Published in Ocular Surface

Treatment Primer for Utilizing Amniotic Membrane Tissue

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19 min read

Read on to get a broad overview of amniotic membranes, including ocular surface conditions optometrists can treat with them, treatment protocols, and more.

Treatment Primer for Utilizing Amniotic Membrane Tissue
Over the years, the treatment of ocular surface disease has evolved. Traditionally, treatments have included topical medications such as corticosteroids, cyclosporine, and artificial tears.
However, long-term use of such medications can result in side effects, including neurotoxicity from preservatives (such as benzalkonium chloride [BAK]) found in non-preservative-free drops.1 Furthermore, there is always a risk of premature cataract development, elevated intraocular pressure (IOP), and potential glaucoma development from long-term corticosteroid use.2
Therefore, amniotic membranes (AM) are now a potential treatment for certain ocular surface diseases due to their minimal risk of rejection and healing properties. This article will review the special qualities of amniotic membranes, the many ocular diseases they can treat, and the various types of membranes and treatment protocols.

Overview of amniotic membranes

An amniotic membrane consists of the innermost layer of the placenta and contains a thick basement membrane and avascular stromal matrix.3 It does not express HLA-A, -B, or -DR antigens and, therefore, presents no risk of immunological rejection.4
Furthermore, it is fairly easy to obtain and essentially has unlimited availability as the tissue is harvested from elective, vetted cesarean sections. The tissue can be stored for several months to a year and is therefore ready to use for surgery and treatment of ocular surface disease when needed.5

Properties of amniotic membranes

Amniotic membranes have demonstrated many beneficial qualities, including anti-inflammatory, anti-scarring, and pro-epithelization effects. Below, we will discuss in more detail the exact mechanism of amniotic membranes.

1. Anti-inflammatory

Amniotic membranes have been shown to promote apoptosis of pro-inflammatory neutrophils and macrophages,6 enhance phagocytosis of apoptotic neutrophils by macrophages,7 and suppress activation of CD4+ cells that can promote inflammation.8

2. Anti-scarring

The stromal matrix of the amniotic membrane has demonstrated an anti-scarring effect by suppressing transforming growth factor (TGF)-beta signaling.9 TGF-beta contributes to the development of scar formation by stimulating the expression of key extracellular matrix (ECM) components and inhibiting matrix metalloproteinases (MMPs) in fibroblasts.10 TGF-beta 1 and TGF-beta 2 transcripts were shown to be downregulated in human corneal and limbal fibroblasts cultured on the stromal side of the amniotic membrane.11

3. Promotes epithelization

An amniotic membrane serves as a transplanted basement membrane that acts as a new healthy substrate to promote epithelialization. Furthermore, it has been discovered that amniotic membranes produce various growth factors, such as basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor, which can all stimulate epithelization.12

History of the amniotic membrane

The first documented use of an amniotic membrane was in 1910 by Dr. John Staige Davis. He attempted to use the lining of an amniotic membrane as a skin graft.13 Later, the first documented use for the eye was by Dr. Andrew de Rötth in 1940 for conjunctival reconstruction.14
Unfortunately, both were unsuccessful in their attempts. Therefore, the amniotic membrane was not seen in ophthalmic literature until around 1995, when research from Drs. Kim and Tseng demonstrated the successful use of amniotic membranes for ocular surface reconstruction in damaged corneas of rabbit models.15 Since then, it has gained rapid popularity in the ophthalmological field.

Ocular uses for amniotic membrane

Since Kim and Tseng’s successful use of an amniotic membrane for ocular surface reconstruction, this tissue has been successful in treating a myriad of ocular diseases.
Some of the main ones are discussed in further detail below:

Ocular graft vs. host disease

Graft vs. host disease (GVHD) is an abnormal immune response following stem cell transplantation for the treatment of various hematological diseases. It can lead to various ocular complications, the most common being dry eye disease (DED), which is seen in 40 to 70% of patients with GVHD.16
There are two forms of GVHD, acute and chronic. Acute GVHD is most commonly seen within the first 100 days after transplantation and resembles a toxic-like syndrome, whereas chronic GVHD is seen after 100 days of transplantation.17
GVHD can affect many ocular structures, including the lacrimal and meibomian glands, conjunctiva, and cornea. The most commonly used therapies for managing ocular GVHD (oGVHD) have been topical immunosuppressants and corticosteroids. However, these treatments have been associated with neurotoxic effects, steroid-induced glaucoma, cataracts, and have shown poor tolerance.18
Due to AM’s anti-inflammatory, anti-scarring, and regenerative properties, it has become a favorable treatment option for ocular GVHD. A molecule found in cryopreserved membranes known as HC-HA/PTX3 was found to preserve tear secretion and conjunctival goblet cells as well as suppress infiltration of inflammatory/immune cells in lacrimal glands in mouse models.19
Later studies have shown that both sutured AM and non-sutured AM have been reported successful in treating acute and chronic oGVHD by accelerating corneal epithelialization and reducing conjunctival inflammation.20,21

