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Amniotic membrane as ophthalmic medical, surgical tool

No significant local immune responses or signs of acute rejection have been seen.

by Leonid Skorin Jr., OD, DO, FAAO, FAOCO

This course is jointly sponsored by PCON, The State University of New York State College of Optometry and Vindico Medical Education. It is COPE-approved for 2 continuing education credits.

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Leonid Skorin Jr.

The amniotic membrane, or amnion, is the innermost layer of the placenta. It is translucent and composed of an inner single layer of epithelial cells overlying a basement membrane that attaches to a thin layer of connective tissue by filamentous strands. The height of the amniotic epithelial cells varies widely between different regions: placental amnion vs. extra-placental amnion.

Although the amnion is adjacent to the chorion (the connective tissue that contains the fetal vessels), it is not completely fused to it, so they can be easily separated by means of blunt dissection, according to Gris and Güell. Amniotic membrane is derived from fetal ectoderm, according to Dua and colleagues.

Immunofluorescent techniques have shown that human amniotic epithelial cells do not express HLA-A, B, C or DR antigens on their surface, according to Adinolfi and colleagues. Various studies have shown that human subjects show no significant local immune response or signs of acute rejection following transplantation with human amniotic membrane, and none of the subjects tested produced antibodies in their blood against the HLA-antigens, according to Akle and colleagues.

Used in ocular surface reconstruction

Because this tissue is relatively immunologically inert, it is now being used in ocular surgery, primarily in ocular surface reconstruction. Preserved amniotic membrane can be used as a graft, a patch or a combination of both, according to Gris and Güell. When implanted as a graft, it covers a tissue defect, replacing the missing stromal matrix and providing a basement membrane on which epithelialization can take place. When used as a patch, it protects the ocular surface from possible external insults and helps reduce inflammation and promotes epithelial regeneration beneath the implant.

The amnion must be separated from the chorion because the chorion has been found to induce considerable inflammation with neovascularization, which leads to rejection, according to Gris and Güell. Because the chorion possesses considerable antigenicity, commercially available fetal membranes contain only the amnion.

In 1940, de Rotth was the first to use fetal membranes in ophthalmology. Unfortunately, de Rotth used freshly obtained fetal membranes that contained both the amnion and chorion to reconstruct the conjunctival surface. This resulted in a high rejection rate because of the chorion’s antigenicity. Later that decade, Sorsby and colleagues used preserved amniotic membranes as a patch for treating acute ocular burns. Sorsby and colleagues reported very good results with the chemically processed dry amniotic membrane (Dua and colleagues, and Solomon and Tseng). Perhaps because of tissue preservation difficulties, amniotic membrane use in ophthalmic surgery was abandoned at that time, according to Gris and Güell.

Corneal reconstruction

Tarsorrhaphy: This photo shows ProKera inserted under a partially sutured tarsorrhaphy. Images: Reprinted with permission from the Ocular Surface Research and Education Foundation and research director, Scheffer C.G. Tseng, MD, PhD.

Interest in amniotic membrane use was renewed in 1995 with an article that showed that 40% of rabbit corneas with limbal stem cell deficiency could be reconstructed by replacing the conjunctival surfaces with preserved human amnion, according to Kim and Tseng.

Today, preparation, preservation and storage methods are more advanced. This has added to the revival of this tissue use. Commercially available tissue and donors are routinely tested for various infectious agents before the tissue is implanted in patients. The placental donors are usually screened against HIV types 1 and 2, human T-lymphocyte virus, hepatitis B and C viruses and syphilis at delivery and 6 months after delivery, according to Solomon and colleagues. Preserved membrane is considered to be inert tissue with no viable cells, according to Dua and colleagues.

Clinical mechanisms of action

In the original studies from the 1940s, amniotic membrane was used as a graft or biological bandage (Sorsby and colleagues and de Rotth). This bandage effect helps promote the healing process of inflamed and exposed ocular surfaces and significantly reduces the pain and discomfort patients experience with these denuded ocular surfaces, according to Dua and colleagues.

