`for Persistent Epithelial Defects
`With Ulceration
`
`SHWU-HUEY LEE, MD, AND SCHEFFER C. G. TSENG, MD, PHD
`
`• PURPOSE: To determine whether preserved hu
`man amniotic membrane can be used as an alterna
`tive substrate for treating persistent corneal epi
`thelial defects with sterile ulceration.
`• METHODS: Amniotic membrane transplantation
`was performed in 11 eyes of 11 consecutive
`patients with corneal ulcers of different causes
`that had persisted for a mean ± SD of 17.5 ± 13.9
`weeks.
`• RESULTS: Ten patients healed in 3.9 ± 2.3
`weeks (P < .01) without recurrence for 9.0 ± 5 .9
`months. One patient failed to heal because of
`preexisting corneal perforation pursuant to severe
`rheumatoid arthritis.
`• CONCLUSION: Amniotic membrane transplan
`tation may be considered an alternative method for
`treating persistent epithelial defects and sterile
`ulceration that are refractory to conventional
`treatment and before considering treatment by
`conjunctival flaps or tarsorrhaphy.
`
`T HE INITIAL EVENT LEADING TO A CORNEAL EPI-
`
`thelial defect that progresses to ulceration is
`the breakdown of the corneal epithelial sur-
`
`Accepted for publication Oct 17, 1996.
`From the Ocular Surface and Tear Center, Department of Ophthal
`mology, Bascom Palmer Eye Institute (Drs Tseng and Lee); and Depart
`ment of Cell Biology and Anatomy (Dr Tseng), University of Miami
`School of Medicine, Miami, Florida. Presented in part at the Second
`Annual Meeting of the Ocular Surface and Tear Workshop, April 1996,
`Miami, Florida. Supported in part by Public Health Service Research
`Grant EY 10900 from Department of Health and Human Services,
`National Eye Institute, National Institutes of Health, Bethesda, Maryland,
`and in part by an unrestricted grant from Research to Prevent Blindness,
`Inc, New York, New York.
`Reprint requests to Scheffer C. G. Tseng, MD, PhD, Bascom Palmer
`Eye Institute, William L. McKnight Vision Research Center, 1638 NW
`10th Ave, Miami, FL 33136; fax: (305) 326-6306; e-mail: stseng®
`mednet.med.miami.edu
`
`face. Surface epithelial breakdown can be caused by
`such exogenous insults as infection, xerosis, trauma,
`or chemicals and can be aggravated by such endoge
`nous conditions as paralytic or neurotrophic exposure,
`keratomalacia, or recurrent corneal erosions. When
`associated with excessive inflammation or failure of
`normal healing responses, such epithelial breakdown
`may eventually progress to persistent sterile corneal
`ulcers.1'3 Therefore, early diagnosis and institution of
`appropriate medical therapies to correct the underly
`ing cause, to suppress the untoward inflammation,
`or to augment tissue healing are often curative in
`the management of corneal epithelial defects and
`ulcers.1,2
`However, for reasons that remain unclear, the
`established ulceration can persist and predispose to
`imminent perforation even after the application of
`these therapies. When persistent epithelial defect
`with ulceration develops, we often resort to such
`surgical therapies as tissue adhesive,4,5 conjunctival
`resection,6 lamellar keratoplasty,7 and penetrating
`keratoplasty.2,3,8 However, when there is poor visual
`potential, treatment with conjunctival flaps9,10 or
`tarsorrhaphy11 is contemplated.
`Recently, Kim and Tseng12 reported that amniotic
`membrane transplantation offers a 40% chance of
`restoring corneal clarity and the epithelial phenotype
`in rabbits (n = 13) in which the entire corneal and
`limbal surface had been experimentally destroyed,
`leading to a condition termed limbal stem cell defi
`ciency. Based on this encouraging finding, we exam
`ined the question whether the preserved human
`amniotic membrane can also be used as an alternative
`substrate to treat persistent epithelial defects with
`ulceration.
