Advances in corneal stem-cell transplantationin rabbits with severe ocular alkali burns

Federico Luengo Gimeno, MD, Victoria Lavigne, Silvia Gatto,J. Oscar Croxatto, MD, Laura Correa, Juan E. Gallo, MD, PhD

PURPOSE: To evaluate the efficacy of autologous corneal epithelial sheet implantation in restoringtransparency of rabbit corneas severely injured by alkaline and the effect of photocoagulation inarresting corneal neovessel ingrowth.

SETTING: Ophthalmology Department, School of Biomedical Sciences, Universidad Austral, BuenosAires, Argentina.

METHODS: Limbal stem-cell deficiency (LSCD) was induced in 14 rabbits by alkali burns. A limbalcell biopsy was done in the contralateral eye, and the cells were cultured on a fibroblast feeder layergrown on autologous clotted platelet-poor plasma or commercial fibrin for 21 days. Anterior kera-tectomy was followed by suturing corneal cell sheets over the stroma. If regrowth of vesselsoccurred, argon laser photocoagulation was applied to them. Rabbits were killed at 30, 60, 90,180, and 360 days and the corneas processed for histopathology and inmunohistochemistry.

RESULTS: A small (2.5 mm2) limbal biopsy achieved stem-cell replication in vitro. Corneal clarityand epithelial defects evolved with a trend toward improvement. There was a significant reductionin corneal neovascularization. Histology showed a multilayered stratified epithelium including sev-eral epithelial-like cells with clear cytoplasm in the deepest part. There were no signs of intraepithe-lial mucin cells on the implanted corneas. Immunohistochemical results showed expression ofcytokeratins 3 and 12 in the central corneal epithelium and an absence of cytokeratin 19.

CONCLUSIONS: Autologous limbal epithelial cell transplantation improved the corneal surface ineyes with LSCD. Photocoagulation of neovessel ingrowth was effective over the 1-year follow-up. Results may facilitate the application of this technique in patients.

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LABORATORY SCIENCE

Corneal stem cell diseases are a major problem in oph-thalmology, and many scientists have been trying todevelop an ideal treatment for this condition.1–8 Cor-neal epithelium is derived from limbal epithelialstem cells; these slow-cycling cells are believed to belocated in the basal epithelial layer of the palisadesof Vogt, at the limbus.9 They are thought to maintaintheir population through a combination of inherentcellular characteristics and the presence of a propitiousmicroenvironment at the limbus.10,11 In the simplestdefinition, stem cells are capable of ‘‘unlimited’’ self-renewal and upon division, give rise to transientamplifying cells (TACs) that have limited renewalcapability.12 Although stem cells divide relatively in-frequently in mature nontraumatic tissues, they havea high proliferative potential13 and can be induced todivide more frequently after wounding or under in vi-tro culture conditions.14 Conversely, TACs dividemore frequently than stem cells and have limited

Q 2007 ASCRS and ESCRS

Published by Elsevier Inc.

1958

growing potential; upon depletion of their prolifera-tive capacity, they undergo terminal differentiationto constitute the superficial cells of the corneal epithe-lium.15 Disease or destruction of the limbus is associ-ated with events that eventually lead to visualimpairment or blindness. Conjunctivalization and vas-cularization of the corneal surface and persistent or re-curring epithelial defects are hallmarks of limbaldeficiency.16

The number of people with corneal blindness is notas small as one may think.17–19 It is estimated that 1.5million people develop corneal neovascularization inthe United States annually, leading to corneal opacifi-cation.20 Among the most frequent causes of total lim-bal stem-cell deficiency (LSCD) are ocular alkali burns,Steven-Johnson syndrome, and ocular pemphigoid.Management of these disorders is complicated.Although many efforts have been made to improvethe prognosis of LSCD, the final outcome is often

0886-3350/07/$dsee front matter

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1959LABORATORY SCIENCE: CORNEAL STEM-CELL TRANSPLANTATION IN ALKALI BURNS

unsuccessful. Damage to the cornea from burns canrange from mild to severe, requiring clinicians to rap-idly assess the level of injury and take appropriatesteps to minimize loss of visual function. Ocular alkaliburns are considered one of the most serious work-related accidents because the alkaline substances canrapidly penetrate cell membranes.21,22 Such burnshave a significant socioeconomic impact because ofthe high cost of treatment and the severe visualimpairment, which often ends in disability.