Recent studies have shown that this tissue covering — when applied to photoastigmatic refractive keratectomy denuded corneal stroma — makes the surface smoother and more spherical, which provides mechanical protection that reduces astigmatism, according to Zhou and colleagues. Amniotic membrane use seems to not only eliminate photoastigmatic refractive keratectomy-induced astigmatism but also may prevent surgically induced astigmatism, according to Young. It also reduces the corneal opacities that may appear after stromal ablation with the excimer laser, according to Wang and colleagues.

An amniotic membrane patch effectively treats persistent corneal epithelial defects that have not responded to medical treatment. It also promotes postoperative epithelialization, according to Gris and colleagues.

Amniotic membranes have been used to manage epithelial defects resulting from infection, persistent ulceration associated with neurotrophic corneas and recurrent corneal erosion; to prevent the formation of scar tissue from chemical injuries; and to provide a substrate in cases of bullous keratopathy (Gris and colleagues, Lee and Tseng, Chen and colleagues, Meller and colleagues).

There is a higher failure rate for these tissues in cases of neurotrophic keratopathy associated with severe dry eye and exposure, according to Solomon and colleagues. In these instances, the tissue patch may offer only a temporary solution until dissolving in approximately 2 weeks, according to Schultze. In eyes that have neurotrophic keratopathy, additional measures such as permanent punctal occlusion, eyelid tarsorrhaphy and the use of a multilamellar transplantation technique may provide a more long-term benefit in refractory patients (Solomon and colleagues, Schultze and Singh).

Does not promote re-epithelialization

Amniotic membranes have not been successful in promoting re-epithelialization in cases of persistent epithelial defects with stromal thinning or in patients with corneal perforation (Lee and Tseng, Azuara-Blanco and colleagues). Failure in these cases occurs because the underlying scaffolding or architectural structure needed to support the amniotic membrane is inadequate. The amniotic membrane is too thin a tissue to supply any significant mass or structural support. Hard tissues, such as cornea, sclera or others are necessary to restore corneal biomechanical properties in these cases, according to Dua and colleagues.

Other mechanisms of action specific to the amniotic basement membrane include facilitating epithelial cell migration, reinforcing epidermal cell-basement membrane adhesion, promoting epithelial differentiation and preventing epithelial cell apoptosis (programmed cell death), according to Solomon and Tseng. The amniotic membrane can also promote non-goblet cell differentiation of the conjunctival epithelium, which in turn helps increase conjunctival goblet cell density (Meller and Tseng, Prabhasawat and Tseng).

Pterygium: On the left, pterygium is encroaching on the visual axis. On the right is the same eye with an amniotic membrane graft, 1 month postoperatively.

Unique matrix component

The stromal side of the membrane contains a unique matrix component that suppresses growth factor, signaling, proliferation and myofibroblast differentiation of normal human corneal and limbal fibroblasts, according to Tseng and colleagues. This explains why amniotic membrane transplantation is responsible for the anti-scarring properties during conjunctival surface reconstruction and how it prevents recurrent scarring after pterygium removal, according to Solomon and colleagues. At least some of the anti-inflammatory and anti-scarring affects are believed to be due to the membrane’s effect on apoptosis, according to Dua and colleagues. The stromal matrix of the membrane excludes inflammatory cells by trapping these cells and rendering them into rapid apoptosis (Dua and colleagues, Solomon and Tseng).

This same membrane stromal matrix contains various forms of protease inhibitors, according to Hao and colleagues. Specifically, the potent anti-angiogenic chemical, thrombospondin-1, and the powerful antiangiogenic and endothelial cell growth inhibitor, endostatin, are found in amniotic membranes and explain how they prevent corneal neovascularization. The membrane may also act as a physical barrier preventing diffusion of or “mopping up” inflammatory mediators and promoters of vascularization, according to Dua and colleagues. Controlling inflammation and neovascularization are critical in preparing a corneal stromal bed for limbal stem cell transplant in cases of corneal surface reconstruction in patients with limbal stem cell deficiency, in patients with recurrent pterygium associated with symblepharon or in the general treatment of any severe ocular surface disorder (Tseng and colleagues, Shimazaki and colleagues, Tsubota and colleagues).