Ocular trauma (chemical burns/abrasions)

Amniotic membranes have been found useful in treating ocular chemical burns. In chemical burns grades I to III, amniotic membranes alone have been sufficient to restore corneal and conjunctival surfaces.22,23
In severe burns, grade IV or higher, AM restored conjunctival surface and reduced stromal inflammation, but did not prevent limbal stem cell deficiency and required limbal stem cell transplantation.24 Note the grading system is based on the Roper-Hall classification system (see Table 1 below).
Table 1 outlines the Roper-Hall classification system for ocular surface chemical burns.
GradePrognosisCorneaConjunctival Limbus
IGoodCorneal epithelial damageNo limbal ischemia
IIGoodCorneal haze, iris details visible<⅓ limbal ischemia
IIIGuardedTotal epithelial loss, stromal haze, iris details obscured⅓ to ½ limbal ischemia
IVPoorCornea opaque, iris and pupil details obscured>½ limbal ischemia
Table 1: Courtesy of Meller et al.
In cases of large and central corneal abrasions, there is an increased likelihood of infection and scarring, which could threaten vision.25 Amniotic membranes have been shown to be extremely effective in corneal re-epithelization and anti-scarring effects, which help to increase final visual outcomes.26

Stevens-Johnson syndrome

Stevens-Johnson Syndrome is a rare disease characterized by the presence of epidermal and mucosal bullous lesions.27 Its exact pathophysiology is unknown, but the majority of cases are due to a delayed drug hypersensitivity reaction to a medication.28
The most common ocular finding is bilateral conjunctival hyperemia with purulent discharge at initial presentation.29 As the disease progresses, inflammatory changes to the ocular surface may lead to the development of conjunctival ulcerations, pseudomembrane formation, corneal ulceration, and epithelial sloughing.30
Amniotic membrane/grafting has been used to successfully treat corneal epithelial defects and lid margin involvement by placing a graft over the entire ocular surface, including the bulbar and palpebral conjunctiva, and securing it to the lid margin.31 Furthermore, AMs have been found to help control immune-mediated inflammation prior to surgery by reducing surface inflammation, decreasing the severity of vascularization, and reducing the risk of recurrent erosions.32

Neurotrophic keratitis (persistent epithelial defects/ulcers)

Neurotrophic keratitis (NK) is a degenerative disease of the corneal epithelium and stroma caused by damage to trigeminal nerve innervation that impairs corneal sensitivity.33 Therefore, tearing and blinking reflexes, as well as wound healing, are compromised as a result of impaired corneal nerve function, and can result in persistent epithelial effects and severe DED.34
Amniotic membranes have proven to help heal persistent epithelial defects (PED) by providing a basement membrane substrate for the migration and adhesion of epithelial cells. Furthermore, it acts as a shield for the epithelium from the abrasive effects of an abnormal palpebral conjunctiva.35
Cryopreserved AM have been successful in treating corneal epithelial defects and ulcers caused by NK. AM grafts demonstrated a success rate of approximately 89% in achieving rapid and complete healing of defects and/or ulcers in an average of 18 days after surgery.36,37
Furthermore, AM has been shown to successfully manage signs and symptoms of moderate-to-severe DED related to NK. After placing an amniotic membrane on the eye for approximately 4 to 5 days, the overall severity of DED was reduced from Dry Eye Workshop (DEWS) classification level 3 to DEWS level 1 at both 1 and 3 months, as well as a significant decrease in corneal staining and increase in corneal nerve density.38,39
Table 2 outlines the DEWS classification system for dry eye severity.
Dry Eye SeverityLevel 1Level 2Level 3Level 4
Discomfort; severity and frequencyMild and/or episodic; occurs under environmental stressModerate, episodic, or chronic; stress or no stressSevere; frequent or constant without stressSevere and/or disabling; constant
Visual symptomsNone or episodic mild fatigueAnnoying and or activity-limiting episodicAnnoying, chronic and/or constant, limiting activityConstant and/or possibly disabling
Conjunctival injectionNone to mildNone to mild+/-+/++
Corneal staining (severity/location)None to mildVariableMarked centralN/A
Corneal/tear signsNone to mildMild debris, I meniscusFilamentary keratitis, mucus clumping, tear debrisFilamentary keratitis, mucus clumping, tear debris ulceration
Lid/meibomian glandsmeibomian gland dysfunction (MGD) variable presentMGD variably presentMGD frequentTrichiasis, keratinization, symblepharon
Tear film break-up time (seconds)Variable≥10≥5Immediate
Schirmer score (measures tear secretion, mm/5 minutes)Variable≥10≥5≥2
Table 2: Courtesy of Dry Eye Workshop.

