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Amniotic Membrane Graft

Editor: Koushik Tripathy Updated: 8/25/2023 3:04:39 AM


The human amniotic membrane grafting is used as an adjunctive procedure across surgical specialties and in translational medicine to cultivate stem cells. It is easily available, and techniques of preparation and storage are relatively inexpensive. Experts use amniotic membrane graft (AMG) in multiple specialties, including ophthalmologists, dentists, urologists, burn specialists, ear, nose, and throat surgeons, gynecologists, and researchers in stem cell technology.[1][2][3]

Anatomy and Physiology

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Anatomy and Physiology

AMGs are Used in Ophthalmology

  • As a substrate for the growth of epithelial cells
  • For tectonic support in cases of small perforations and thinning of the cornea or sclera
  • To cover large areas of corneal and conjunctival epithelial defects

AMG has multiple advantages over other similar materials, including pericardium, fascia, and mucous membranes.

AMG is:

  • Easily available in large quantities[2][3]
  • Transparent/semi-transparent
  • Nonimmunogenic
  • Good substrate for epithelial cell growth
  • Cosmetically acceptable
  • AMG provides a smooth surface to cover raw areas

Disadvantages of AMG include:

  • Biological membrane
  • Tectonically, it is not very strong
  • Possibility of infection
  • Possible transmission of hepatitis, HIV (human immunodeficiency virus), and coronavirus disease (COVID-19)

Properties of the Human Amniotic Membrane

The outer chorion and inner amnion make up the human placenta. The amnion is 20 to 50 microns thick and has an outer cuboidal epithelium, basement membrane, and inner stroma. The stroma has an inner compact layer, a middle fibroblastic layer, and an outer spongy layer. The basement membrane of the amniotic membrane has similar properties to the conjunctival basement membrane but differs from the corneal subepithelial basement membrane and can be an effective substitute for the conjunctival basement membrane.[4]

Lack of immunogenicity and the promotion of epithelial growth are favorable properties of AMG. AMG expresses anti-inflammatory, anti-microbial, and anti-fibrotic factors.[4][5] The use of AMG in regenerative medicine as a scaffold has been studied, and it has been found to be effective in many clinical trials.[1]

The structural and regenerative features of AMG have been discussed in detail in the literature.[6] However, the lack of tensile strength and probable transmission of infection can limit its usage.[7]

Anti-inflammatory properties of AMG are due to the secretion of many factors such as:

  • Transforming growth factor-beta (TGF β)
  • Fibroblast growth factor
  • granulocyte colony-stimulating factor (GCSF)
  • Interferon-gamma (IFNγ)
  • Interleukin 1 alpha (IL-1α) and beta (IL-1β)
  • Interleukin 4 (IL-4)
  • IL-12p40
  • IL-12p70
  • IL-15
  • IL-16
  • IL-17[8][9]

AMG also contains regulators of soft tissue healing (growth factor or GF), including:

  • Basic fibroblast GF (bFGF)
  • Beta nerve GF (β-NGF)
  • Epidermal GF (EGF)
  • Fibroblast GF 4 (FGF-4)
  • Hepatocyte GF (HGF)
  • Growth hormone (GH)
  • Insulin-like GF 1 (IGF-I)
  • Insulin-like GF-binding protein 1, 2, 3, 4, and 6 (IGFBP-1,2,3,4,6)
  • Platelet-derived GF AA (PDGF-AA) and BB (PDGF-BB)
  • Vascular endothelial GF (VEGF)
  • Transforming GF-alpha (TGF-α) and beta 1 (TGF-β1)[8][9]


Use of AMG in Ophthalmology

The uses of AMG in ophthalmology include[3]:

1) Corneal diseases:

  • Chemical injuries:
    • Acute chemical injuries with a corneal epithelial defect[10]
    • As a basement membrane for allo-limbal graft /SLET (simple limbal epithelial transplant) in acute chemical burns[11]
    • As a basement membrane for auto-limbal graft /SLET in chronic cases for LSCD (limbal stem cell deficiency)[12]
  • In the acute stage of Stevens-Johnson syndrome (SJS)[13]
  • For surface healing/ perforations/ thinning in long-standing SJS
  • Bullous keratopathy
    • Aphakic/pseudophakic bullous keratopathy for pain relief with or without anterior stromal puncture[14]
    • Epithelial defects
      • Persistent epithelial defects[15]
      • Neurotrophic ulcers
      • Recurrent corneal erosions
      • Non-healing ulcers [16]
  • Small perforations and thinning
    • Multi-layered AMG for tectonic purposes[7]
  • After superficial keratectomy for
    • Salzmann nodular degeneration 
    • Climatic droplet keratopathy[17]
    • Superficial corneal scars [18]

2) Conjunctiva

  • Chemical and thermal injuries
    • To help the healing of epithelial defect in acute burns[19]
    • Surface reconstruction in chronic cases[16]
  • After trabeculectomy to repair leaking cystic bleb with bleb-thinning using subconjunctival AMG draping with the advancement of the conjunctiva[20]
  • Surface healing in acute SJS[13]
  • After pterygium excision to cover the bare sclera[21]
  • After OSSN (ocular surface squamous neoplasia) excision to cover the bare sclera[22]
  • Fornix and eyelid margin reconstruction[23]
  • Symblepheron release[23]