As a result of advances in tissue engineering and celltherapy, there is new hope for patients with alkaliburns.6 The success of corneal stem-cell transplantationrelies on the achievement of a clear cornea or a betterocular surface that allows a later penetrating cornealgraft (2-stepprocedure).23 Transplantationof epithelialstem cells has been used in animals.24,25 In most cases,cell sheets were grown on amniotic membranes orcommercial fibrin gels. Clinical reports included fewerpatients and differed in results.3,23,26–29 The main com-plication in these reports is ingrowth of bloodvessels inthe cornea, which causes a reduction in transparencythat is associated with decreased and even loss of vi-sion; this in turn leads to graft failure.20,30

Platelet-poor plasma (PPP) is an autologous sub-stance able to hold tissues or materials in a requiredconfiguration. Its biocompatible and biodegradableproperties prevent the PPP from inducing foreign-body reactions, tissue necrosis, and extensive fibro-sis.31 Its use in cell cultivation varies.32

The use of argon laser photocoagulation for cornealneovascularization has been described.33,34 The re-gression of neovessels has been achieved in some

Accepted for publication July 9, 2007.

From the Ophthalmology Department (Luengo Gimeno, Gallo),School of Biomedical Sciences, and Transfusional Service Depart-ment (Gatto), Hospital Universitario Austral, Universidad Austral,Pilar, and the Tissue Engineering Department (Lavigne, Correa),Laboratorios Craveri, and the Ocular Pathology Department(Croxatto), Fundacion Oftalmologica Argentina Jorge Malbran,Buenos Aires, Argentina.

No author has a financial or proprietary interest in any material ormethod mentioned.

Presented at the XXVII International Congress of Eye Research,Buenos Aires, Argentina, November 2006.

Supported by Universidad Austral grant 05v001, Buenos Aires,Argentina.

Corresponding author: Juan E. Gallo, MD, PhD, Decanato, Facultadde Ciencias Biomedicas, Avenida Peron 1500, Pilar (B1629AHJ),Buenos Aires, Argentina. E-mail: jgallo@cas.austral.edu.ar,jgallo06@gmail.com.

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cases30; however, this therapy has not been used forcorneal neovascularization after limbal epithelial celltransplantation. It is not known whether it can havea harmful effect on the grafted stem cells.

We evaluated the efficacy of autologous corneal ep-ithelial sheet implantation in restoring the transpar-ency of rabbit corneas severely injured by alkalineand the potential benefit of photocoagulation in arrest-ing corneal neovessel ingrowth.

MATERIALS AND METHODS

Fourteen New Zealand White rabbits weighing approxi-mately 3000 g eachwere used. The rabbits were treated in ac-cordance with the guidelines of the ARVO Statement for theUse of Animals in Ophthalmic and Vision Research.35 Allsurgical procedures were performed using general anesthe-sia comprising a combination of intramuscular 1 mg/kgmidazolam and 70 mg/kg ketamine and topical anesthesiacomprising sub-Tenon’s capsule injection of 1 mL of lido-caine 4% and proparacaine drops.

Animal Model of Total Limbal Stem-Cell Deficiency

A solution of 1 M sodium hydroxide was used on the cor-neal surface of the left eye under general and topical anesthe-sia. After 1 minute, the eye was gently washed with saline.Clinical signs of LSCD were evaluated during the following2 months and immediately before transplantation. Alkaliinjuries were recorded according to the International LifeSciences Institute classification described by Bagley et al.,36

which was slightly modified (Tables 1A, 1B, and 1C).

Biopsy, Culture, and Expansion of Corneal LimbalEpithelial Cells

A small biopsy of corneal limbal epithelial cells was ob-tained from the superior temporal limbus of the contralateralhealthy eye using a 1.8 mm diameter trephine and a delicateforceps. This was preceded by gentle debridement of theconjunctiva and rinsing of the zone with saline to diminishcell contamination in the culture. The sample was immedi-ately placed in a tube with Ham’s F-12 containing 100 U/mLpenicillin, 100 mg/mL streptomycin, 2.5 mg/mL amphoteri-cin B, and 20 mg/mL gentamicin and then transported tothe laboratory.

The sample was washed 3 times in antibiotic–antimycoticsolutions. A fourth wash removed undesirable antimicro-bials. The sample was enzymatically processed with trypsinto obtain isolated cells. The rabbit limbal epithelial cells wereseeded on an irradiated murine 3T3 fibroblast feeder layerand cultured in an epithelial medium composed of a 3:1 mix-ture of Dulbecco’s modified Eagle medium and Ham’s F-12supplemented with 10% fetal bovine serum, 0.4 mg/mL hy-drocortisone, 5 mg/mL transferrin, 5 mg/mL insulin, 10�10 Mcholera toxin A, 1.8 � 10�4 M adenine, 2 � 10�11 M triio-dothyronine, 100 U/mL penicillin, and 100 mg/mL strep-tomycin and then placed in a 37�C 5% carbon dioxideincubator.