Conjunctival/pterygium surgery

AM have been used as an alternative to conjunctival grafts during pterygium surgery. Although conjunctival grafts have been shown successful in reducing recurrent rates,40,41 concerns exist about the potential risk of affecting the outcome of any future glaucoma-filtering surgery.40
Furthermore, for those with advanced pterygia with wide conjunctival involvement, conjunctival grafts may be limited by a lack of remaining healthy cells.40 Prabhasawat et al. found that although AM were less proficient than conjunctival autografts in reducing recurrence rates, the recurrence rate was still significantly lower than that of primary closure and was capable of achieving a higher rate of satisfactory final appearance.42
As mentioned earlier, Drs. Kim and Tseng were one of the first to demonstrate that amniotic membranes could be used for ocular diseases.15 They discovered a high success rate of corneal surface reconstruction in rabbit corneas. This was later supported by Paridaens et al., who used an amniotic graft to cover a conjunctival wound and provide a substrate for migration of conjunctival epithelium cells.43

Dry eye disease

Amniotic membranes are now being used to treat dry eye disease. It not only helps to keep the eye moist and act as a bandage lens against blinking, but it can reduce ocular inflammation, which is one of the major underlying causes of DED.
The DREAM study found that in patients with moderate-to-severe dry eyes, the overall DEWS score was significantly reduced after treatment with AM, even after 3 months of treatment. Ocular discomfort and corneal staining improved at 3 months post-treatment.39
Furthermore, amniotic membranes were found a year earlier in a study by John et al. to significantly increase corneal nerve density and improve corneal sensitivity. This is an important finding since the corneal nerve plexus is responsible for regulating corneal sensitivity, blink reflex, and tear production. Corneal nerve regeneration was shown to restore the corneal surface as evidenced by a decrease in corneal punctate staining and improvement of tear break-up time (tear film stability).38

Types of amniotic membrane: dehydrated vs. cryopreserved

There are two major categories of amniotic membranes: dehydrated (AmbioDisk, AcellFX, Eclipse, Biovance 3L Ocular, Aril) and cryopreserved (PROKERA, AmnioGraft/AmnioGuard). To date, Prokera is the only amniotic membrane that is FDA-cleared with an FDA designation as being anti-inflammatory, anti-fibrotic, and anti-angiogenic along with supporting epithelial adhesion and differentiation.

There is a new type of AM emerging (XcellerEYES) known as lyophilized AM, which has characteristics of both dehydrated and cryopreserved membranes.
Although all types have been utilized for ocular diseases, it is important to understand the key differences between them before implementation.

Preserving and storing amniotic membranes

Dehydrated membranes are preserved using a vacuum with low-temperature heat, which helps to retain devitalized cellular components. They can be stored at room temperature and have a 5-year shelf life.5
Cryopreserved membranes involve slow freezing at -80° Celsius using DMEM/glycerol preservation media to allow for slow-rate freezing. This type of membrane must be stored at -80° Celsius and, therefore, requires a special freezer set at this temperature. The shelf life is approximately 2 years.44
Lyophilized membranes’ preservation method consists of removing water from a tissue by sublimation. This results in the inhibition of chemical reactions that result in tissue alteration, thus keeping the structure intact. These membranes can be stored at room temperature for a couple of years.45

