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`PERSPECTIVE
`
`Amniotic membrane transplantation
`
`Harminder S Dua, Augusto Azuara-Blanco
`
`In 1910 Davis was the first to report the use of fetal mem-
`branes as surgical material in skin transplantation.1 Since
`then the use of amniotic membrane in surgery has been
`expanded.1–9 It is now utilised as a biological dressing for
`burned skin, skin wounds, and chronic ulcers of the leg,9–16
`as an adjunctive tissue in surgical reconstruction of
`artificial vagina,9 17–19 and for repairing omphaloceles.9 20 It
`has also been used to prevent tissue adhesion in surgical
`procedures of the abdomen, head, and pelvis.9 21 22 In the
`1940s several authors reported the beneficial role of amni-
`otic membrane in treating a variety of ocular surface
`disorders.5–7 23 However, its use was abandoned for decades
`until recently, when it was reintroduced to ophthalmolo-
`gists. Several studies have addressed this subject and the
`scope of the application of amniotic membrane transplan-
`tation (AMT) in the management of ocular surface disor-
`ders is ever increasing.
`Certain characteristics make the amniotic membrane
`ideally suited to its application in ocular surface recon-
`struction. It can be easily obtained and its availability is
`nearly unlimited. The tissue can be preserved at −80(cid:176)C for
`several months, allowing suYcient time to plan surgery or
`consider a trial of other options. Amniotic membrane does
`not express HLA-A, B, or DR antigens and hence
`immunological rejection after its transplantation does not
`occur.24–26
`It
`is also believed to have antimicrobial
`properties, reducing the risks of postoperative infection.27
`Antifibroblastic activity28–30 and cell migration/growth pro-
`moting activity31–33 have also been demonstrated with
`regard to the amniotic membrane.
`The purpose of this paper is to review the characteristics
`of amniotic membrane that make it potentially useful to
`treat ocular surface abnormalities and to discuss the
`current
`indications,
`the surgical
`technique, and the
`outcome of AMT.
`
`Histology and physiology
`Mammalian embryos lie within a fluid filled sac (fetal
`membranes) that arises from extraembryonic tissues. At
`full term of gestation, the fetal membranes are composed
`of two principal layers. The outer layer or chorion which
`forms the outer aspect of the sac and is in contact with
`maternal cells. It consists of compressed trophoblastic
`tissue of chorion laeve and mesenchymal tissue. The inner
`layer or amniotic membrane consists of a single layer of
`ectodermally derived columnar cells firmly fixed to an
`underlying layer of mesenchyme which contains large
`amounts of collagen.34 35 The amniotic membrane is
`bathed by amniotic fluid. According to Shimazaki et al,31
`the epithelium of the amniotic membrane survives for up
`to 70 days after preservation. We have noted that after
`freezing the amniotic membrane at −70(cid:176)C for 6 months to
`a year the epithelial cells appear very vacuolated but
`remain attached to the underlying basement membrane
`and mesenchyme (unpublished data).
`
`The apical surface of amniotic cells have many microvilli
`(Fig 1). At the base, cell processes or pedicels extend into
`the basement membrane in podocyte fashion. The basal
`cell processes have a hemidesmosome type of attachment
`to the basal membrane with tonofilaments, and the subja-
`cent basement membrane substance is partly amorphous
`and partly microfibrillar. The cytoplasm contains many
`pinocytic vesicles, abundant organelles including cisternal
`endoplasmic reticulum, and Golgi apparatus. The nucleus
`has a very irregular configuration, with a number of inden-
`tations of the nuclear membrane. The nucleolus is often
`large and homogeneous suggesting nucleolar activity.
`Overall, the ultrastructure of the epithelium suggests that
`the amnion has multiple specialised functions. It has been
`specifically adapted to perform three major functions—as a
`covering epithelium, as an active secretory epithelium, and
`for intense intercellular and transcellular transport.34 35
`
`Amniotic membrane in ophthalmology
`MECHANISM OF ACTION
`Promoter of epithelialisation
`The presence of a normal substrate in the cornea is essen-
`tial for normal proliferation and diVerentiation of epithelial
`cells. Basement membrane facilitates migration of epithe-
`lial cells33 36; it also reinforces adhesion of basal epithelial
`cells,37 promotes epithelial diVerentiation,38 39 and prevents
`epithelial apoptosis.40 The amniotic membrane, by serving
`as a “transplanted basement membrane”, acts as a new
`healthy substrate suitable for proper epithelialisation.
