March 2012
Volume 53, Issue 3
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Cornea  |   March 2012
Transplantation of Cultivated Oral Mucosal Epithelium Prepared in Fibrin-Coated Culture Dishes
Author Affiliations & Notes
  • Masatoshi Hirayama
    From the Department of Ophthalmology, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan; and
    the Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
  • Yoshiyuki Satake
    From the Department of Ophthalmology, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan; and
  • Kazunari Higa
    From the Department of Ophthalmology, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan; and
  • Takefumi Yamaguchi
    From the Department of Ophthalmology, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan; and
    the Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
  • Jun Shimazaki
    From the Department of Ophthalmology, Ichikawa General Hospital, Tokyo Dental College, Chiba, Japan; and
    the Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
  • Corresponding author: Jun Shimazaki, Department of Ophthalmology, Ichikawa General Hospital, Tokyo Dental College, 5-11-13, Sugano, Ichikawa, Chiba, 273-0041, Japan; jun@eyebank.or.jp
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1602-1609. doi:10.1167/iovs.11-7847
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      Masatoshi Hirayama, Yoshiyuki Satake, Kazunari Higa, Takefumi Yamaguchi, Jun Shimazaki; Transplantation of Cultivated Oral Mucosal Epithelium Prepared in Fibrin-Coated Culture Dishes. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1602-1609. doi: 10.1167/iovs.11-7847.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To compare the clinical results of cultivated oral mucosal epithelial cell sheet transplantation (COMET) of substrate-free sheets with those of COMET of amniotic membrane (AM)-based sheets.

Methods.: Sixteen eyes receiving COMET of substrate-free sheets (substrate-free group) were studied retrospectively and compared with disease-, age-, and ocular surface status–matched eyes undergoing COMET with AM serving as the substrate (AM group). Each group consisted of six eyes with chemical injury, six with pseudo-ocular cicatricial pemphigoid, two with Stevens-Johnson syndrome, and two with ocular cicatricial pemphigoid. Graft survival rate, best-corrected visual acuity (BCVA), and neovascularization (NV) were assessed.

Result.: In all 32 eyes, the entire corneal surface on which the cultivated autologous oral mucosal epithelium sheet had been placed was free of epithelial defects at postoperative day 2. The success rates of COMET at 12 months after surgery were 62.5% in the substrate-free sheet group and 43.8% in the AM group. A Kaplan-Meier curve revealed that the graft survival rate in the substrate-free group was significantly superior to that in the AM group (P = 0.046). Mean postoperative BCVA improved significantly at 1, 3, and 6 months in the substrate-free sheet group, and BCVA was significantly better than that in the AM group at all time points. Postoperative NV improved significantly in the substrate-free group at all time points.

Conclusions.: A better midterm clinical outcome was achieved with COMET of a substrate-free cell sheet than with COMET of AM as a substrate for treating severe stem cell deficiency.