3) Socket reconstruction[23]

4) Cultivation of limbal stem cells[24][25]

  • In vivo as SLET[24]
  • In vitro as CLAT (conjunctival limbal autograft transplantation)[25] or COMET (cultured oral mucosal epithelial transplant)[26]

5) Retinal disorders

  • To aid in the closure of recurrent macular holes, post-traumatic chronic macular holes, and recurrent macular holes in pathological myopia[27][28][27][29]
  • To promote the repair of retinal breaks[27]


In cases of large corneal perforations, AMG will not be able to provide tectonic support.[30]

Iritis with hypopyon has been reported after amniotic membrane grafting, which may be sterile in nature and may respond to topical steroids.[31] Repeat amniotic membrane graft from the same donor can cause hypersensitivity reactions. Hence if AMG is required to be repeated, it preferable to get AMG from a different donor.[32]


Equipment and tissue needed for the preparation and transport of AMG include:[33]

  • Dulbecco's modified Eagle's medium
  • Nitrocellulose paper
  • -80 degrees C refrigeration
  • Human placenta


The human amniotic membrane is prepared by a technician trained in the procedure under the laminar flow hood or in a sterile environment like the operating room, and AMG is stored in a -80 degrees C refrigerator.


The placenta is obtained after elective cesarean section from mothers who have been tested negative for HIV (human immunodeficiency virus), HBsAg (hepatitis B surface antigen), HCV (hepatitis C virus), syphilis, and during the COVID era, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). The placenta is taken under a laminar flow hood with all sterile precautions. Blood clots are removed by rinsing it with a balanced salt solution (BSS), which contains 100 µg/ml of neomycin, 50 µg/ml of streptomycin, 50 µg/ml of penicillin, and 2.5 µg/ml of amphotericin B.[34][33][30][35]

Separation of the amniotic membrane from the chorion is performed by blunt dissection. It is then placed on nitrocellulose paper with the epithelial surface up.[34] The membrane is cut into 4 × 4 cm pieces and stored in sterile vials containing Dulbecco's modified Eagle's medium and glycerol at a ratio of 1 to 1 (volume/volume).[33] The vials are frozen at −80 degrees C. Other techniques of preparation are also described in the literature.[36] Instead of BSS, physiological saline or 0.01 M phosphate-buffered saline (PBS) containing 100 mg of dibekacin sulfate can be used to rinse the amniotic membrane.[33] Further rinsing may be done after cutting AMG into pieces with 0.5 M DMSO (dimethyl sulfoxide) dissolved in PBS, then in 1.0 M, and 1.5 M DMSO in PBS, for 5 minutes each.[33] Cryopreservation, freeze-drying of the membrane, and gamma irradiation are other methods to preserve the amniotic membrane.[37][36][38]

Technique or Treatment

Amniotic membrane graft is placed epithelial side up on the ocular surface and cornea. The identification of the stromal side is by noting the stickiness. The membrane may be placed as inlay to fill the areas of stromal thinning as one layer or as a multilayer graft. An amniotic membrane may also be used as a bandage to cover epithelial defects as an onlay. The membrane may be spread across the ocular surface and tucked into the fornices in cases of chemical injury, and held in place by a conformer. The amniotic membrane may be used as a substrate for the placement of limbal stem cells.[3][19]

In all these procedures, the amniotic membrane may be fixed with 10-0 nylon sutures or 8-0 polyglactin 910 sutures to the ocular surface. Fibrin glue may also be used to anchor the membrane.


Early loss of membrane may occur if it is not anchored securely. Keratitis due to contamination of the membrane may also occur. Sterile hypopyon has been reported after amniotic membrane grafting in a patient with pseudophakic bullous keratopathy.[31] The hypopyon and iritis in this patient responded well to periocular steroids.[31] On the whole, there are no major complications known to occur with amniotic membrane graft. Rarely HIV, HBsAg, or HCV may be transmitted by amniotic membrane graft.[31] 

Clinical Significance

The membrane is taken out from the refrigerator, left to thaw for 10 minutes, rinsed in BSS, and used.[16] Wet membranes are preferred over dry membranes.[34][37] Freeze-dried, gamma-irradiated, or cryopreserved membranes need to be thawed before usage.

Amniotic membranes can be glued or sutured to the surface. The use of fibrin glue has made the use of an amniotic membrane simpler. A bandage contact lens may be placed on the ocular surface to prevent the slippage of the membrane.

