Background The Stevens–Johnson syndrome, ocular pemphigoid,and thermal or chemical burns can cause scarring and opacificationof the cornea and loss of vision. Transplantation of epithelialcells from the limbus of the contralateral cornea can restoreuseful vision. However, this procedure requires a large limbalgraft from the healthy eye and is not possible in patients whohave bilateral lesions.
Methods We took specimens of limbal epithelial cells from thehealthy contralateral eyes of six patients with severe unilateralcorneal disease. The epithelial cells were cultured and expandedon amniotic membrane. The amniotic membrane, together with thesheet of limbal epithelial cells, was transplanted to the denudedcorneal surface of the damaged eye after superficial keratectomyto remove fibrovascular ingrowth. The mean (±SD) follow-upperiod was 15±2 months.
Results Complete reepithelialization of the corneal surfaceoccurred within two to four days of transplantation in all sixeyes receiving transplants. By one month, the ocular surfacewas covered with corneal epithelium, and the clarity of thecornea was improved. In five of the six eyes receiving transplants(83 percent), the mean visual acuity improved from 20/112 to20/45. In one patient with a chemical burn who had total opacificationof the cornea, the acuity improved from the ability to countfingers at 40 cm to 20/200. No patient had recurrent neovascularizationor inflammation in the transplanted area during the follow-upperiod.
Conclusions Transplantation of autologous limbal epithelialcells cultured on amniotic membrane is a simple and effectivemethod of reconstructing the corneal surface and restoring usefulvision in patients with unilateral deficiency of limbal epithelialcells.
The normal ocular surface is covered by corneal, limbal, andconjunctival epithelial cells that, together with a stable preoculartear film, maintain its integrity. Severe damage to the limbalepithelial cells from chemical or thermal burns, the Stevens–Johnsonsyndrome, ocular cicatricial pemphigoid, contact lenses, severemicrobial infection, or multiple surgical procedures or cryotherapyin the limbal region may lead to loss of the limbal epithelialcells.1 Limbal-cell deficiency is usually manifested by vascularizationand chronic inflammation of the cornea, ingrowth of fibroustissue, and corneal opacification.2,3,4,5
Patients with limbal epithelial-cell deficiency in one eye only,or in both eyes but affecting different areas of the limbusin the two eyes, may be treated by transplantation of autologouslimbal tissue.6,7,8,9 A serious limitation of transplantationof autologous limbal epithelial cells is that one or two limbal-cellgrafts, spanning two to three clock hours of the limbus, haveto be removed from the healthy contralateral eye. Although noreports have described complications in donor eyes in humans,limbal-cell deficiency can occur in rabbits if the central cornealepithelium is subsequently removed from donor eyes.10,11 Toavoid this potential risk, limbal epithelial cells from a smalllimbal-biopsy specimen may be expanded in vitro. Transplantationof corneal epithelial cells expanded in vitro on 3T3 fibroblastsresulted in reconstruction of the corneal surface in two patientswith total loss of limbal epithelial cells.12
Transplantation of amniotic membrane to provide a substratefor regenerating epithelial cells has been found to be effectivein reconstructing the corneal surface in rabbits.13 Transplantationof limbal-tissue allografts,14 together with amniotic membrane,15,16,17,18in patients with partial or total limbal deficiency resultedin rapid reepithelialization of the reconstructed corneal surface,but prolonged immunosuppression was required.14 In this study,we took a small limbal-biopsy specimen from the normal contralateraleyes of six patients with unilateral limbal epithelial-celldeficiency and expanded the specimen on amniotic membrane toform an epithelial-cell sheet. We then transplanted the limbalepithelial-cell sheet, together with the amniotic-membrane substrate,to the damaged eye of the same patient.
Methods
Subjects
This study was approved by the Ophthalmologic Society and theDepartment of Health, Executive Yuan, Taiwan, and all the patientsgave oral informed consent. All surgical procedures were performedby the same surgeon at Chang Gung Memorial Hospital, Taoyuan,Taiwan.