All of these biological properties shown by the amniotic membrane can be summarized as a series of clinical effects, which can be expected after transplantation of the amniotic membrane onto the ocular surface, according to Gris and Güell:

• It facilitates epithelialization of the tissues (cornea or conjunctiva) using the healthy surrounding epithelium, maintaining the epithelial phenotype present in the area.
• It reduces tissue inflammation under the implant and in the surrounding area.
• It reduces neovascularization in the corneal stroma.
• It reduces residual scarring following tissue regeneration, both in the cornea and in the conjunctiva.

Amniotic membrane as medical therapy

Recently a medical device has become available that can be used in eyes in which ocular surface cells have been damaged or the underlying corneal stroma is inflamed or scarred. According to Tseng, it is most commonly indicated in the following cases:

• epithelial defects, recurrent erosion or non-infectious ulceration
• chemical and thermal burns if in the acute stage of injury
• after the removal of corneal lesions such as band keratopathy
• chronic recalcitrant keratitis from herpes zoster ophthalmicus, herpes simplex virus or vernal keratitis
• Stevens-Johnson syndrome (acute stage)
• in conjunction with socket or fornix reconstruction to prevent lid or lash rubbing
• miscellaneous conjunctival or limbal lesions

Severe symblepharon: The polycarbonate ring-set system of the ProKera device functions like a symblepharon ring.

The device is known as ProKera (Bio-Tissue Inc., Miami) and consists of an ophthalmic conformer that incorporates cryopreserved amniotic membrane, according to the product insert. The ophthalmic conformer is composed of a dual polycarbonate ring-set system in which the amniotic membrane is clipped in between.

The original device was composed of polymethylmethacrylate, but the polycarbonate has been found to be more flexible (for easier insertion and removal) and less irritating to the patient. The amniotic membrane is preserved in a validated storage medium made of Dulbecco’s modified Eagle medium and glycerol (1:1) containing ciprofloxacin and amphotericin B to retain the membrane’s natural biological properties, according to the product insert.

Donors screened for infections

The amniotic membrane tissue is recovered aseptically from a donated placenta through elective Caesarean section delivery. The donor is screened for infectious, malignant, neurological and autoimmune diseases and other exposures or habits before they are determined to be suitable to donate tissue for human transplantation. Donors for ProKera are tested at the time of delivery and found to be non-reactive for HIV types 1 and 2 antibodies, hepatitis B surface antigen and core antibody, hepatitis C antibody, human T-lymphocyte 1 and 2 antibodies and syphilis, according to the product insert.

ProKera is assembled so that the stromal (sticky) side of the tissue is in contact with the corneal surface, according to Tseng. This product acts like a “natural” bandage contact lens. The polycarbonate ring-set system functions like a symblepharon ring. During the acute phase of various anterior segment diseases or injuries, such as Stevens-Johnson syndrome or chemical eye burns, conjunctivitis produces raw conjunctival surfaces that adhere to one another or to the globe, often causing massive symblepharon, according to Kaufman and colleagues.

Adhesions, shortening of the fornices and cul-de-sacs may all continue to occur even after daily lysis of the symblepharon if the raw edges of the opposing mucosal surfaces touch each other before healing is complete. A symblepharon ring or shell with a bandage soft contact lens or, in the case of ProKera, an affixed amniotic membrane, can prevent the raw bulbar and palpebral conjunctival surfaces from touching each other, according to Kaufman and colleagues.

ProKera is available in two sizes. The patient’s interpalpebral width should help guide the selection of the appropriate size. For small eyes, a 15-mm ring is available, while for normal or large eyes the 16-mm ring is available, according to the product insert. The 15 mm and 16 mm refer to the inner diameter of the ring-set holding the amniotic membrane. ProKera is not appropriate for patients with unusually tight eyelids, which may make it difficult or painful to insert.