The membrane disintegrates partially and is adsorbed on to the surface. It is known to reduce surface scarring, angiogenesis, and surface inflammation.[39]

For sealing perforations, multi-layered AMG may be used. For persistent corneal epithelial defect, it is used as an onlay. It may be anchored to the surface by sutures or fibrin glue.[7]

In translational medicine, AMG is used as a substrate in vitro to grow limbal stem cells.[40][41] Epithelial cells are seeded as explants on the amniotic membrane. The epithelial cell explants may be autologous or allogenic. They may be limbal stem cells (cultivated limbal epithelial stem cell transplantation or CLET) or oral mucosal cells (cultivated oral mucosal cells or COMET).[42] Following 360° conjunctival peritomy and pannus removal from the corneal surface in cases of bilateral or unilateral chemical burns, Sjogren syndrome, and other causes of limbal stem cell deficiency, the amniotic membrane with cultured explants (allogenic in bilateral or autologous in unilateral cases) is placed on the ocular surface and sutured or glued.

Results of Amniotic Membrane Grafts

AMG was done after the excision of primary pterygium in 80 eyes of 71 patients by Ma and colleagues.[43] It was compared with retrospective charts of 54 patients who underwent conjunctival autograft and 50 patients who received topical mitomycin C (MMC). After 6 months of follow up, recurrence was 3.8 % in the AMG group, 5.4% in the autograft group, and 3.7% in the MMC group.[43]

Tejwani and coauthors did a retrospective study of 24 acute and 48 chronic cases of chemical and thermal injuries who had undergone amniotic membrane transplantation with an average follow up of 7.8 months.[10] Epithelial defect healed in 94.3 % of cases. Symptomatic relief was noted in 88% of cases, and 55% of cases had improved limbal stem cell function.[10]

Gregory published a series of 79 patients with acute SJS who were hospitalized.[13] He classified them into mild, moderate, severe, or very severe ocular involvement based on the amount of epithelial sloughing of the cornea, bulbar conjunctival congestion, staining with fluorescein dye, and lid margin involvement. Twenty-eight patients who had severe and 10 patients who had very severe ocular involvement had received an amniotic membrane graft. Some patients needed AMG more than once. Patients were followed up for 3 months. They showed minimal symptomatic of dry eyes and good visual acuity. He proposed that cases classified as severe or very severe ocular SJS must have AMG within 7 to 10 days of hospitalization to avoid long term sequelae.[13] 

When used for pain relief in cases of pseudophakic bullous keratopathy, Pires and colleagues reported freedom from pain in 50 out of 55 patients for 33 weeks after AMG.[14]

The amniotic membrane has also been used to help in sealing small corneal perforations and aid in healing persistent corneal epithelial defects.[7][15]

Shimazaki et al. used living related donors for allogenic CLET in six patients and cadaveric donors in seven patients.[25] Re-epithelization of the surface was noted in 8 eyes within a mean period of 19.9 days. They did not find any difference in success rates between the living-related and the cadaveric donors in severe limbal stem cell deficiency cases[25]

In a similar study, Daya et al. reported results of ex vivo cultured limbal epithelial cells from corneoscleral rims discarded after taking the donor button for penetrating keratoplasty. There were 10 patients with severe limbal stem cell deficiency in this study, after an average follow-up of 28 months. The parameters of LSCD improved in 7 eyes, and vision improved in 4 eyes.[44]

Cultivated oral mucosal epithelial cells (COMET) and cultured allogenic limbal stem cells were noted to have a similar clinical improvement in bilateral LSCD.[45] The advantages of COMET include no need for immunosuppression as it is autologous. However, the rates of graft failure and persistent corneal epithelial defects may be higher compared to allogenic CLET.[45]

Recently, in vivo cultivation of limbal stem cells in the form of simple limbal epithelial stem cell transplantation (SLET) has been used in unilateral cases of LSCD.[46] The technique involves harvesting one clock hour of limbal tissue from the same patient's donor eye and, after pannus removal and conjunctival peritomy, gluing bits of the limbal tissue on an amniotic membrane graft placed on the cornea. This technique removes the necessity of an expensive stem cell laboratory. It is reproducible and has a high success rate in terms of reepithelialization of the cornea and conjunctiva, and the restoration of the transparency of the cornea and visual acuity with or without the need for corneal transplantation.[12]

Enhancing Healthcare Team Outcomes

Amniotic membrane grafting needs interprofessional collaboration. Gynecologists provide the placenta after routine uncomplicated lower segment cesarian section delivery. The preparation of the membrane is by the nursing staff or eye bank technicians under the laminar flow hood. The membrane is stored in an -80 degree C refrigerator. Transportation has to maintain a cold chain. Interprofessional collaboration between various specialists, including gynecologists, nurses, eye bank technicians, pharmacists, ophthalmologists, translational medicine specialists, dentists, ear nose and throat specialists, and urologists, is needed to ensure optimum patient care.

Nursing, Allied Health, and Interprofessional Team Interventions

 Storage, distribution, and hygienic maintenance of the human amniotic membrane is done by the nursing staff and eye bank professionals under the guidance of the ophthalmologist or clinician.

Nursing, Allied Health, and Interprofessional Team Monitoring

The temperature of the refrigerator where the membrane is stored in the freezer compartment must be monitored. The amniotic membrane is stored for long term use in a -80°C freezer before distribution to various centers. The maintenance of the temperature and sterility of the AMG must be ensured before use for patient safety.



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