`
`VOL.123, No. 3
`
`© AMERICAN JOURNAL OF OPHTHALMOLOGY 1997;123:303-312
`
`303
`
`MTF Ex. 1040, pg. 1
`
`
`
`Table. Demographic and Clinical Data
`
`Patient No.,
`Age (yrs),
`Sex
`
`Eye
`
`Primary
`Diagnosis
`
`Secondary
`Diagnosis
`
`Previous
`Pertinent
`Treatments
`
`PED
`(wks)
`
`Healed
`(wks)
`
`Treatments
`With
`AMT
`
`Follow-up
`(mos)
`
`Status of
`Amniotic
`Membrane
`
`1, 69, M LE Postinfection
`2, 76, F RE Postinfection
`
`HSK, glaucoma
`Glaucoma
`
`3, 79, F RE Postinfection
`4, 81, M LE Neurotrophic
`
`Aplastic anemia
`HZO, PKP
`
`5, 30, M RE Neurotrophic
`6, 86, F RE Neurotrophic
`7, 73, M RE Neurotrophic,
`exposure,
`exophthalmos
`
`PKP
`Glaucoma
`Graves thyroid
`ophthalmopathy
`
`8, 75, M LE Recurrent
`corneal
`erosion
`9, 79, F RE Cicatricial
`
`PBK, glaucoma
`
`Entropion
`
`10, 72, M LE Cicatricial
`
`Glaucoma
`
`11,49, F RE Corneal
`perforation
`
`RA, SS
`
`Mean 69.9 ±
`± SD 16.3
`
`SCL, patching
`
`Pressure patch, SCL,
`tarsorrhaphy, orbital
`decompression,
`hyperbaric oxygen,
`EGF
`SCL, scraping,
`puncture
`
`PO, eyelid eversion,
`tarsorrhaphy
`
`6
`13
`
`3
`13
`
`32
`12
`52
`
`10
`
`24
`
`12
`
`16
`
`SCL
`SCL
`
`SCL
`
`SCL
`
`SCL, patching
`
`19
`6
`
`13
`3.4
`
`14
`3.7
`2.1
`
`Intact
`Dissolved in
`2 mos
`Intact
`Three layers,
`intact
`Dissolved
`Dissolved
`Two layers,
`intact
`
`SCL
`
`14
`
`Intact
`
`3
`
`8
`
`No
`
`SCL
`
`SCL
`
`—
`
`12.5
`
`Dissolved
`
`2
`
`9*
`
`Two layers
`intact
`Dissolved
`
`17.5 ±
`13.9
`
`3.9 ±
`2.3
`
`9.0 ±
`5.9
`
`*Not counted in the mean follow-up time.
`AMT = amniotic membrane transplantation; cicatricial = cicatricial keratoconjunctivitis without specific diagnosis; EGF = epider
`mal growth factor; HSK = herpes simplex keratouveitis; HZO = herpes zoster ophthalmicus; PBK = pseudophakic bullous keratoplasty;
`PED = persistent epithelial defect; PKP = penetrating keratoplasty; PO = punctal occlusion; RA = rheumatoid arthritis; SCL = bandage
`soft contact lens; SS = Sjogren syndrome; — = no treatment.
`
`PATIENTS AND METHODS
`
`THE STUDY WAS CONDUCTED UNDER PROTOCOL 93/363,
`approved by the Medical Science Subcommittee for
`the Protection of Human Subjects in Research of the
`University of Miami School of Medicine. From 1994
`to 1996 at the Bascom Palmer Eye Institute, amniotic
`membrane transplantation was performed consecu
`tively in 11 eyes of 11 patients with corneal epithelial
`defects and ulcers that had persisted for a mean ± SD
`of 17.5 ± 13.9 weeks. These corneal problems had
`diverse causes and had developed after severe micro-
`bial infection (n = 3), neurotrophic keratopathy (n =
`4), recurrent corneal erosion caused by bullous kera
`topathy (n = 1), cicatricial keratoconjunctivitis (n =
`
`2), or severe keratoconjunctivitis sicca with corneal
`perforation (n = 1) (Table).