The study subjects were two men and four women, with a mean(±SD) age of 24±9 years. All had partial or totalunilateral limbal epithelial-cell deficiency due to chemicalburns (Patients 1, 4, and 6), pseudopterygium after excisionof a dermoid cyst (Patient 2), congenital pterygium (Patent3), or recurrent chronic inflammation with phlyctenular degeneration(Patient 5) (Table 1). Before this study, four of the patientshad undergone surgery on their damaged eye. Patient 2 had undergoneautologous conjunctival transplantation, Patients 3 and 5 hadundergone amniotic-membrane transplantation, and Patient 1 hadundergone both procedures. All the previous transplantationshad failed and had resulted in recurrent fibrovascular ingrowtharound the limbal areas, indicating that the remaining limbalepithelial cells were unable to restore the corneal surface.Chemical burns had damaged 270 degrees of the limbus in Patient4 and 360 degrees of the limbus in Patient 6, resulting in fibrovascularingrowth around the injured limbal area and recurrent ulcerationof the central cornea. Patients 5 and 6 had total deficiencyof limbal epithelial cells, as judged by fibrovascular ingrowthon 360 degrees of the limbus.
Table 1. Characteristics of the Patients Receiving Limbal Epithelial-Cell Transplants.
Preparation of Human Amniotic Membrane
Amniotic-membrane tissue was obtained, processed, and preservedas reported by Lee and Tseng.19 The amniotic membranes, withthe basement-membrane side up, were placed on a culture plateand incubated at 37°C in an atmosphere of 5 percent carbondioxide and 95 percent air overnight before they were used.
Limbal Biopsy and Culture of Explanted Tissue
Limbal biopsy was performed on the normal contralateral eye(Figure 1). The eyelid was sterilized with povidone–iodine,and a piece of limbal tissue 1 by 2 mm that contained epithelialcells and part of the corneal stroma was separated from thelimbal margin and excised from the superficial corneal stromaby lamellar keratectomy. The tissue was placed in a 35-mm dishcontaining 1.5 ml of supplemental hormonal epithelial mediumin 5 percent fetal-calf serum and immediately sent to the laboratoryfor culture. The medium consisted of Dulbecco's modified Eagle'smedium and Ham's F12 (in a 1:1 ratio), supplemented with 0.5percent dimethyl sulfoxide, 2 µg of mouse epidermal growthfactor per milliliter, 1 µg of bovine insulin per milliliter,and 0.1 µg of cholera toxin per milliliter.
Figure 1. Transplantation of Autologous Limbal Epithelial Cells Cultured on Amniotic Membrane.
Limbal tissue (1 by 2 mm) was removed by lamellar keratectomy from the superior limbus of the healthy contralateral eye (Panel A). The explanted tissue was placed on amniotic membrane in a 35-mm dish containing 1.5 ml of culture medium (Panel B). After two to three weeks, the epithelial cells had grown and spread to form a circular sheet of cells with a diameter of 2 to 3 cm (Panel C). The cultured limbal epithelial cells with amniotic membrane were then transplanted to the diseased eye (Panels D and E).
The limbal epithelial cells were cultured as previously described,with some modifications.20,21 The limbal epithelial-cell explantswere inoculated onto the basement-membrane side of the amnioticmembrane. The medium was changed every two days and the culturewas maintained for two to three weeks, by which time the epithelialcells had grown and spread to form a cell layer covering anarea 2 to 3 cm in diameter (Figure 1).
Histologic Findings
The limbal epithelial-cell sheets on the amniotic membrane wereexamined by both light and electron microscopy. Samples werefixed and processed with use of standard histologic procedures.For light microscopy, 4-µm sections were cut and stainedwith hematoxylin and eosin or periodic acid–Schiff reagentand Alcian blue. For electron microscopy, ultrathin sectionswere examined under a transmission electron microscope (Jeol-1200CX, Jeol, Tokyo, Japan) for the presence of characteristic structuresof the basement membrane.