`Additionally, the amniotic membrane produces various
`growth factors such as basic fibroblast growth factor,
`hepatocyte growth factor, and transforming growth factor
`♢, that can stimulate epithelialisation.31 41 However, it has
`been demonstrated that cryopreservation of amnion results
`in a decrease of growth factors.41 Amniotic membrane also
`inhibits protease activity.30 42 It has also been shown that in
`some instances the amniotic membrane, rather than
`providing a substrate, acts as a “bandage contact lens”
`allowing epithelialisation to occur under its cover.43
`
`Inhibitor of fibrosis
`Several factors are involved in the antifibrotic eVect of the
`amniotic membrane.28 29 It has been shown that amniotic
`membrane induces a downregulation of
`transforming
`growth factor ♢ signalling, responsible for fibroblastic acti-
`vation in wound healing.28 The amniotic membrane may
`also function as an anatomical barrier, keeping the poten-
`tially adhesive surfaces apart. The stroma of the amniotic
`membrane is normally avascular and is believed to inhibit
`the incursion of new vessels.
`
`Indications for AMT in ophthalmology
`Amniotic membrane transplantation has been successfully
`used in patients with persistent epithelial defects unre-
`sponsive to medical treatment,43 44 and as an alternative to
`conjunctival flaps, botulinum toxin injection, or tarsor-
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`Amniotic membrane transplantation
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`Figure 1 Transmission electron microscopy of the amnion. The apical border of the amniotic epithelial cells contains a great number of microvilli. The
`cytoplasm contains numerous vesicles. Basal cell processes (pedicels) extend into the basement membrane. The underlying connective tissue has a
`homogeneous structure.
`
`rhaphy. The frequency of success in two recent series was
`10 of 11 cases44 and four of five cases,43 respectively. In our
`experience, the amniotic membrane, by virtue of its trans-
`parency, allows the patient navigational vision and is
`particularly useful if the aVected eye is the better seeing
`eye. The use of more than one layer may be eVective in
`covering ulcers with substantial stromal depth. However,
`the use of AMT as a tectonic procedure in cases with
`impending or recent perforation appears to be unsatisfac-
`tory, and failure was reported in five consecutive cases.43
`Amniotic membrane has been used as an alternative to
`conjunctival autograft during the removal of pterygia. The
`recurrence rate of pterygium after AMT (10.9% for
`primary pterygia) was lower than the bare sclera technique
`(45%), but higher than autologous conjunctival graft
`(2.6%).45 Multiple surgical approaches have been used to
`treat pterygium. Although conjunctival autograft is consid-
`ered to be the most eYcient, AMT appears to be a reason-
`able option in cases with diVuse conjunctival involvement
`and patients in whom the bulbar conjunctiva must be pre-
`served for a prospective glaucoma filtering procedure.
`AMT has been successfully used in the treatment of
`recurrent pterygium associated with severe symblepharon
`and diplopia.31 In Shimazaki’s series, all four patients had a
`favourable functional and anatomical outcome after AMT.
`Similarly, AMT has been used successfully in 13 of 16 eyes
`in the reconstruction of conjunctival defects created during
`surgical removal of large conjunctival lesions.33
`Corneal stem cell deficiency is associated with conjunc-
`tivalisation of the cornea and can be complicated with per-
`sistent epithelial defects, vascularisation, scarring, calcifi-
`cation, ulceration, melting, and perforation of the cornea.
`Patients with these abnormalities are poor candidates for
`conventional corneal transplantation. Lamellar or pen-
`etrating keratoplasty provides only a temporary replace-
`ment of the host’s corneal epithelium and does not perma-
`nently restore limbal function. In cases with diVuse corneal
`stem cell deficiency, limbal transplantation (allo or auto)
`is now considered essential
`for
`corneal
`surface
`
`reconstruction.46–53 AMT combined with limbal transplan-
`tation has been successfully used in patients with diVuse
`limbal stem cell deficiency and severe ocular surface
`disease, including Stevens–Johnson syndrome, advanced
`ocular cicatricial pemphigoid, chemical and thermal
`burns.32 46 Alternatively, autologous limbal-corneal epithe-
`lium can be cultured on amniotic membrane and used for
`corneal surface reconstruction.54
`Notwithstanding the encouraging and successful results
`reported thus far it is important to caution against the
`overenthusiasm in the use of amniotic membranes that is
`beginning to emerge of late. The beneficial eVect of amni-
`otic membrane in the management of ocular surface disor-
`ders has not always been validated with controlled clinical
`trials. In some series the favourable outcomes could well be
`attributed to concurrent surgical procedures. Shimazaki et
`al 46 stated that “we do not know exactly in which case the
`amniotic membrane should be used and how much the
`current procedure (AMT and limbal transplantation) is
`superior to the simple limbal autograft transplantation.”