`Human amniotic membrane was prepared and
`preserved using our previously described method with
`minor modification.12 The human placenta was ob
`tained shortly after elective cesarean delivery when
`human immunodeficiency virus, hepatitis virus type
`B, hepatitis virus type C, and syphilis had been
`excluded by serologic tests. Under a lamellar-flow
`hood, the placenta was cleaned of blood clots with
`sterile Earle's balanced saline solution containing 50
`|JLg/ml of penicillin, 50 |xg/ml of streptomycin, 100
`(xg/ml of neomycin, and 2.5 |ig/ml of amphotericin B.
`The amnion was separated from the rest of the
`chorion by blunt dissection through the potential
`
`304
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`AMERICAN JOURNAL OF OPHTHALMOLOGY
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`
`1997
`
`MTF Ex. 1040, pg. 2
`
`
`
`spaces situated between these two tissues and flat
`tened, with the epithelium/basement membrane sur
`face up, onto nitrocellulose paper having a pore size of
`0.45 |xm. The paper, with the adherent amniotic
`membrane, was then cut into 3 cm X 4 cm disks and
`stored before transplantation at —80 C in a sterile vial
`containing Dulbecco modified Eagle medium and
`glycerol at the ratio of 1:1 (v/v).
`After retrobulbar anesthetic injection in eyes with
`persistent epithelial defect and ulceration, the base of
`the ulcer was debrided with a microsponge and 0.12
`forceps, and the poorly adherent epithelium adjacent
`to the edge of the ulcer was removed to the area where
`the epithelium became adherent. The amniotic mem
`brane was then removed from the storage medium,
`peeled from the nitrocellulose filter paper, transferred
`to the recipient eye, and fitted to cover the defect by
`trimming off the excess edges. This fashioned mem
`brane was then secured to the edge of the defect by
`interrupted 10-0 nylon sutures and, in some cases, by
`a running 10-0 nylon suture. After the knots had
`been buried, the corneal surface became smooth as a
`result of the well-approximated amniotic membrane
`filling in the ulcer bed. Except for deep ulcers, one
`layer of membrane was generally sufficient. A bandage
`contact lens was applied with topical Maxitrol oint
`ment (neomycin sulfate, polymyxin B sulfate, and
`dexamethasone).
`
`RESULTS
`
`MEAN AGE OF THESE 11 PATIENTS (SIX MEN, FIVE WOMEN)
`was 69.9 ± 16.3 years (Table). Except for the bandage
`contact lens used for 4 months by Patient 2, lenses
`were discontinued in all patients after epithelializa-
`tion. Amniotic membrane transplantation failed in
`Patient 11, who had a preexisting corneal perforation
`caused by severe rheumatoid arthritis; corneal epithe
`lial defects in the remaining 10 patients healed within
`a mean of 3.9 ± 2.3 weeks (P < .01) (compared with
`17.5 ± 13.9 weeks before the transplantation, exclud
`ing Patient 11), and none of the 10 experienced any
`recurrence during the 9.0 ±5.9 months of follow-up.
`The transplanted amniotic membrane dissolved in
`five patients (2, 5, 6, 9, and 11), of whom four (except
`Patient 11) remained healed. The dissolution was
`readily identified because the membrane became
`
`more opaque before dissolution. Following are case
`reports for four selected patients.