Treatment protocols for amniotic membranes

Dehydrated/lyophilized lenses involve removing the membrane from the packaging. There is no ring to hold the membrane in place on the cornea, so a bandage lens is placed overtop. Some brands, such as AmbioDisk and XcellerEYES, must be placed with a certain side down. Others, such as AcellFx, Eclipse, and Biovance 3L Ocular are bidirectional.
Cryopreserved membranes (Prokera) require rinsing with saline for about 5 to 10 seconds to remove the potentially irritating storage solution. The membrane is held in place by a PMMA ring. The concave side of the ring is placed face down onto the cornea by first sliding the membrane under the upper lid and then under the inferior eyelid. Some practitioners choose to tape the upper lid to help with discomfort.
The membrane can stay on the eye anywhere from 1 to 7 days, depending on severity of injury. The membrane will dissolve over time and the ring is later removed. AmnioGraft/AmnioGuard is a type of cryopreserved amniotic membrane that does not have a ring and comes in various sizes. It is mainly reserved for conjunctival tumors, reconstructions, and glaucoma blebs.44

Structural and biological differences between amniotic membranes

Due to the difference in the preservation process of dehydrated versus cryopreserved membranes, the biological and structural components may be altered. It has been found that the extracellular matrix structure (ECM) was compromised by dehydration but not by cryopreservation. This is important since the ECM acts as a reservoir and modulator of cytokines and growth factors.46
Rodríguez-Ares et al. found that lyophilization maintained the histological structure of AM, but seemed to cause greater reductions in total protein amount and growth factor concentration than cryopreservation.45
A complex unique to cryopreserved membranes known as “HC-HA/PTX3” was isolated by Drs. He and Tseng in 2009. This molecule was found to be the main source of healing properties of amniotic membranes.47 The lack of HC-HA/PTX3 in dehydrated membranes is thought to contribute to the decreased effectiveness in reducing inflammation and fibrosis.46 Whether or not HC-HA/PTX3 is found in lyophilized amniotic membranes has yet to be found in literature.

Side effects and patient education

Although there are very few side effects/reactions to the amniotic membrane itself, one of the major side effects is reduced vision. Therefore, it is important to only treat one eye at a time. Prokera is contraindicated in patients with allergies to ciprofloxacin and/or amphotericin B as they are used in the storage solution.
Furthermore, with cryopreserved membranes with rings, patients may experience mild to moderate discomfort. If extreme burning, pain, and/or irritation are experienced by the patient, it is important to educate the patient to immediately call the office.
It is also important to explain where the membrane has come from in case of religious or ethical concerns. Also, the patient must be made aware that the membrane may not be covered by insurance and of any potential out-of-pocket cost (usually varies between $1,200 and $1,500).

Early intervention with amniotic membranes

Lastly, the timing of intervention is crucial for the effectiveness of the membrane healing process. In acute ocular disorders, such as corneal abrasions and ocular chemical burns, same-day placement of the amniotic membrane is ideal to reduce the risk of scarring and improve final visual outcomes.
Dr. Tseng recommends that the amniotic membrane be placed within 2 weeks of the injury, and the first week is better than the second. After 2 weeks, the corneal damage is irreversible, and amniotic membrane transplantation would have a limited effect.34
Furthermore, it is much easier to treat DED in the mild stages compared to moderate-to-severe stages. Amniotic membranes have been shown to be effective in treating all levels of dry eye stages.47,48

In conclusion

Amniotic membranes are becoming more widely accepted as a beneficial and viable choice for certain ocular surface conditions. There are multiple types of AM to choose from, and they are readily available for use as they can be stored for many years in-office.
So next time a patient walks in with one of the ocular diseases mentioned above, strongly consider using an amniotic membrane; you might just save their vision.
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Laura Goldberg, OD, MS, FAAO, Dipl ABO
About Laura Goldberg, OD, MS, FAAO, Dipl ABO

Dr. Goldberg is currently an associate optometrist at Woolf Eye Lab in Pasadena, MD. She completed a residency in Primary Care & Ocular Disease at VAMC Wilmington, DE, and graduated from New England College of Optometry, Class of 2016. For her MS in Vision Science, she studied possible causes of developmental progression of myopia.

Myopia control has become a passion of hers, and she offers myopia control therapy to patients in-clinic. In addition to her passion for optometry, she enjoys traveling and experiencing many cultures and customs. Ultimately she envisions her career unfolding at the nexus of all three optometric specialties; clinical work, research, and teaching, in order to facilitate continuing advancements in patient care.

Laura Goldberg, OD, MS, FAAO, Dipl ABO
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