`Similarly, Tseng et al 32
`recently reported successful
`management of patients with sector limbal stem cell
`deficiency treated with removal of conjunctiva-like epithe-
`lium from the corneal surface combined with AMT. They
`did not, however, have any controls, where amniotic mem-
`brane was not used. In a similar group of patients Dua55
`and Dua et al 56 also reported excellent outcome following
`removal of conjunctiva-like epithelium, without AMT,
`suggesting that AMT is probably not required in such
`patients. Following observations on the healing of corneal
`epithelial defects involving the limbus, Dua and Forrester57
`had observed the migration of conjunctival epithelium on
`to the corneal surface and reported that mechanical debri-
`dement could prevent the manifestation of conjunctivalisa-
`tion of the cornea (partial stem cell deficiency).
`Fujishima et al 58 recently used amniotic membrane in
`guarded filtration procedures supplemented with mitomy-
`cin C to inhibit scarring and promote filtration. Amniotic
`membrane was placed underneath the scleral flap. In this
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`small series the mid term outcome of trabeculectomy was
`satisfactory in 13 of 14 eyes. After filtering operations the
`extraocular changes (that
`is, subconjunctival fibrosis)
`account for the majority of failures. Antifibrotic agents,
`mitomycin C and 5-fluorouracil, are currently used to
`improve the chances of success, although the complication
`rate also rises.59 A controlled clinical trial will be needed to
`evaluate whether amniotic membrane helps to improve the
`outcome of filtration procedures.
`Bleb leaks after filtration surgery can be associated with
`hypotony, shallow flat anterior chamber, and choroidal
`detachment and may increase the chances for bleb
`infection and subsequent endophthalmitis. Leaking filter-
`ing blebs usually require prompt treatment. Therapeutic
`options include bandage contact lens, Simmons’ shell,
`injection of autologous blood, cryopexy, thermal Nd:YAG
`laser, cyanoacrylate glue, fibrin tissue glue, and surgical
`revision.59 Budenz et al 60 recently reported favourable use
`of human AMT for
`revision of
`leaking blebs after
`glaucoma surgery in five patients. AMT compared favour-
`ably with conjunctival advancement.
`Anecdotally, AMT has been successfully used in three
`patients to treat myopic regression with corneal opacity
`after photorefractive keratectomy (PRK) in high myopia.61
`Excessive corneal haze and myopic regression are associ-
`ated with excessive healing response, which might be
`inhibited by amniotic membrane. In rabbits the corneal
`haze was
`reduced
`by AMT in
`excimer
`laser
`photoablation.62 63
`
`Surgical principles
`PREPARATION OF AMNIOTIC MEMBRANE
`Amniotic membrane is obtained under sterile conditions
`after elective caesarean delivery from a seronegative donor
`(see below). Two similar methods of tissue preparation
`have been described.