Transplantation of Limbal Epithelial Cells Cultured on Amniotic Membrane
After periotomy at the limbus, the perilimbal subconjunctivalscar and inflamed tissues were removed to the bare sclera. Cornealfibrovascular tissue was removed by lamellar keratectomy ina manner similar to that described for allograft limbal transplantation.14For eyes with limbal and corneal damage but with a normal centralcornea, the limbal epithelial cells and the amniotic membranewere used as a sectorial limbal and corneal graft or a limbalequivalent, fashioned according to the size of the recipienteye, and transplanted to the corresponding recipient limbalarea (from 90 to 360 degrees) (Figure 2A). For eyes with damageto the entire limbal and corneal surface, the limbal epithelialcells and the amniotic membrane were transplanted as a sheetto cover the damaged area (Figure 2B).
For eyes with limbal and corneal damage but with a normal central cornea (Panel A), the cultured limbal epithelial cells, together with the amniotic-membrane substrate, were transplanted to the corneal surface as a sectorial graft (white lines) or a limbal equivalent (red and white lines), fashioned according to the size of the diseased area. For eyes with damage to the entire limbal and corneal surface (Panel B), the limbal epithelial cells with the amniotic membrane were transplanted as a sheet to cover the damaged area.
In all patients, the entire defect was covered with a sheetof cultured limbal epithelial cells together with the amnioticmembrane substrate, with the epithelial side up. The graft wasthen secured to the corneal side by interrupted 10-0 nylon suturesand to the surrounding conjunctival edge by interrupted 8-0Vicryl sutures. During the procedure, the cultured limbal epitheliumwas protected by sodium hyaluronate (Healon, Pharmacia &Upjohn, Uppsala, Sweden). After surgery, the eye was coveredovernight with a pressure patch. A therapeutic contact lenswas placed on the eye for one week, beginning on the day aftersurgery. A 1 percent solution of prednisolone acetate was appliedtopically four times a day for the first week and twice a dayfor the next two weeks, followed by 0.1 percent fluorometholonetwice a day for two to three months, depending on the severityof inflammation and conjunctival congestion around the surgicalfield.
Results
In two to three weeks, the limbal epithelial cells grew to forma sheet 2 to 3 cm in diameter on the amniotic membrane (Figure 3A).In flat-mount preparations, the epithelial-cell layer didnot stain with periodic acid–Schiff reagent or Alcianblue, and the bare amniotic membrane was stained purple by bothreagents (Figure 3B). On histologic examination, the epithelialsheet was seen to be composed of four to five cell layers atthe margin (Figure 3C) and one to four cell layers in the areabetween the margin and the original explanted tissue. Ultrastructuralexamination revealed the presence of a basement-membrane structure,ranging from rudimentary to developed, at the junction of thebasal cells and the amniotic membrane (Figure 3D).
Figure 3. Morphologic Features of Limbal Epithelial Cells Cultured on Amniotic Membrane.
Explanted limbal tissue was placed on amniotic membrane (encircled by the white line in Panel A) in a 35-mm dish containing 1.5 ml of culture medium. A flat-mount preparation of the epithelial-cell sheet (Panel B) showed that the cell layer did not stain with periodic acid–Schiff reagent and Alcian blue, and that the bare amniotic membrane stained purple. Histologic examination showed that the epithelial sheet was composed of four to five layers of cells at the margin (Panel C). The scale bar represents 200 µm. An electron micrograph shows the presence of a rudimentary-to-developed basement membrane with focal condensation of electron-dense ground substance at the junction between the basal cells and the amniotic membrane (Panel D, arrows). The scale bar represents 200 nm.
Clinical Results
After a mean follow-up period of 15±2 months, visionhad improved in five of the six eyes that received transplants(83 percent). The improvement was by more than 10 lines in oneeye, 2 to 3 lines in two eyes, and 1 line in two eyes accordingto the Snellen visual-acuity scale. In the one eye that didnot improve after receiving a transplant, the visual acuitywas maintained at 20/20 (Table 1). Complete reepithelializationoccurred within 2 to 4 days (mean, 3±1) in all eyes thatunderwent surgery. Inflammation was reduced and vascularizationregressed in the reconstructed corneal surfaces within one totwo weeks. By one month after operation, corneal clarity wasimproved and the surface was smooth and wettable. There hasbeen no recurrent neovascularization or inflammation in thetransplanted areas during the follow-up period. A minimal scarformed at the donor site, but there was no neovascular growthinto the cornea. Depending on the area of limbal and cornealdamage, cultured limbal epithelial cells with an amniotic-membranesubstrate can be used as a limbal equivalent or as a sheet coveringthe entire limbus and cornea.