Autologous PPP or commercial fibrin glue (Tissucol) wasclotted in circular culture chambers. Approximately 4 � 104

rabbit limbal epithelial cells/cm2were seeded on a fibroblastfeeder layer grown in those chambers, and cells formeda monolayer after a few days. To achieve cell stratification,

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Table 1A. Classification of preoperative limbal blood vessel ischemia and evolution after limbal epithelial cell transplantation basedon modification of Bagley et al.36 system.

Animals Affected/Total Number of Animals (%)

PeriodClass 0 Z No

IschemiaClass I Z !25%

Circumference AffectedClass II Z 25% to 50%Circumference Affected

Class III Z 50% to 75%Circumference Affected

Class IV Z 75% to 100%Circumference Affected

Preoperative d d 1/14 (7.14) 2/14 (14.28) 11/14 (78.57)Postoperative (d) d d d d d

30 d d 6/14 (42.85) 6/14 (42.85) 2/14 (14.28)60 d 2/8 (25.00) 1/8 (12.50) 3/8 (37.50) 2/8 (25.00)90 d 2/5 (40.00) 2/5 (40.00) 1/5 (20.00) d

180 1/4 (25.00) 1/4 (25.00) 2/4 (50.00) d d

360 1/2 (50.00) 1/2 (50.00) d d d

Table 1B. Classification of preoperative corneal stroma and evolution after limbal epithelial cell transplantation based on modificationof Bagley et al.36 system.

Animals Affected/Total Number of Animals (%)

PeriodClass 0 Z Iris

Details Clearly VisibleClass II Z Opacity

Causes Blurriness of Iris DetailsClass III ZPupil Discernible

Despite Opacity

Class IV Z Unableto Discern Pupil;Complete Opacity

Preoperative d 1/14 (7.14) 1/14 (7.14) 12/14 (85.71)Postoperative (d) d d d d

30 d 2/14 (14.28) 5/14 (35.71) 7/14 (50.00)60 d 2/8 (25.00) 1/8 (12.50) 5/8 (62.50)90 d 2/5 (40.00) 1/5 (20.00) 2/5 (40.00)180 3/4 (75) d 1/4 (25.00) d

360 1/2 (50) d d d

Table 1C. Classification of depth of corneal injury based on modification of Bagley et al.36 system.

Animals Affected/Total Number of Animals (%)

Period Class 0 Z Corneal EpitheliumClass II Z !1/3 IncludingEpithelium and Stroma

Class III Z O1/3 IncludingEpithelium and Stroma Class IV Z Entire Cornea

Preoperative d d d 14/14 (100)

epithelial growth factor was added to the culture medium toa final concentration of 10 ng/mL 4 days before surgery. Inall cases, culture media were supplemented with 50000 kal-licrein inhibitory units aprotinin/mL to prevent fibrinolysis.At the time of surgery, all sheets were easily detached fromthe chambers with a forceps.

Transplantation and Postoperative Care

Topical iodine–povidone 5% was instilled in the left eyepreoperatively after the anesthesia procedure explainedabove. A 360-degree peritomy was made. A complete ante-rior keratectomywas done under an S5-Pro operatingmicro-scope (Zeiss), and the corneal fibrovascular tissue wasremoved. Delicate blades, a forceps, and Vannas and

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Westcott scissors were used to perform a one-third cornealthickness keratectomy including the limbus using the Mal-bran peeling-off technique.37 The corneal sheet containinglimbal stem cells was then placed over the stromal surface.The sheet was sutured using 8 interrupted 10-0 nyloncorneoscleral sutures. Suture knots were rotated and buriedin the recipient stroma. Finally, a two-third partial tarsorrha-phywas done using 5-0 silk sutures. After awakening, all an-imals received 21% oxygen for 1 hour.

The tarsorrhaphy sutures were removed on day 3. Postop-erative topical therapy included ciprofloxacin 0.3% 5 timesa day for 2 weeks, dexamethasone 0.1% 4 times a day,and cyclosporine 1% once a day for 1 month. Clinical exami-nation was periodically performed (3, 7, 15, 30, 60, and90 days, 6 months, and 1 year) under an operating

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microscope and photographically documented with a Cool-pix 5700 digital camera (Nikon).