Maintaining a healthy ocular surface (OS) is crucial for acquiring good visual acuity and barrier function. The corneal epithelium is multilayered and nonkeratinized. The stem cells of the corneal epithelium, which are believed to reside in the limbal region, 1,2 differentiate into mature corneal epithelial cells and form part of the OS. 3 Severe OS disorders, such as chemical and thermal injuries, Stevens-Johnson syndrome (SJS), and ocular cicatricial pemphigoid (OCP), lead to limbal stem cell deficiency (LSCD), which causes conjunctival epithelial invasion of the corneal surface with chronic inflammation, persistent epithelial defects (PED), stromal opacity, and neovascularization (NV). 4,5  
Recently, transplantation of ex vivo cultivated epithelial cell sheets has been developed to treat LSCD. Autologous limbal epithelium is considered to be the best source of donor material. 6 However, allogenic cell sheets are inferior because of the risk of immunologic rejection and steroid use-related complications. Various groups have investigated the use of autologous ectopic tissue, including conjunctiva, oral mucosa, nasal mucosa, and dental pulp as potential cell sources. Cultivated oral mucosal epithelial transplantation (COMET) has been demonstrated to be promising in both basic and clinical research. 7 10 The benefits of COMET include availability in cases with bilateral LSCD and no need for long-term immunosuppression. 
Various methods of preparing cell sheets have been investigated, including varying the culture medium or conditions and the use of feeder cells and alternative cell sources. Each method has produced a different type of cell sheet, and such variations may affect clinical outcomes. 9,11 For example, use of a substrate in cell culture influences cell growth and differentiation. 12,13 However, no studies to date have compared the clinical outcome of COMET using cell sheets produced by different methods. Our institute has prepared oral mucosal cell sheets on amniotic membrane (AM) as a carrier and on fibrin-coated culture dishes. 13 The latter technique was first reported by Rama et al. 11 and Itabashi et al., 14 and our group modified the technique by adjusting the degree of fibrin degradation and by administering a proteinase inhibitor. 13 Briefly, oral mucosal epithelial cells were cultivated on fibrin-coated dishes with proteinase inhibitors such as aprotinin for 2 weeks. The underlying fibrin was then degraded by discontinuing the aprotinin, thus allowing removal of the cells as a substrate-free cell sheet. 
We present clinical data on 16 eyes that underwent COMET of substrate-free cell sheets prepared on fibrin-coated dishes. The results were compared with those obtained with an AM substrate in a case–control study. 
Methods
This report describes the clinical outcome of COMET as a retrospective, case–control study. Sixteen consecutive eyes that underwent COMET of substrate-free sheets for total LSCD in our hospital between April 2004 and September 2009 were included in the study (substrate-free sheet group). Total LSCD was clinically diagnosed in all patients on the basis of a complete absence of the Palisades of Vogt and conjunctivalization. All study protocols and procurement of human tissue complied with the Declaration of Helsinki. Informed consent was obtained from all participants after a full explanation of the risks and benefits of the study. 
Sixteen age-, disease-, and OS status–matched eyes (AM sheet group) that underwent COMET with sheets prepared on an AM substrate were chosen from 40 eyes (all AM group) that had undergone surgery during the same period. We conducted a case–control study, because we found significant differences in both original diseases (more SJS and OCP in the all AM group; P = 0.039) and preoperative visual acuity (1.68 ± 0.83 in the substrate-free group and 2.17 ± 0.55 in the all AM group; P = 0.020) between eyes that had undergone substrate-free and AM-based COMET when all consecutive cases were analyzed. We matched preoperative OS status, such as persistent epithelial defects and symblepharon, to further minimize selection bias. 
There were no eyes in the substrate-free group that had a history of epithelial transplantation such as cultivated limbal epithelial transplantation or COMET. Eyes in AM groups were excluded if they had a history of previous corneal epithelial transplantation, as such procedures may have affected the clinical outcome. 
Preoperative characteristics of the patients in the substrate-free and AM sheet groups are shown in Tables 1 and 2. The substrate-free sheet group consisted of 10 men and 5 women with a mean ± SD age of 60.1 ± 15.4 years. The mean follow-up period was 109.8 ± 47.0 weeks. The preoperative diagnosis was chemical injury in six eyes, pseudo-ocular cicatricial pemphigoid (POCP) in six (trachoma in three, Behçet's disease in one, and postkeratitis in two), Stevens-Johnson syndrome (SJS) in two, and ocular cicatricial pemphigoid (OCP) in two. A Schirmer I test result of >4 mm was obtained before surgery in 15 of 16 eyes. Four eyes manifested PED, seven eyes had conjunctivalization of the corneal surface, and nine eyes had symblepharon at the preoperative examination. 
Table 1.
 
Preoperative Characteristics of Patients with Total LCSD
Table 1.
 