`
`CASE REPORTS
`
`• CASE l: A 69-year-old man developed an epithelial
`defect and glaucoma following herpes simplex kera-
`touveitis in the left eye 1.5 years before he developed a
`pseudomonas corneal ulcer, which was successfully
`treated with fortified topical antibiotics. However, the
`epithelial defect progressed into stromal ulceration,
`which persisted for 1 month despite various treat
`ments (Figure 1, a and b). His visual acuity was
`reduced to LE, light perception (Table). After amni
`otic membrane transplantation, epithelialization en
`sued and was detected readily 6 days later by fluoresce-
`in staining (Figure 1, c and d). The epithelial defect
`over the amniotic membrane healed within 2 weeks
`(not shown). Interestingly, the transplanted amniotic
`membrane became more transparent 3 months after
`surgery (Figure le). Four months after the amniotic
`membrane transplantation, he underwent successful
`combined penetrating keratoplasty, cataract extrac
`tion, and lens implantation, resulting in visual acuity
`of LE, 1/200 and a clear graft, which remained stable
`for 19 months thereafter (Figure If).
`
`• CASE 5: A 30-year-old man underwent penetrating
`keratoplasty in his right eye for corneal scarring
`secondary to a neurotrophic corneal ulcer presumably
`caused by long-standing type I diabetes. Immediately
`after, an epithelial defect with ulceration developed
`and persisted for 8 months (Figure 2, a and b) despite
`topical medications, patching, and trials of bandage
`soft contact lens (Table). His visual acuity was RE,
`2/200, corneal sensitivity was decreased, and blink
`rate was reduced. Amniotic membrane transplanta
`tion resulted in early evidence of epithelialization in 1
`week (Figure 2, c and d), which was complete in 1
`month, during which a soft contact lens was used and
`the
`transplanted amniotic membrane dissolved.
`There was no recurrence for 14 months thereafter;
`the patient's final visual acuity was RE, 2/200 (Figure
`2, e and f).
`
`• CASE 7: A 73-year-old man developed exposure
`keratopathy as a result of exophthalmos and eyelid lag
`
`VOL.123, No. 3
`
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`
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`
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`
`
`
`Figure 1. Patient 1. Persistent epithelial defect with ulceration secondary to postinfectious ulcer and herpes simplex
`keratouveitis. The large area of ulceration was noted (a) without and (b) with fluorescein staining. Compared with the
`postoperative appearance 6 days after surgery (c and d), the amniotic membrane became more transparent 3 months
`later (e). A clear graft was noted after combined penetrating keratoplasty, extracapsular cataract extraction, and
`intraocular lens implantation, which were performed 4 months after amniotic membrane transplantation (f).
`
`306
`
`AMERICAN JOURNAL OF OPHTHALMOLOGY
`
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`
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`
`
`
`m
`
`Figure 2. Patient 5. Neurotrophic corneal ulcer following corneal transplantation. The preoperative corneal epithelial
`defect and ulcer by rose bengal staining (a) and fluorescein staining (b). At postoperative week 1 (c), epithelial healing
`had taken place, as shown by fluorescein staining (d). At postoperative month 5 (e and f), the amniotic membrane had
`dissolved, and no recurrence of defect was noted for 11 months thereafter.
`
`VOL.123, N o. 3
`
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`
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`
`
`
`secondary to long-standing Graves thyroid ophthal-
`mopathy. His left eye had been enucleated after severe
`ocular trauma 2 years before our initial examination
`of him. In his right eye he had worn an aphakic
`contact lens for 17 years, and he had developed a
`lens-induced corneal epithelial defect 16 months
`before initial examination but which healed in 1
`month with a bandage soft contact lens. A second
`corneal epithelial defect (4 mm X 6 mm), however,
`had persisted for 1 year despite trials of bandage soft
`contact lens, pressure patching, several attempts of
`partial
`tarsorrhaphy, and one attempt of orbital
`decompression surgery. During this period, he experi
`enced two episodes of bacterial infection. Because of
`the refractory nature of the ulceration, he had under
`gone, without success, a course of hyperbaric oxygen
`treatment (Table). Compassionate use of
`topical
`drops containing epidermal growth factor, together
`with advancement of tarsorrhaphy, reduced the de
`fect to 2 mm X 2.5 mm, but it returned to its original
`size upon discontinuation of the eyedrops and re
`opening of the eyelid fissure to its original position,
`that
`is, with one-third
`tarsorrhaphy (Figure 3a).