Transplantation of a Limbal Equivalent
Cultured limbal epithelial cells with an amniotic-membrane substratewere transplanted to cover about 180 degrees of the limbus inPatients 1 and 3 and about 90, 270, and 360 degrees of the limbusin Patients 2, 4, and 5, respectively (Figure 4).
Figure 4. The Eyes before and after They Received Transplants.
The lower photographs were taken after a mean postoperative follow-up of 15±2 months. Patients 1, 2, 3, 4, and 5 received sectorial limbal and corneal grafts. In Patients 1 and 3, the area of transplantation encompassed about 180 degrees of the limbus, and in Patients 2, 4, and 5, it encompassed about 90, 270, and 360 degrees of the limbus, respectively. Patient 6 received a transplant over the whole corneal surface.
Patient 5, a 23-year-old woman, had had recurrent inflammationin both eyes, due to phlyctenular disease, since the age of10 years. Slit-lamp examination showed hyaline degeneration,and there was fibrovascular growth into the limbal area, whichwas worse in the right eye than in the left eye. She had undergoneseveral operations in the right eye, including superficial keratectomyand amniotic-membrane transplantation. Before this study, hervisual acuity was 20/200 in the right eye and 20/20 in the lefteye. At surgery, the fibrovascular membrane over the entireperipheral cornea of the right eye (about 2 mm from the limbus)and over the limbus was removed by superficial keratectomy,leaving the central cornea untouched. The cultured limbal epitheliumwith amniotic-membrane substrate was transplanted onto the limbaland peripheral corneal area. Three days after transplantation,the limbal area was covered with transparent, normal-lookingepithelium, which did not stain with fluorescein. During theensuing 12 months, the patient's best corrected visual acuityimproved to 20/50, the corneal and limbal epithelium remainedstable, and there was no inflammation or neovascularization(Figure 4).
Transplantation of a Sheet Covering the Whole Limbus and Cornea
Patient 6, a 29-year-old woman, had had a chemical burn on herleft eye 18 months previously. After the injury, she receivedemergency treatment with normal saline irrigation and was referredto us one month later. Slit-lamp examination of her left eyerevealed a corneal opacity with a central epithelial defectand neovascular growth extending 4 to 6 mm into the entire cornea(Figure 5A). Her best corrected visual acuity was counting fingersat 40 cm with the left eye and 20/20 with the right eye. Biopsyof her right upper limbal area (1 by 2 mm) and culture of explantedlimbal cells were performed as described above. Three weekslater, the epithelial-cell sheet was about 2.5 by 3 cm in size.Lamellar keratectomy was performed to remove the entire opacifiedlimbal and corneal area to a thickness of about one third ofthe corneal layer, and a sheet of limbal epithelial cells withamniotic membrane was transplanted onto the denuded area. Thegraft was extended to the conjunctival area about 3 mm fromthe limbus and sutured with 8-0 Vicryl. The corneal portionwas secured with interrupted 10-0 nylon mattress sutures ontothe peripheral cornea. Total reepithelialization was noted 4days after surgery, and the cornea started to become clear about30 days later (Figure 5D). During a 15-month follow-up period,the patient's best corrected visual acuity improved to 20/200,and the cornea and limbus were covered by a stable epitheliumwith no signs of inflammation or neovascularization (Figure 4and Figure 5).
Figure 5. Serial Photographs of the Eye of Patient 6 before and after Transplantation.
Initial examination (Panel A) revealed corneal opacity with central corneal erosion and neovascular growth extending into the entire cornea for 4 to 6 mm. Lamellar keratectomy was performed to remove the entire opacified limbal and corneal area to a thickness of about one third of the corneal layer (Panel B). Limbal epithelial cells with the amniotic-membrane substrate were transplanted onto the denuded limbal and corneal area. Photographs were taken 1 day (Panel B), 7 days (Panel C), 30 days (Panel D), and 450 days (Panel E) after the operation.