Preoperative Characteristics of Patients with Total LCSD
Case No. Age/Sex Diagnosis Eye OS Condition Schirmer's Test without Topical Anesthesia (mm)
PED* Conjunctivalization† Symblepharon‡ NV§ (Grade)
Patients Receiving a Substrate-free Transplant
1 70/M CI Left + 3 9
2 29/M CI Left + 1 10
3 69/M CI Right 2 35
4 66/M CI Left + 7
5 54/M CI Left + + 13
6 39/M CI Left + + 3 4
7 49/M SJS Right + 3 Unknown
8 55/M SJS Left + + 2 5
9 69/F OCP Right + 3 5
10 75/F OCP Left + 2 12
11 68/F POCP Right + + 2 15
12 67/F POCP Left + + 3 9
13 69/M POCP Left + + 2 16
14 80/F POCP Right + 2 18
15 71/F POCP Right + 3 4
16 33/M POCP Left 3 20
Patients Receiving an AM-based Transplant
1 71/F CI Left 3 12
2 37/M CI Left + + 3 12
3 64/M CI Left 3 31
4 57/M CI Right + 2 Unknown
5 54/M CI Right + 10
6 39/M CI Left + Unknown
7 48/M SJS Left + + 3 5
8 53/M SJS Left + + 2 Unknown
9 72/M OCP Left + + + 3 Unknown
10 74/F OCP Right + 1 11
11 59/F POCP Right + + 3 Unknown
12 65/M POCP Right + 3 1
13 65/F POCP Right + + 3 9
14 81/M POCP Left + 3 13
15 81/F POCP Left + 6
16 14/M POCP Right + + 3 34
Table 2.
 
Patient Demographics
Table 2.
 
Patient Demographics
Characteristics Substrate-Free Group AM Group P
Age, y
    Mean (SD) 60.1 (15.4) 58.4 (17.7) 0.32
    Range 29–80 14–81
Sex
    Male:female 10:6 11:5 1.0
Disease
    CI 6 6
    POCP 6 6
    SJS 2 2
    OCP 2 2
Follow-up period, wk
    Mean (SD) 109.8 (47.0) 146.6 (74.1) 0.20
The AM sheet group consisted of 11 men and 5 women with a mean age of 58.4 ± 17.7 years. The mean follow-up period was 146.6 ± 74.1 weeks. The preoperative diagnosis was chemical injury in six eyes, POCP in six (trachoma in one, Behçet's disease in one, thermal burn in one, and postkeratitis in three), SJS in two, and OCP in two. Total LSCD was clinically diagnosed in all these patients. Seven eyes manifested PED, four eyes had conjunctivalization of the cornea, and 11 eyes had symblepharon. 
Preparation of Cultivated Oral Mucosal Epithelial Sheets
AM was donated with written informed consent by mothers seronegative for the human immunodeficiency and hepatitis B and C viruses at the time of cesarian section. The AM was stored in 15% dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO) and phosphate-buffered saline (PBS) at −30°C until use. Denuded AM was prepared as described previously. 15 Membranes were rinsed in PBS, spread onto the upper chambers of a six-well insert (Transwell; Corning, Corning, NY), and air dried at room temperature. Fibrin-coated inserts were prepared as described previously. 13 Briefly, cell culture inserts (Corning) were coated with 250 μL fibrin (3 mg/mL; Bolheal; Astellas, Tokyo, Japan) and stored at 4°C. After the fibrin had polymerized 2 hours later, the inserts were kept in storage until use. 
The procedures for harvesting and culturing the oral mucosal tissues were as described by Satake et al. 16 All tissue donors were informed of the purpose of the study and received advice on oral hygiene and treatment for tooth decay before the oral mucosal biopsy. After sterilizing the oral cavity, the inferior buccal mucosa was excised with an 8-mm-diameter biopsy punch (KAI Industries Co., Ltd., Gifu, Japan) with the donor under local anesthesia. Specimens were removed from the submucosal connective tissue by dissection with scissors. The mucosal epithelium was cut into small pieces and then washed several times in sterile Ca2+ and Mg2+-free PBS to remove blood and adipose tissue. The specimens were then submerged in Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 mixture at a ratio of 1:1 (vol/vol) (Invitrogen Corp., Carlsbad, CA) with 5 g/mL gentamicin (Invitrogen), 0.25 g/mL amphotericin B (Sigma), 100 μg/mL streptomycin (Wako Pure Chemical, Osaka, Japan), and 100 units/mL penicillin (Wako). The basal cells of the oral mucosal epithelial cells were harvested after enzymatic treatment with 1.2 IU Dispase II (Roche Diagnostics, Indianapolis, IN) at 37°C for 1 hour and 0.05% trypsin-0.53 mM EDTA solution (Invitrogen-Gibco) at room temperature for 10 minutes. The cell suspension was washed in DMEM/F12 medium (1:1 mixture) with 10% fetal bovine serum (FBS), to remove debris and small pieces of residual material. A single-cell suspension of basal cells from the oral mucosal epithelium was resuspended in conditioned medium for oral mucosal epithelium (DMEM/F12 supplemented with 10 ng/mL epidermal growth factor [EGF], Invitrogen-Gibco; 10 μg/mL insulin, Wako; 0.5 μg/mL hydrocortisone, Sigma; 1 μM isoproterenol, Sigma; 2 nM triiodothyronine, Sigma; 100 μg/mL streptomycin, Wako; 100 units/mL penicillin, Wako; and 4% autologous serum), followed by seeding (1.0–2.0 × 105 cells/well) onto human denuded AM- or fibrin-coated culture plate inserts in a six-well plate (Transwell; Corning) containing mitomycin-C (MMC; Sigma) treated with 3T3 fibroblasts (2.5 × 105 cells/well). The culture was submerged in this medium until confluence, after which the medium was changed (DMEM/F12 supplemented with 10 ng/mL EGF, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 100 μg/mL streptomycin, 100 units/mL penicillin, and 4% autologous serum). The culture was exposed to air by lowering the level of the medium at the end of the culture period. Aprotinin (300 KIU/mL; Wako) was added to the medium to prevent fibrin digestion during culture. A histopathologic examination revealed that the cultivated oral mucosal epithelial sheets consisted of five to six cell layers. Aprotinin was discontinued in conjunction with cell growth (Fig. 1). 
Figure 1.
 