`(A) Under a lamellar flow hood, the placenta is first
`washed free of blood clots with balanced saline solution
`containing 50 µg/ml of penicillin, 50 µg/ml of streptomy-
`cin, 100 µg/ml of neomycin, and 2.5 µg/ml of amphotericin
`B. The inner amniotic membrane is separated from the rest
`of the chorion by blunt dissection (through the potential
`spaces between these two tissues). The membrane is then
`flattened onto a nitrocellulose paper, with the epithelium/
`basement membrane surface up. The membrane with the
`paper is cut into 4 · 4 cm pieces and placed in a sterile vial
`containing Dulbecco’s modified Eagle’s medium and
`glycerol at a ratio of 1:1 (vol/vol). The vials are frozen at
`−80(cid:176)C. The membrane is defrosted immediately before
`use by warming the container to room temperature for 10
`minutes.33
`(B) After washing with physiological saline or 0.01 M
`phosphate buVered saline (PBS) containing 100 mg of
`dibekacin sulphate, the amniotic membrane with the cho-
`rion is separated from other uterus tissue by blunt dissec-
`tion. The membrane is then cut into pieces measuring 5 ·
`5 cm and rinsed three times in 0.01 M PBS. Each piece is
`rinsed in 0.5 M DMSO dissolved in PBS, then in 1.0 M
`and 1.5 M DMSO in PBS, for 5 minutes each. The mem-
`brane is placed in a plastic container and preserved at
`−80(cid:176)C until use. The container with amniotic membrane
`is warmed to room temperature preoperatively, and the
`membrane is rinsed three times in saline, then once in
`saline containing 100 mg of dibekacin sulphate. At the
`time of surgery the amniotic membrane is separated
`bluntly from the underlying chorion with forceps.53
`
`MICROBIOLOGICAL SAFETY
`Transmission of infectious agents is one of the risks associ-
`ated with transplantation of human organs and tissues.
`
`Safety criteria applied to organ transplantation should be
`applied even more strictly to tissue transplantation such as
`amniotic membrane.64
`All recipients of organs and tissues need to be advised
`that the donors have been tested for some infections, but
`that there is a very slight possibility that a donor may be
`infected, for instance, because of the “window period”,
`when the donor is already infected but the screening tests
`are not yet positive. Where donor tissue can be stored for
`180 days without impairing its fitness for use, as is the case
`with the amniotic membrane, it is considered essential that
`serological testing of the donor be carried out at the time of
`procurement and 6 months later, to allow for the window
`period. Tissue should be used for transplantation only
`when both samples are negative. This does not, however,
`exclude the possibility of infection with pathogens for
`which no test is available such as the Creutzfeldt–Jakob
`disease (CJD) prion protein. Infection may also occur as a
`results of procurement procedures and tissue processing.
`There is therefore a need for microbiological quality
`control of tissue procurement and any associated banking
`as well as for prevention of transmission of infection from
`the donor.64
`After the identification of a potential donor, a detailed
`social and medical history needs to be obtained. The clini-
`cian responsible for collecting the tissue must ensure that
`the donor has been asked about any activities or other risk
`factors which may suggest that the donor is unsuitable.
`Donors at risk of having HIV (human immunodeficiency
`virus), HBV (hepatitis B virus), HCV (hepatitis C virus),
`and CJD must be excluded. Consent for donation and
`subsequent use, and for screening for infection must also
`be obtained at
`this stage. Local research and ethics
`committee approval may also be considered necessary in
`some institutions.
`Microbiological testing should be undertaken by labora-
`tories competent to do the tests. Records of tests and
`results together with serum samples must be kept for 11
`years post transplantation and donor anonymity must be
`maintained. Serum samples from all donors must be tested
`for anti-HIV-1 and 2, hepatitis B surface antigen (HBsAg),
`and anti-hepatitis C virus (anti-HCV). Testing for syphilis
`is also required. Donors infected with HIV-1 or 2, HCV,
`HBV, or
`syphilis
`should be excluded. Testing for
`cytomegalovirus and toxoplasma is not relevant for most
`tissue transplantation including amniotic membrane.
`Tissue must be handled, processed, and transported at
`all times in a way that minimises contamination from
`exogenous sources as far as practically possible. Donor
`material should be collected under clean and aseptic con-
`ditions. When the tissue is stored, a registered medical
`practitioner must take overall responsibility for the micro-
`biological safety of the tissue and maintenance of records.
`All tissue must be traceable from the donor via any
`intermediaries to the recipient and from the recipient back
`to the donor.64
`
`SURGICAL TECHNIQUE
`The membrane is always sutured to the ocular surface with
`its epithelial side up and the mesenchymal surface in con-
`tact with the eye, to facilitate adherence of the membrane
`to the ocular surface. For this reason it is important to be
`able to distinguish its two surfaces. This is easiest when the
`membrane is fresh, but when dealing with membranes that
`have been thawed after storage at −70(cid:176)C it becomes di Y-
`cult. Most surgeons have developed a technique that suits
`them best—for example, mounting the membrane on
`nitrocellulose paper, the right way up, so that the correct
`side can be determined when the membrane is thawed.