This device is not an option if the patient is unable to close his or her eyelids fully. Treat exposure problems prior to implantation or they will diminish the effects of the membrane. A partial temporary sutured tarsorrhaphy should be performed in patients with inadequate blink, lagophthalmos, floppy eyelids, ectropion or exophthalmos, according to Tseng.

Because ProKera is stored in a preserved solution that contains ciprofloxacin and amphotericin B, do not use it in patients with a known history of allergic reaction to any of the fluoroquinolones or the antifungal amphotericin B. The manufacturer also warns that although all screening and microbial testing results were satisfactory for the donor, the membrane may still transmit infectious agents.

To prevent tissue degradation, the membrane must be stored in a specific manner after the clinician receives it in the office. Thaw the device at room temperature for 5 to 10 minutes prior to its use. It should not be “heated up” in a microwave or hot water bath.

Device in place: ProKera is inserted in the eye. The photo at right shows the device with fluorescein staining.

Using the device

Because the device is sterile, the clinician should use an aseptic technique to handle it. ProKera is packaged in a dual pouch system. The outer aluminum foil pouch is not sterile, but the inner, clear pouch is sterile. Therefore, sterile forceps should be used to handle this inner pouch, which may then be placed in a sterile field. Use sterile scissors to cut the inner package, forming an opening that is large enough to be able to remove the ProKera device comfortably. The membrane portion of the device is very slippery, thus the clinician should use either a blunt sterile forceps or his or her fingers, clad in a sterile glove, to grab and lift the device out of the inner pouch.

To avoid having the membrane slip out of the clinician’s hand and to prevent any inadvertent damage or tearing of the membrane, the clinician should grab only at the plastic ring rim. Extra membrane may be hanging over the sides of the ring, but this excess tissue should not be cut off or trimmed down.

Before placing the ProKera device in the patient’s eye, apply topical anesthetic drops. Use an eyelid speculum to open the patient’s eye to the maximum. Rinse the device with an eyewash or balanced salt solution prior to insertion as some patients experience burning from any residual storage media.

Insert the ProKera underneath the upper lid first and then tuck the inferior edge of the device under the lower lid. The speculum can help manually lift the lid away from the globe for easier device insertion. Place prophylactic antibiotic eye drops in the patient’s eye after removing the speculum.

Patient instructions

The manufacturer requires that a Donor and Recipient Information Form be filled out and returned to the company immediately. Instruct patients to not rub their eyes, excessively blink or move or handle the device with their fingers. Patients should not swim or soak their face in water without protective eye wear, and they should keep their eyes closed tightly during showering. Because the membrane is not transparent, it will blur the patient’s vision. Therefore, caution the patient about driving, operating heavy machinery or doing any other task that requires unobstructed vision or good depth perception.

The patient should use nonpreserved artificial tears three or four times a day, especially if there is a concern about dry eye or exposure. The patient can also use antibiotic eye drops if necessary, or the membrane can be soaked with antibiotic eye drop medication before insertion; all of these solutions penetrate the membrane.

The optometrist can easily examine the eye behind a slit lamp. Fluorescein staining allows easy monitoring of healing beneath the device. There is no need to remove it during fluorescein staining. Intraocular pressure can also be assessed with a Tonopen (Medtronic, Jacksonville, Fla.) while the device is still on the eye. If temporary removal is required, again, handle the device aseptically and store it in a sterile container with balanced salt solution before reinsertion.

Healing, removal

The manufacturer recommends that the device be used for no longer than 30 days or until the ocular surface has healed or the membrane has dissolved, according to the product insert. In many cases, healing is complete in 1 or 2 weeks. For cases with severe inflammation (e.g., acute chemical burns), it is beneficial to insert a new ProKera device every 5 days to avoid polymorphonuclear leukocyte cells becoming trapped on the membrane, which may lessen its therapeutic effect, according to Tseng.