Culturing scheme for substrate-free sheets.
Figure 1.
 
Culturing scheme for substrate-free sheets.
COMET Surgical Procedure
After a 360° conjunctival peritomy, we scraped the area of the epithelial defect, removing a thin layer of conjunctivalized tissue by superficial keratectomy. In patients with symblepharon, we lysed the adhesion with scissors and formed the conjunctival fornix. The subconjunctival space was treated by placing microsponges containing 0.04% MMC for 5 minutes, followed by a saline wash (300 mL). The cultivated autologous oral mucosal epithelial sheet on the AM substrate was then secured to the corneal surface with 10-0 nylon sutures. The substrate-free sheet was transferred to the patient's eye via the donut-shaped filter paper that was used to detach the sheet from the culture insert (Fig. 1). The filter paper was removed easily with forceps without damaging the cell sheets. 10 No sutures were used when a substrate-free sheet was transplanted. The OS was protected with a therapeutic contact lens. After surgery, topical antibiotics (0.5% levofloxacin, Cravit; Santen Pharmaceutical Co., Ltd., Osaka, Japan) and steroids (0.1% betamethasone; Sanbetason; Santen Pharmaceutical) were initially applied five times per day and then tapered to three times per day. At approximately 1 month after surgery, topical corticosteroid administration was switched from 0.1% betamethasone to 0.1% fluorometholone (Fluometholon; Santen Pharmaceutical). Preservative-free artificial tears were applied frequently. 
Clinical Assessment
Preoperative and postoperative BCVAs were measured with Landolt acuity charts, and OS manifestations were inspected with a slit lamp biomicroscope and fluorescein staining. PED was defined as a corneal epithelial defect of at least 1 week's duration for which conventional therapy had failed. Conventional therapy included antibiotic ointment; eyelid closure procedures, such as tarsorrhaphy; and wearing therapeutic contact lens. NV was categorized into four grades by slit lamp biomicroscopy: grade 0, no NV on cornea; grade 1, NV reaching the peripheral cornea; grade 2, NV reaching the area between grades 1 and 3; and grade 3, NV reaching the central area of the cornea. We also determined whether the OS was stable. The criteria for a stable OS included: a clear appearance with no epithelial defect, decreased fibrovascular tissue invasion of the cornea, and no or minimal symblepharon. We regarded the graft as having failed when a stable OS was lost. We quantified and conducted a comparative study of each patient's factors to assess the OS status. We scored OS status as: PED, 1 point; conjunctivalization, 1 point; and symblepharon, 1 point. The mean total score of each patient was calculated. 
Statistical Analysis
Statistical analyses were conducted using Landolt visual acuity values that were converted to logMAR. The logMAR of hand motion was defined as 3, according to the visual acuity measurements by Holladay. Failure was analyzed separately and was investigated in terms of age, sex, cause, and type of procedure (substrate-free sheet or AM sheet). Differences between groups were evaluated by Mann-Whitney U test, Wilcoxon signed-rank test, or Mantel-Cox test. P < 0.05 was considered statistically significant (Excel Statistics 2010; Social Survey Research Information Co., Ltd. Tokyo, Japan). 
Results
Graft Sheet Survival
In the substrate-free group, one eye showed a small epithelial defect at the edge of the cell sheet on postoperative day 1; however, it improved by the next day. No other eyes developed epithelial defects during the patients' hospitalization. Cell sheets on the corneal surface were stable, with no epithelial defects in the AM group. 
In the substrate-free sheet group, a successful OS was achieved in 10 eyes (62.5%) at the last follow-up visit. PED developed in four eyes, and fibrovascular tissue invasion developed in two eyes. A stable OS was achieved in 6 of 16 eyes (37.5%) in the AM sheet group at the last follow-up visit. However, PED developed in six eyes and fibrovascular tissue invasion in four. A Kaplan-Meier analysis revealed that the graft survival rate was significantly better in the substrate-free sheet group than in the AM sheet group (P = 0.046; Mantel-Cox test; Fig. 2). 
Figure 2.
 