`Because of the patient's single-eye status, amniotic
`membrane transplantation was performed with a
`bandage contact lens and additional patching, which
`resulted in rapid epithelialization as early as 1 week
`after surgery (Figure 3, b and c). The remaining defect
`continued to heal in the following 3 weeks, except for
`an area where the membrane dissolved, leaving a 2
`mm X 2 mm defect (Figure 3d, outlined by arrows). A
`second layer of membrane was placed, which resulted
`in complete healing in 1 week (Figure 3, e and f).
`
`• CASE 8: A 75-year-old man had been treated with
`antiglaucoma eyedrops and laser trabeculoplasties for
`long-standing glaucoma, but complications arose in
`his left eye, with painful bullous keratopathy and
`recurrent corneal erosion for 2.5 months, despite
`trials of bandage contact lens, two scrapings, and
`corneal stromal puncture (Table). His visual potential
`was poor. Intraocular pressure was LE, 30 mm Hg.
`Amniotic membrane transplantation was performed
`limbus to limbus. Within 1 week, epithelialization on
`the epithelium-free membrane from the surrounding
`limbal epithelium began (Figure 4, a and b) and was
`complete 6 weeks after surgery. Interestingly,
`the
`transplanted amniotic membrane became more trans
`
`parent 2 months postoperatively (Figure 4, c and d).
`The corneal surface remained stable, and the patient
`remained asymptomatic for 14 months thereafter.
`
`DISCUSSION
`
`THREE BASIC SURGICAL STRATEGIES ARE USED TO TREAT
`persistent corneal epithelial defects and ulcers. 2'13,14
`First, mechanical insults to the ocular surface caused
`by misdirected lashes or incomplete closure can be
`eliminated by plastic surgery. Second, the ocular
`surface defense can be augmented by punctal occlu
`sion to treat keratoconjunctivitis sicca or by tarsorrha
`phy to prevent exposure and/or neurotrophic keratop
`athy. Finally, the conjunctival flap can be used as a
`new substrate to replace the damaged corneal stroma.
`Amniotic membranes have been used for other
`indications15: as a graft for burned skin,16 artificial
`vagina, and conjunctival symblepharon 17; as a dress
`ing for skin wounds18 and for repairing omphalo
`celes19; or as a substrate to prevent tissue adhesion in
`surgeries of the head, abdomen, 20 and pelvis. Except
`in rare situations, all of these applications use living
`rather than preserved amniotic membranes, and this
`might cause undesirable allograft rejection.
`As indicated here, persistent corneal epithelial
`defects with ulceration can also be effectively man
`aged by being covered with a layer of preserved
`human amniotic membrane. The amniotic mem
`brane contains a thick basement membrane and an
`avascular stromal matrix,21'24 and we theorize that
`these features are crucial to successful transplanta
`tions. The basement membrane facilitates migration
`of epithelial cells,25 reinforces adhesion of basal epi
`thelial cells,26,27 and promotes epithelial differentia
`tion.28'31 The basement membrane is also important
`in preventing epithelial apoptosis. 32,33 Collectively,
`these actions explain why the amniotic membrane
`effectively permits rapid epithelialization: here, in a
`mean of 3.9 ± 2.3 weeks after surgery (P < .01) in 10
`of 11 consecutive patients with persistent sterile
`corneal epithelial defects with ulceration. The result
`ing corneal surface was stable, as shown by the lack of
`recurrence for 9.0 ± 5 .9 months postsurgery and by
`the ability of the treatment to withstand additional
`surgery, for example, penetrating keratoplasty (Figure
`If). Because only the substrate without live cells was
`
`308
`
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`
`
`
`Figure 3. Patient 7. Persistent epithelial defect with ulceration secondary to a neurotrophic corneal ulcer and
`penetrating keratopathy caused by Graves thyroid ophthalmopathy. The large area of ulceration was noted in the central
`cornea (a). At postoperative week 1, epithelial healing had taken place (b), as shown by fluorescein staining (c). The
`remaining defect decreased to a smaller size, and the membrane had dissolved by week 4 after surgery (d, outlined by
`arrows). After a second membrane was applied, this defect was completely healed in 1 week (e and f).