Argon laser photocoagulation connected to an indirectbinocular ophthalmoscope (Ophthalas Eye Lite, Alcon Labo-ratories) and a 20.0 diopter lens were used to occlude theneovessels invading the cornea postoperatively. Laser shotsof 550 mV and 0.3-second exposure were applied on thescleral part of the blood vessels near the limbus, with noshots on the cornea. The photocoagulation session was re-peated if regrowth of vessels occurred. No more than 150shots were used per session.

Histological Examination

Animals were killed for histological and immunohisto-chemical examination at 30, 60, 90, 180, and 360 days. Theglobe was enucleated and placed in 10% paraformaldehydefor 24 hours. The cornea was extracted from the globe usinga delicate forceps and scissors. Histological specimens werestained with hematoxylin–eosin (H&E) and examined underan Eclipse E800 microscope (Nikon).

Immunohistochemical Analysis

Primary monoclonal antibodies against cytokeratins K3and K12 (K3/12) (Mab AE5, Chemicon) and cytokeratin

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K19 (Mab 1675,Chemicon)wereused to identify the presenceof different cytokeratins in the corneal epithelium. The sameimmunohistochemical analysiswas performed in the rabbit’scontralateral eye,which had not had surgery (control group).

Corneal samples were cut at 10 mm thickness in a cryostatand processed for immunohistochemistry technique. Briefly,sections were incubated with the first monoclonal antibody(to K3/12 or to K19) and then with biotinylated goat anti-mouse immunoglobulin (Vector Laboratories) followed byavidin–biotin–peroxidase (Vectastain, Vector Laboratories).Finally, they were revealed with diaminobenzidine tetrahy-drochloride enhanced by ammonium nickel sulfate.

RESULTS

A limbal biopsy of 1.8 mm diameter was sufficient toachieve in vitro stem-cell replication. Overall, it took3 weeks to develop a corneal sheet ready to be grafted.Matrices of PPP showed very good elasticity and werehighly transparent. In contrast, commercial gels wereopaque and considerably stiff (Figures 1 and 2). Bythe first week, there was total reepithelialization ofthe small ulcer produced by the biopsy and no side ef-fects were seen on the biopsied eye.

Figure 1. Macroscopic features ofcultured corneal sheets. A and C:Corneal sheet using Tissucol. Band D: Corneal sheet using autolo-gous PPP. The Tissucol sheets areopaque, while the PPP sheets aretransparent.

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Figure 2. Histological characteris-tics of corneal sheets. A: Cornealsheet using autologous PPP. B: Cor-neal sheet using Tissucol. Both im-ages show a stratified epitheliumconsisting of 4 layers. Inferior cellsare polygonal and have a prominentnucleolus. The middle layers showcells with irregular morphology,and the upper layer is formed byflattened cells with compact ovoidnucleus ofhomogeneous chromatin.

All alkali injuries resulted in preoperative clinicalsigns of total LSCD (corneal neovascularization, sur-face irregularities, and opacity). Figure 3 shows theclinical progress of the rabbits that had been followedfor 1 year. Corneal opacities evolved differently be-tween rabbits; however, there was a general tendencytoward improvement over time (Table 1B). A clear re-duction in epithelial defects was seen in most cases,even in those with neovessel ingrowth. The cornealgrafts did not develop neovascularization during thefirst postoperative month. From the second month on-ward, argon laser photocoagulation was performed in9 animals according to the onset of neovessels.

Histological corneal sections showed a multilayerstratified epithelium at all times. Epithelial-like cellswith clear cytoplasm were found in the deepest partof the epithelium at day 30 and remained visible 1year postoperatively (Figure 4). There were no signsof vascular pannus or avascular intraepithelial mucincells on the treated corneas (Figure 4). Several eyeshad posterior corneal surface damagewith Descemet’smembrane replication due to the alkaline burn (Fig-ure 4). Corneal damage caused by laser photocoagula-tion was not seen.

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Immunohistochemical results showed expression ofK3/12 in the central part of the corneal epithelium. Ex-pression of K19 was not found. The results are similarto those seen on normal corneas (Figure 5).

Five animals developed corneal ectasia and/or en-dophthalmitis 1 month after the alkali burn procedureand did not have limbal stem-cell transplantation.They were excluded from the study and replaced byother animals.