Graft survival rate. Kaplan-Meier analysis revealed that the graft survival rate was significantly better in the substrate-free sheet group than in the AM sheet group (P = 0.046; Mantel-Cox test).
Figure 2.
 
Graft survival rate. Kaplan-Meier analysis revealed that the graft survival rate was significantly better in the substrate-free sheet group than in the AM sheet group (P = 0.046; Mantel-Cox test).
Postoperative Visual Acuity
As shown in Figure 3, mean postoperative BCVA improved significantly at 1 (P = 0.016), 3 (P = 0.0061), 6 (P = 0.041), and 12 (P = 0.0090) months in the substrate-free group. Eleven eyes in the substrate-free group (68.8%) showed BCVA improvements by more than two lines during follow-up. In the AM sheet group, mean BCVA improved significantly at 1 (P = 0.024) and 3 (P = 0.023) months, but not at 6 or 12 months. Seven eyes (43.8%) showed improvements in BCVA of more than two lines (Table 3). Postoperative mean BCVA in the substrate-free group was significantly better than that in the AM group at all time points. 
Figure 3.
 
Preoperative and postoperative BCVA. *Wilcoxon test; P < 0.05 compared the difference with preoperative BCVA, †Mann-Whitney U test; P < 0.05 compared the difference with the value of AM group.
Figure 3.
 
Preoperative and postoperative BCVA. *Wilcoxon test; P < 0.05 compared the difference with preoperative BCVA, †Mann-Whitney U test; P < 0.05 compared the difference with the value of AM group.
Table 3.
 
Proportion of Eyes with an NV Grade >2
Table 3.
 
Proportion of Eyes with an NV Grade >2
Substrate-Free Group AM Group P
Before surgery 7/16 10/16 0.48
Postoperative month 1 0/16 0/16 1.0
P 0.0024* 0.00020*
Postoperative month 3 1/16 1/16 1.0
P 0.037* 0.0021*
Postoperative month 6 1/16 5/15 0.083
P 0.037* 0.16
Postoperative month 12 2/16 8/15 0.023*
P 0.11 0.72
Neovascularization
At the preoperative examination, grade 3 NV was present in 7 (43.8%) eyes in the substrate-free group and in 10 (62.5%) eyes in the AM group. Grade 3 NV was observed after surgery in both groups (Table 2). The incidence of an NV grade >2 decreased significantly at 1, 3, and 6 months in the substrate-free group, and at 1 and 3 months in the AM group. At 12 months, the incidence of eyes with an NV grade >2 was significantly less in the substrate-free group than in the AM group (P = 0.023). 
OS Status
No significant differences were observed in preoperative OS scores between the substrate-free and AM groups (P = 0.68; Mann-Whitney U test). Postoperative OS status was significantly improved in both groups at any time points. The OS score at 1 month in the substrate-free group was significantly less than that in the AM group. There were no significant differences between the substrate-free and AM groups in postoperative OS status at 3, 6, and 12 months (Mann-Whitney U test; Fig. 4). 
Figure 4.
 
