Ophthalmology. 2002;109:1285–1290. 39. Sharma S, Tandon R, Mohanty S, et al. Phenotypic evaluation of severely damaged ocular surface after reconstruction by cultured limbal epithelial cell transplantation. Ophthalmic Res. 2013;50:59–64. 40. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2000; 343:86–93. 41. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology. 2001; 108:1569–1574. 42. Schwab IR. Cultured corneal epithelial for ocular surface disease. Trans Am Ophthalmol Soc. 1999;97:891–986. 43. Grueterich M, Espana EM, Tseng SC. Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche. Surv Ophthalmol. 2003;48:631–646. 44. Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349:990–993. 45. Ramaesh K, Dhillon B. Ex vivo expansion of corneal limbal epithelial/stem cells for corneal surface reconstruction. Eur J Ophthalmol. 2003;13: 515–524.
46. 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;52:483–502. 47. Shortt AJ, Secker GA, Lomas RJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelia cells. Biomaterials. 2009;30:1056–1065. 48. Sangwan VS, Matalia HP, Vemuganti GK, et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J Ophthalmol. 2006;54:29–34. 49. SM Sharma, Fuchsluger T, Ahmad S, et al. Comparative analysis of human-derived feeder layers with 3T3 fibroblasts for the ex vivo expansion of human limbal and oral epithelium. Stem Cell Rev. 2012;8:696–705. 50. Li W, He H, Kuo CL, et al. Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane. Invest Ophthalmol Vis Sci. 2006;47:2381–2389. 51. Rendal-Vázquez ME, San-Luis-Verdes A, Yebra-Pimentel-Vilar MT, et al. Culture of limbal stem cells on human amniotic membrane. Cell Tissue Bank. 2012;13:513–519. 52. Baharvand H, Heidari M, Ebrahimi M, et al. Proteomic analysis of epithelium-denuded human amniotic membrane as a limbal stem cell niche. Mol Vis. 2007;13:1711–1721. 53. Li W, Hayashida Y, He H, et al. The fate of limbal epithelial progenitor cells during explant culture on intact amniotic membrane. Invest Ophthalmol Vis Sci. 2007;48:605–613. 54. Li W, Hayashida Y, Chen YT, et al. Air exposure induced squamous metaplasia of human limbal epithelium. Invest Ophthalmol Vis Sci. 2008; 49:154–162. 55. Rama P, Matuska S, Paganoni G, et al. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363:147–155. 56. O1 Baylis, Figueiredo F, Henein C, et al. 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. J Cell Biochem. 2011; 112:993–1002.
600
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
| www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