The amniotic membrane does not fuse with the underlying host epithelium, but it does thin and dissolve as the host epithelium heals. The membrane should not dissolve in less than 1 week. As it dissolves, mucous debris may accumulate in the eye. This mucous should be gently irrigated with eyewash. If the membrane dissolves after adequate healing has taken place, remove the device. It is easiest to remove the device using blunt forceps with or without the help of a lid speculum. An eye ointment can facilitate the removal.

Occasionally, as the healing progresses, the membrane may thin out to the point that it detaches from the ring-set. It is not practical to try to reassemble the device; just remove it. Another ProKera may be inserted if healing was not yet complete. In most instances, if the membrane dissolves before healing has occurred, it is probably due to an exposure problem. Address this by using either nonpreserved artificial tears to increase eye lubrication or by adding a temporary tarsorrhaphy before inserting another ProKera.

Amniotic membrane as surgical therapy

Amniotic membranes have numerous therapeutic actions. These include anti-scarring, anti-inflammation and anti-angiogenesis. These properties make this tissue an ideal homologous graft for external ocular wound repair and healing.

Pterygia are triangular-shaped abnormal overgrowths of the conjunctival tissue onto the corneal surface. They occur nasally most commonly but can be seen temporally as well, according to Koranyi and colleagues.

The pterygium base is attached to vascularized conjunctival tissue that grows toward the corneal surface and forms an apex. As the apex continues to encroach onto the cornea, blood vessels are dragged onto the corneal surface, according to Buratto and colleagues. This leads to ocular irritation, adverse cosmetic features, decreased visual acuity from corneal opacification and induced irregular corneal astigmatism, according to Donaldson and Alfonso.

Modern applications of pterygium surgery have been extensively documented (Koranyi and colleagues, Buratto and colleagues, Donaldson and Alfonso, Skorin and colleagues).

Key surgical steps

Topical anesthesia is appropriate for this surgery. The entire pterygium is removed from the sclera and the cornea, with the subconjunctival fibrovascular tissue meticulously removed. The corneal surface is polished using a diamond-encrusted burr. Mitomycin C, an antimiotic agent that inhibits cell proliferation and fibrosis, is applied to the conjunctival fornix.

The amniotic membrane graft can be affixed to the underlying bare sclera with Tisseel fibrin glue (Baxter, Westlake Village, Calif.), which is a sealant composed of thrombin and fibrinogen. This obviates the need for sutures, making the postoperative recovery significantly more comfortable for the patient.

Tseng and Kheirkhah recommended using mitomycin C in all surgical cases of pterygium excision because the amniotic membrane graft contains no live cells. The healing depends on migration of host cells into the membrane. The surrounding host tissue, if not treated with mitomycin C, may still retain abnormal tissue and give rise to recurrence upon invasion into the membrane.

The recurrence rates from pterygium excision surgery using amniotic membrane grafts in conjunction with adjunctive therapy have been improving. A study by Solomon and colleagues that incorporated a larger removal of subconjunctival fibrous tissue with the injection of long-acting steroid (triamcinolone acetonide) showed that amnion grafts achieved a low 3% recurrence rate. Ma and colleagues found a 3.7% recurrence rate.

Surgery for pinguecula is nearly identical to pterygium except that a smaller amount of subconjunctival fibrovascular tissue is removed, according to Tseng and Kheirkhah. Although pinguecula are usually of no concern to most patients, in some they may produce an annoying redness and irritation or interfere with contact lens wear. They are formed by hyaline degeneration and proliferation of elastic fibers of the underlying substantia propria.

Conjunctivochalasis

Conjunctivochalasis: This condition often interferes with proper blinking and can create an unstable tear film.

Conjunctivochalasis is a redundant, loose, non-edematous conjunctiva between the globe and eyelid that tends to be bilateral, occurs in older patients and is often an overlooked cause of ocular irritation, according to Meller and Tseng. Although conjunctivochalasis usually involves the infra-temporal conjunctiva, it can spread to the superior bulbar conjunctiva giving rise to superior limbic keratoconjunctivitis, according to Yokoi and colleagues.