Preoperative and postoperative OS score. OS scores were assessed as PED, 1 point; conjunctivalization, 1 point; symblepharon, 1 point. Mean of total scores of each patient were described. There were no significant differences in postoperative OS status at 3, 6, and 12 months between in substrate-free group and in AM group (Mann-Whitney U test). *Wilcoxon test; P < 0.05 compared the difference with preoperative OS status.
Figure 4.
 
Preoperative and postoperative OS score. OS scores were assessed as PED, 1 point; conjunctivalization, 1 point; symblepharon, 1 point. Mean of total scores of each patient were described. There were no significant differences in postoperative OS status at 3, 6, and 12 months between in substrate-free group and in AM group (Mann-Whitney U test). *Wilcoxon test; P < 0.05 compared the difference with preoperative OS status.
Complications
No intraoperative complications were observed in either group. Except for postoperative ocular hypertension in three eyes, no major postoperative complications, such as a microbial infection, developed in any eyes. 
Discussion
The results of this study suggest that substrate-free sheets are a promising option during COMET, to reconstruct the OS in eyes with total LSCD. This study is the first to report a comparison of 12-month surgical outcomes with the use of different cell sheet preparation methods. Use of a substrate-free sheet allowed for a stable OS in 10 (62.5%) of 16 eyes during a mean follow-up period of 109.8 weeks (Fig. 5). Apart from ocular hypertension, no remarkable postoperative complications, such as rejection or infection, were observed. 
Figure 5.
 
Slit lamp photographs of a patient with a successful outcome in the substrate-free group taken before surgery (A), at postoperative day 1 with fluorescein staining (B), and at the last follow-up visit both without fluorescein staining (C) and with fluorescein staining (D). This case showed total limbal stem cell deficiency preoperatively. The corneal surface was covered with oral mucosal epithelial cells after surgery and maintained a stable OS during follow-up.
Figure 5.
 