`Others will use a suture, with the knot as the marker or
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`Figure 2 Surgical technique. The diagram illustrates the amniotic
`membrane sutured to the cornea and covering a paracentral corneal
`epithelial defect.
`
`Figure 3 Surgical technique. The amniotic membrane is sutured to
`perilimbal episclera and to the edge of the conjunctiva (after peritomy)
`covering the whole corneal surface.
`indelible marker pen, to mark one side of the membrane.
`We have developed a method that we find useful. After
`spreading the membrane on the ocular surface we apply
`the tips of a blunt fine forceps to one surface of membrane
`and pinch lightly with the forceps and lift. A fine strand of
`“vitreous-like” substance can usually be drawn up from the
`mesenchymal but not the epithelial (basement membrane)
`side of the amniotic membrane.
`The amniotic membrane is spread on to surface of the
`eye and cut to appropriate size and shape, keeping the final
`piece slightly larger than the size of the defect to be
`covered. It is usually sutured to the cornea with 10-0
`Nylon sutures and to the episclera/conjunctiva with 9-0
`Vicryl sutures. After surgery a bandage contact lens is put
`in place, and topical steroids and antibiotics are used.
`Sutures can be removed at 3 weeks. The membrane stains
`with fluorescein stain and like the cornea, attracts
`ciprofloxacin deposits,
`if
`the drug is used topically,
`postoperatively.43
`In cases of persistent epithelial defects, the base of the
`ulcer and loose epithelium adjacent to the edge of the ulcer
`are debrided before applying the membrane. The amniotic
`membrane is trimmed and fitted to cover the epithelial
`defect and sutured to the edge of the defect (Fig 2).44 If the
`epithelial defect is large, a 360 degree peritomy is done and
`the membrane sutured to cover the cornea from limbus to
`limbus (Fig 3). In pterygium or symblepharon surgery, the
`membrane is applied to cover areas of conjunctival defects
`after removal of fibrotic tissue (Fig 4).30
`Several problems can occur after amniotic membrane
`transplantation. The amniotic membrane may disintegrate
`before epithelialisation, in some cases within 2 weeks after
`transplantation. Necrosis of the amniotic membrane may
`
`Figure 4 Surgical technique. The amniotic membrane can be used to
`cover a conjunctival defect after releasing adhesions during symblepharon
`surgery, and in a similar manner (nasally or temporally) after excision of
`pterygium.
`
`be related to collagenases present on the ocular surface as,
`in our experience, it occurs more often in cases with
`intense inflammation (unpublished data). The amniotic
`membrane will not remain attached to the ocular surface if
`the mesenchymal surface is not facing the host.
`
`Summary
`Amniotic membrane has unique properties that can be
`helpful to treat diVerent ocular surface diseases. AMT is
`useful in promoting normal epithelialisation of cornea and
`conjunctiva. It is also eVective in preventing excessive
`fibrosis during ocular surface reconstruction. Future
`possible indications are being investigated. Because of the
`potential risk of infection strict safety criteria must be
`applied. The procedure is still evolving and, not surpris-
`ingly, is being tried in a variety of diverse conditions. Some
`of these may prove inappropriate. Controlled clinical trials
`will be needed to establish the role of AMT in ocular sur-
`face reconstruction.
`
`We are grateful to Vision Express UK Ltd for supporting Dr Azuara-Blanco’s
`fellowship. Dr Azuara-Blanco was the Vision Express fellow in cornea and con-
`tact lenses, at the Queen’s Medical Centre, University Hospital, Nottingham
`from July 1997 to December 1998. We are also grateful
`to April
`Powell-Richards for her technical assistance.
`
`HARMINDER S DUA
`Department of Ophthalmology, University of Nottingham, Queen’s
`Medical Centre, University Hospital, Nottingham
`AUGUSTO AZUARA-BLANCO
`Department of Ophthalmology, University of Nottingham, Queen’s
`Medical Centre, University Hospital, Nottingham and the Princess
`Alexandra Eye Pavilion, The Royal Infirmary of Edinburgh,
`Edinburgh
`
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`

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`Amniotic membrane transplantation
`ARMINDER S DUA
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`(cid:160)H
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` 1999 83: 748-752Br J Ophthalmol
`
`doi: 10.1136/bjo.83.6.748
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