DISCUSSION

In our study, autologous limbal epithelial cell trans-plantation improved the corneal surface of most eyeswith total LSCD, as documented by the absence of orreduction in recurrent epithelial defects, symblephar-on, and total corneal neovascularization. Cases that de-veloped partial neovessel ingrowth after somemonthsof transplantationwere treatedwith argon laser photo-coagulation, resulting in different degrees of regres-sion. Photocoagulation was well tolerated by graftedstem cells. The phenotype of the corneal epithelium us-ing antibodies against cytokeratins was in accordancewith previous studies.23,38–41 Transplantation of

Figure 3. Clinical evolution during1-year follow-up.A: Presence of cor-neal opacification and vasculariza-tion before surgery. B: Applicationof autologous corneal sheet.C: Clin-ical appearance 30 days after trans-plantation. D: Presence of cornealneovascularization 90 days aftergraft, later treated with argon laserphotocoagulation. E: Corneal clar-ity and absence of signs of LSCD12 months after transplantation.

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Figure 4. Histopathological changes at the 1-year follow-up (H&E stain). A: Rabbit cornea with alkali burn and transplantation of autologouscorneal epithelial cells expanded ex vivo 30 days after surgery shows postinflammatory evidence of cicatricial ectasia with regenerated endo-thelium. The corneal surface has a non-keratinizated–stratified epithelium with normal morphology and configuration. Epithelial-like cellswith clear cytoplasm are seen in the deepest part of the epithelium. B: Rabbit cornea with alkali burn and transplantation of autologous cornealepithelial cells expanded ex vivo 1 year after surgery. The cornea had normal thickness and a loss of keratocytes. The original Descemet’s mem-brane is covered with fibrocollagenous tissue proliferation that shows endothelial cells and a new Descemet’s membrane toward the anteriorchamber. The 5-layer stratified epithelium has normal morphology.

autologous limbal corneal epithelial cells expanded exvivo was an effective procedure for treating LSCD.Most eyes had no vascularization in at least 6 clockhours of the cornea. Corneal tissue opacification wasassociated with stromal fibrosis or Descemet’s replica-tion. Nevertheless, all cases presented a normal cor-neal surface. The preoperative eye situation could becompared to legal blindness in humans.36 Undoubt-edly, in the clinical situation, the lesions may involvenot only the globe but also the conjunctiva and eye-lids; however, the prognosis depends on the initial le-sions, which can be variable. In our model, there was

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total preoperative corneal opacity, chronic conjuncti-val inflammation, and symblepharon, which may sug-gest abnormal tear-film integrity and resistance toinfection. These situations were not seen aftertreatment.

After grafting, the eyes made good progress and thedegree of injury diminished. It seems reasonable tothink these cases would benefit from a full-thicknesscorneal transplantation later.

The limbal biopsy is a safe procedure without com-plications.23,25,26 The stem cell–containing limbal epi-thelium is in proximity with conjunctiva, a highly

Figure 5. Immunohistochemicalanalysis of cytokeratins at 12months. A and C: Immunoreactiv-ity of K19 (A) and K3/12 (C) ina normal rabbit cornea. B and D:Immunoreactivity of K19 (B) andK3/12 (D) in a rabbit cornea withalkali burn and subsequent trans-plantation of autologous limbal ep-ithelial cells expanded ex vivo. Inboth corneas, there is negative im-munoreactivity to K19 and positiveimmunoreactivity to K3/12. Theexpression of the K3 and K12 pairin the implanted cornea shows dif-ferent degrees of intensity, whereasthe normal cornea shows homogen-eous staining.

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vascularized tissue, so preventive measures aimed atreducing contamination of stem-cell cultures fromerythrocytes or conjunctival cells are of paramount im-portance.42 We were able to perform this proceduresuccessfully in our study. We believe that the use ofPPP, an autologous substance, is a good alternativefor cell support. In this way, the corneal sheet can beeasily handled during in vitro manipulation or sur-gery, facilitating the formation of noncontinuous su-tures. Also, the transparency of the PPP allowstechnicians to follow the process of epithelial strat-ification. In addition, immunological reactions are un-common using autologous substances. This is anadvantage over the commercially available fibrin orthe human amniotic membrane.

Argon laser therapy emerged as a promising treat-ment for corneal neovascularization after corneal lim-bal cell transplantation, reducing the risk for graftfailure. There was a marked tendency toward neoves-sel reduction over time. Rabbits that had several ses-sions of photocoagulation at 1 year of follow-up stillachieved a substantially clear cornea, which suggeststhat the argon laser applied on vessels over the scleracaused no side effects to the corneas.

Results in this experimental studymay facilitate thede-velopment and application of the technique in patients.

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First author:Federico Luengo Gimeno, MD

Ophthalmology Department, School ofBiomedical Sciences, Universidad Austral,Pilar, Argentina

- VOL 33, NOVEMBER 2007