Slit lamp photographs of a patient with a successful outcome in the substrate-free group taken before surgery (A), at postoperative day 1 with fluorescein staining (B), and at the last follow-up visit both without fluorescein staining (C) and with fluorescein staining (D). This case showed total limbal stem cell deficiency preoperatively. The corneal surface was covered with oral mucosal epithelial cells after surgery and maintained a stable OS during follow-up.
The success rate of COMET using AM sheets in this study was similar to that reported in an earlier study. 11 We suspected that the substrate-free sheets may be more fragile than the AM sheets; however, the Kaplan-Meier analysis revealed that the graft survival rate in the substrate-free group was significantly better than that in the AM sheet group. In addition, eyes in the substrate-free group showed better improvements in BCVA than those in the AM group during postoperative follow-up. This outcome was consistent with the results of our previous animal study, in which optical clarity was higher in the substrate-free sheet transplantation group than in the AM sheet transplantation group. 13 These results are also consistent with the results of another study showing better corneal transparency with the use of substrate-free sheets than with AM sheets in a rabbit model, suggesting that substrate-free sheets contribute to better postoperative BCVA. 9,13 Because substrate-free cell sheets are in direct contact with the corneal stroma, transplanted epithelial cells directly interact with keratocytes in the corneal stroma without carrier interference. 17 This epithelial–stromal interaction may result in comparable graft survival and better BCVA in the substrate-free sheet group than in the AM sheet group. 9,17  
The space reduction caused by such direct adhesion of the cell sheets may have influenced the development of NV. In this study, a greater amount of eyes with an NV grade >3 was observed in the AM group than in the substrate-free group. Although it is believed that oral mucosal epithelial cells secrete angiogenic factors that induce NV of the graft, keratocytes in the corneal stroma may limit peripheral vascularization by producing antiangiogenic factors such as thrombospondin. 18 A decrease in the amount of available space may limit vascular invasion of the corneal center. 
However, several advantages have been raised regarding the use of AM as a substrate during cell sheet transplantation, including its avascularity and mechanical strength, its antiangiogenic and anti-inflammatory properties, its incorporation of growth factors, and its antifibrotic properties. 19 25 It has also been reported that AM is involved in the maintenance of “stemness” of the epithelium. 26 28 Sudha et al. 29 reported that ex vivo expansion of human limbal epithelial progenitor cells on AM is mediated by the PI3K/Akt/FKHRL1 pathway, which governs cell survival, and the mitogen-activated protein kinase pathway, which controls cell mitosis. Previous studies have shown that cell-cycle kinetics and cell phenotypic characteristics of limbal epithelial progenitor cells are preserved during ex vivo expansion on AM. 30,31 Moreover, more BrdU-labeled cells and fewer Ki67-labeled cells are observed in AM sheets than in carrier-free sheets after in vitro transplantation. 13 Taken together, these results suggest that AM affects cell differentiation and maintenance of stemness in vitro. However, this observation must be confirmed in a clinical study investigating long-term graft stability. Restoration of a clear OS was observed for at least 1 year after autologous oral mucosal epithelial cell sheet transplantation, suggesting that progenitor cells, which have the potential to differentiate into a corneal epithelial phenotype, exist in cell sheet transplants. 32 A longer observation period may be necessary to study ongoing OS stability in the substrate-free and AM sheet groups. 
Another major benefit of the substrate-free cell sheets is that no sutures or fibrin glue is needed to repair the graft. Although the reason for this phenomenon remains to be fully clarified, Nishida et al. 9 suggested that intact basement membrane substrates and adhesion molecules play a major role. In the substrate-free sheets, Higa et al. 13 confirmed the presence of β1 integrin, which may help prevent the sheets from sloughing off the OS. 13 In contrast, AM transplantation using sutures for a corneal injury causes ingrowth of cells, as observed under the AM carrier in a rabbit model; however, ingrowth of cells is prevented when fibrin glue is used. 33 These results indicate that the donor tissue must be strongly attached to the underlying stroma to maintain OS health. 
The limitations of this study include patient selection bias. To reduce selection bias, we carefully considered preoperative status in addition to preexisting diseases and patient age. We matched OS status such as PED, conjunctivalization, and symblepharon in both groups as much as possible. Although similar results were obtained by comparing all consecutive case series in both groups, we believe the case–control analysis conducted in the present study produced more convincing results. Further study, preferably a randomized clinical study with an increased number of cases and longer follow-up period, is necessary to substantiate the clinical outcomes. 
In summary, the results of the present study indicate that autologous oral mucosal epithelial cell transplantation using substrate-free sheets allows for midterm clinical outcomes better than those obtained with sheet grafts with AM as the substrate. Although further long-term follow-up studies are needed to assess the differences between these two approaches, we believe that the use of substrate-free sheets offers a new option to reconstruct a stable OS and treat severe OS diseases. 
Footnotes
 Supported in part by a Grant-in-Aid for scientific research (C) from the Japan Society for the Promotion of Science (KAKENHI: 22591951); and Oral Health Science Center Grant hrc8 from Tokyo Dental College, and by a Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology of Japan, 2010–2012. The sponsor and funding organization had no role in the design or conduct of this research.
Footnotes
 Disclosure: M. Hirayama, None; Y. Satake, None; K. Higa, None; T. Yamaguchi, None; J. Shimazaki, None
The authors thank Jeremy Williams, Tokyo Dental College, for assistance with the English in the manuscript. 
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Figure 1.
 
Culturing scheme for substrate-free sheets.
Figure 1.
 
Culturing scheme for substrate-free sheets.
Figure 2.
 
Graft survival rate. Kaplan-Meier analysis revealed that the graft survival rate was significantly better in the substrate-free sheet group than in the AM sheet group (P = 0.046; Mantel-Cox test).
Figure 2.
 
Graft survival rate. Kaplan-Meier analysis revealed that the graft survival rate was significantly better in the substrate-free sheet group than in the AM sheet group (P = 0.046; Mantel-Cox test).
Figure 3.
 
Preoperative and postoperative BCVA. *Wilcoxon test; P < 0.05 compared the difference with preoperative BCVA, †Mann-Whitney U test; P < 0.05 compared the difference with the value of AM group.
Figure 3.
 
Preoperative and postoperative BCVA. *Wilcoxon test; P < 0.05 compared the difference with preoperative BCVA, †Mann-Whitney U test; P < 0.05 compared the difference with the value of AM group.
Figure 4.
 