Discussion
In the past 10 years, therapeutic techniques for reconstructionof the ocular surface have been greatly advanced by the introductionof limbal epithelial-cell transplantation and amniotic-membranetransplantation. The therapeutic effectiveness of these twoprocedures lies in their ability, respectively, to replenishthe limbal epithelial-cell population and to restore the limbalstroma that supports the epithelial cells.1,4,15,22 In thisstudy, we have demonstrated that the combination of both methods— that is, transplantation of limbal epithelial cellscultured on amniotic membrane — offers additional advantages.
In the four patients who had partial limbal deficiency (Patients1, 2, 3, and 4), this new technique successfully restored thedamaged limbus and the adjacent peripheral cornea, resultingin a noninflamed limbus with a stable, nonvascular cornea. Ourresults suggest that limbal epithelial cells, together withan amniotic-membrane substrate, are sufficient to restore acorneal surface in which previous amniotic-membrane transplantationhas failed. Partial deficiency of limbal epithelial cells hasbeen successfully treated by transplanting amniotic membrane,which promotes the growth of residual limbal stem cells.15 Inour study, Patient 1 (with a chemical burn) and Patient 3 (withcongenital pterygium) had partial limbal deficiency and hadpreviously undergone amniotic-membrane transplantation, butboth patients had persistent inflammation at the margins ofthe prior surgical field. The inflammation could have been dueto incomplete removal of subconjunctival fibrovascular tissue(in Patient 3) or persistent stromal inflammation (in Patient1). Stromal inflammation has been shown to impede the successof limbal grafting.4
In two patients with total limbal deficiency, transplantationof either a 360-degree limbal equivalent (in Patient 5) or theentire limbal and corneal sheet (in Patient 6) successfullyrestored the damaged corneal and limbal surfaces. In both patients,the reconstructed cornea started to become clear about 30 daysafter surgery. Monthly follow-up examinations for more than12 months in both patients revealed no neovascularization orconjunctivalization, indicating that the grafted limbal epithelialcells and their amniotic-membrane substrates were functioningwell.
Limbal deficiency of the donor eye due to removal of a relativelylarge piece of limbus for transplantation has been reportedin rabbits.10,11 Our method substantially reduces the likelihoodof these complications, because only a small piece of limbusis removed. Moreover, this method can also be used when botheyes have limbal deficiency but the location of the deficiencydiffers in the two eyes. Ex vivo expansion of autologous limbalepithelial cells on amniotic membrane provides sufficient limbalepithelial cells for transplantation in two to three weeks.For patients with total bilateral limbal deficiency, use ofheterologous limbal grafts from cadavers14,18,23,24,25 or livingdonors23,26,27,28 has been reported. Our approach is still advantageouswhen living donors are selected, because the surgical techniqueis easier and less tissue is removed from the donor.
The use of autologous limbal epithelial cells grown on amnioticmembrane for transplantation also has all the benefits of amniotic-membranetransplantation, including the facilitation of epithelialization,19,20reduction of inflammation and scarring,29,30 and replacementof substrate when the underlying stromal tissue is destroyed.This approach differs from that reported by Pellegrini et al.,12in that the presence of amniotic membrane simplifies handlingand suturing and eliminates the risk of infection associatedwith the use of mouse cells. Most important, amniotic membraneprovides a natural substrate on which limbal epithelial stemcells can survive and proliferate,31 forming an autologous cellmass sufficient for corneal reconstruction. Moreover, becauseonly autologous cells are transplanted, immunosuppression isnot required.
Supported by grants from the National Health Research Institute(DOH 83-HR-303) and the National Scientific Council, Taiwan(NSC 88-2314-B182-006).
We are indebted to Dr. Scheffer Tseng of the University of Miamifor advice.
Source Information
From the Department of Ophthalmology, Chang Gung Memorial Hospital (R.J.-F.T., L.-M.L.), and the Department of Physiology, Chang Gung University (J.-K.C.) — both in Taoyuan, Taiwan. Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Fla., May 10–15, 1998.
Address reprint requests to Dr. Tsai at 2F 350 Section 4 Cheng Kung Rd., Taipei, Taiwan, or at raytsai{at}ms4.hinet.net.
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