Conjunctivochalasis often interferes with proper blinking and can create an unstable tear film, according to Meller and Tseng. It can lead to exposure-related problems such as nocturnal lagophthalmos and dellen formation and to subconjunctival hemorrhages, according to Di Pascaule and colleagues.

Mild cases of conjunctivochalasis may respond to lubrication with artificial tears. For more refractory cases, antihistamine eye drops, topical corticosteroids or patching the eye shut at bedtime to reduce nocturnal exposure may all be advised. Small areas of redundant tissue may be excised. For excised areas that leave larger defects, an amniotic membrane may be used to reconstruct the conjunctival surface. In a study by Meller and colleagues, 40 patients (47 eyes total) reported improvement in most symptoms including epiphora and ocular irritation after amniotic membrane grafting for conjunctivochalasis

Conjunctival reconstruction

The conjunctiva is an integral part of the ocular surface. In some disease states and trauma cases a considerable amount of conjunctiva is damaged or lost. In such cases, amniotic membrane grafts may help rehabilitate the ocular surface.

When implanted as a graft in areas with conjunctival tissue defects, amniotic membrane helps restore a normal stroma and provides a suitable basement membrane for new epithelial proliferation and differentiation, according to Gris and Güell.

In addition to being used successfully to treat pterygium, pinguecula and conjunctivochalasis, amniotic membrane has been used for conjunctival fornix reconstruction after the excision of large conjunctival tumors, palpebral surgery, orbital surgery, conjunctival scarring and symblepharon (Dua and colleagues, Gris and Güell).

Symblepharon is commonly seen in cases of Stevens-Johnson syndrome and after thermal or chemical globe injuries (Meller and colleagues, Tseng and Blanco). When symblepharon develops in the superotemporal fornix, the blockage of tears from the lacrimal gland can cause severe dryness. If symblepharon occurs in the inferior fornix, nocturnal corneal exposure may develop due to loss of the Bell’s phenomenon during sleep.

Other pathogenic elements of symblepharon include cicatricial entropion with trichiasis, dry eye because of disrupted lid blinking, restricted ocular motility, cicatricial ptosis and socket contraction, which can interfere with contact lens wear, according to Tseng and Blanco.

Acute severe chemical or thermal burns can also cause scleral thinning and melting secondary to scleral ischemia. Scleral melt can also occur after pterygium excision if excessive cautery is applied to the sclera, or if adjunctive therapies such as beta-irradiation or mitomycin C are used, according to Tsai and colleagues.

Amniotic membrane is effective for reconstructing the conjunctiva even over ischemic sclera, provided the surrounding conjunctiva is viable and vascularized, according to Gris and colleagues. If there is severe scleral melting to the point of impending globe perforation, the clinician can combine the amniotic membrane with a scleral graft to add scleral reinforcement as necessary, according to Gris and colleagues.

Miscellaneous indications

Amniotic membrane can be used to close leaking blebs after glaucoma filtering surgery, according to Budenz and colleagues. Leaking blebs are most common in glaucoma surgery cases if mitomycin C was also used during the procedure.

Amniotic membrane has been found to be effective for managing descemetoceles, various noninfectious corneal ulcers and as a patch over the puncta to act as a punctal occluder (Dua and colleagues, Solomon and colleagues).

For more information:

• Leonid Skorin Jr., OD, DO, FAAO, FAOCO, practices in Albert Lea, Minn., and writes and lectures on ocular disease and neuro-ophthalmic disorders. He underwent fellowship training in neuro-ophthalmology. He may be contacted at the Albert Lea Eye Clinic, Mayo Health System, 1206 W. Front St., Albert Lea, MN 56007; (507) 373-8214; fax: (507) 373-2819; e-mail: skorin.leonid@mayo.edu. Dr. Skorin has no financial interest in the products mentioned in this article, nor is he a paid consultant for any companies mentioned.

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