Preoperative and postoperative OS score. OS scores were assessed as PED, 1 point; conjunctivalization, 1 point; symblepharon, 1 point. Mean of total scores of each patient were described. There were no significant differences in postoperative OS status at 3, 6, and 12 months between in substrate-free group and in AM group (Mann-Whitney U test). *Wilcoxon test; P < 0.05 compared the difference with preoperative OS status.
Figure 4.
 
Preoperative and postoperative OS score. OS scores were assessed as PED, 1 point; conjunctivalization, 1 point; symblepharon, 1 point. Mean of total scores of each patient were described. There were no significant differences in postoperative OS status at 3, 6, and 12 months between in substrate-free group and in AM group (Mann-Whitney U test). *Wilcoxon test; P < 0.05 compared the difference with preoperative OS status.
Figure 5.
 
Slit lamp photographs of a patient with a successful outcome in the substrate-free group taken before surgery (A), at postoperative day 1 with fluorescein staining (B), and at the last follow-up visit both without fluorescein staining (C) and with fluorescein staining (D). This case showed total limbal stem cell deficiency preoperatively. The corneal surface was covered with oral mucosal epithelial cells after surgery and maintained a stable OS during follow-up.
Figure 5.
 
Slit lamp photographs of a patient with a successful outcome in the substrate-free group taken before surgery (A), at postoperative day 1 with fluorescein staining (B), and at the last follow-up visit both without fluorescein staining (C) and with fluorescein staining (D). This case showed total limbal stem cell deficiency preoperatively. The corneal surface was covered with oral mucosal epithelial cells after surgery and maintained a stable OS during follow-up.
Table 1.
 
Preoperative Characteristics of Patients with Total LCSD
Table 1.
 
Preoperative Characteristics of Patients with Total LCSD
Case No. Age/Sex Diagnosis Eye OS Condition Schirmer's Test without Topical Anesthesia (mm)
PED* Conjunctivalization† Symblepharon‡ NV§ (Grade)
Patients Receiving a Substrate-free Transplant
1 70/M CI Left + 3 9
2 29/M CI Left + 1 10
3 69/M CI Right 2 35
4 66/M CI Left + 7
5 54/M CI Left + + 13
6 39/M CI Left + + 3 4
7 49/M SJS Right + 3 Unknown
8 55/M SJS Left + + 2 5
9 69/F OCP Right + 3 5
10 75/F OCP Left + 2 12
11 68/F POCP Right + + 2 15
12 67/F POCP Left + + 3 9
13 69/M POCP Left + + 2 16
14 80/F POCP Right + 2 18
15 71/F POCP Right + 3 4
16 33/M POCP Left 3 20
Patients Receiving an AM-based Transplant
1 71/F CI Left 3 12
2 37/M CI Left + + 3 12
3 64/M CI Left 3 31
4 57/M CI Right + 2 Unknown
5 54/M CI Right + 10
6 39/M CI Left + Unknown
7 48/M SJS Left + + 3 5
8 53/M SJS Left + + 2 Unknown
9 72/M OCP Left + + + 3 Unknown
10 74/F OCP Right + 1 11
11 59/F POCP Right + + 3 Unknown
12 65/M POCP Right + 3 1
13 65/F POCP Right + + 3 9
14 81/M POCP Left + 3 13
15 81/F POCP Left + 6
16 14/M POCP Right + + 3 34
Table 2.
 
Patient Demographics
Table 2.
 
Patient Demographics
Characteristics Substrate-Free Group AM Group P
Age, y
    Mean (SD) 60.1 (15.4) 58.4 (17.7) 0.32
    Range 29–80 14–81
Sex
    Male:female 10:6 11:5 1.0
Disease
    CI 6 6
    POCP 6 6
    SJS 2 2
    OCP 2 2
Follow-up period, wk
    Mean (SD) 109.8 (47.0) 146.6 (74.1) 0.20
Table 3.
 
Proportion of Eyes with an NV Grade >2
Table 3.
 
Proportion of Eyes with an NV Grade >2
Substrate-Free Group AM Group P
Before surgery 7/16 10/16 0.48
Postoperative month 1 0/16 0/16 1.0
P 0.0024* 0.00020*
Postoperative month 3 1/16 1/16 1.0
P 0.037* 0.0021*
Postoperative month 6 1/16 5/15 0.083
P 0.037* 0.16
Postoperative month 12 2/16 8/15 0.023*
P 0.11 0.72
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