`
`VOL.123, No. 3
`
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`
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`
`
`
`Figure 4. Patient 8. Recurrent corneal erosion secondary to glaucoma-induced bullous keratopathy. Amniotic
`membrane transplantation was performed limbus to limbus. Compared with the postoperative appearance at 1 week,
`which showed early epithelialization on the epithelium-free membrane from the surrounding limbal epithelium (a and
`b), the amniotic membrane became more transparent 2 months later (c and d).
`
`transplanted, no adverse effect, such as allograft
`rejection, was noted in any patient.
`We are intrigued that two patients (1 and 8)
`showed gradual transparency
`in the
`transplanted
`amniotic membrane several months after transplanta
`tion (Figures 1 and 4). A similar finding was also
`noted in experimental rabbit corneas 12 and recently in
`corneal surface reconstruction together with limbal
`allografts in patients with advanced ocular cicatricial
`pemphigoid and Stevens-Johnson syndrome. 34 This
`finding is important and suggests that this procedure
`can be used to restore vision. Studies are needed to
`explore the question whether reorganization of the
`amniotic membrane stroma during wound healing is
`unique and able to reduce commonly noted fibrotic
`
`responses. We were equally intrigued that four pa
`tients (2, 5, 6, and 9) showed epithelialization despite
`the fact that the transplanted membrane had dis
`solved (Figure 2e). The dissolution came after the
`opaque appearance of the membrane and was identi
`fied in patients 2, 5, 6, 9, and 11. Except for Patient
`11, epithelialization lasted in all other patients. We
`have no explanation for this at present.
`Studies are also needed to investigate whether the
`amniotic membrane might contain a growth factor or
`growth factors, liberated from the dissolved mem
`brane, that stimulate wound healing. Because of the
`failure by topical epidermal growth factor to heal the
`recurrent corneal epithelial defect in Patient 7, we
`wonder whether the amniotic membrane contains
`
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`
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`
`
`
`growth factors other than epidermal growth factor, or
`alternatively whether the effect of epidermal growth
`factor needs to be augmented with additional matrix
`components. The use of more than one layer of
`amniotic membrane was also effective in covering
`smoothing ulcers of substantial stromal depth (Pa
`tients 4, 10, and 11). However, this technique might
`be ineffective for treating those with frank corneal
`perforation, as shown by Patient 11.
`In conclusion, amniotic membrane transplantation
`may be considered an alternative substrate for treating
`persistent corneal epithelial defects with sterile ulcer-
`ation that are refractory to conventional medical
`treatment. Based on this and other reports1234 and the
`protocol described herein, it is feasible to obtain
`preserved human amniotic membrane. This new
`procedure can be considered before conjunctival flaps
`or tarsorrhaphy, as shown in the majority of our
`patients, which is especially meaningful in that this
`approach can achieve a better cosmetic appearance
`and potential visual acuity. It can also be considered
`an option even after failed partial tarsorrhaphy, as
`shown by our Patients 7 and 9. We recognize that our
`series is relatively small and that a more extensive
`study is necessary to delineate the influences of
`causation and of spontaneous amniotic tissue dissolu
`tion, and the effect of postsurgical mediations. Also,
`further investigations and trials may suggest other
`potential uses for human amniotic membrane trans
`plantation.
`
`ACKNOWLEDGMENTS
`The authors wish to express their deep gratitude to Dr
`Juan Batle of Santo Domingo for introducing us, at
`the Annual Meeting for Residents and Alumni of the
`Bascom Palmer Eye Institute in 1992, to the use of
`amniotic membranes, and to Dr Gene Burkett for
`obtaining donor placentas.
`
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