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Introduction

Amniotic membrane consists of a combination of tissue and cells, which when used as a biological dressing, promotes re-epithelisation and wound healing by acting as a foundation for re-growth of soft tissue. Biologically active cells in the epithelial and stromal layers deliver growth factors and cytokines with anti-inflammatory, anti-bacterial, anti-immunogenic and anti-fibrotic properties1.
Amniotic membrane is used in a surgical setting and is attached to the surgical site with sutures or tissue glue.    

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Anatomy and Physiology of the Amniotic Membrane

Fig. 01 Right: Origin of the human Amniotic Membrane6

The amniotic membrane develops from the extra-embryonic tissue consisting of foetal and maternal components which are kept together by the chorionic villi. The foetal component consists of the amniotic and chorionic foetal membranes, which separates the foetus from the endometrium4. The amniotic membrane (AM) is the innermost layer of the foetal membranes of the placenta. It is avascular and has an epithelial layer with a sub-adjacent avascular stromal layer5.

The amniotic membrane is one of the thickest membranes in the human body.

The membrane consists of five layers: (I) the cuboidal epithelium layer; (II) the basement membrane; (III) compact layer; (IV) fibroblast layer; and (V) intermediate (spongy) layer.7

Origin-of-the-amniotic-membrane
Cross section of the human Amniotic Membrane indicating the biochemical characteristics of the section

Fig. 02: Cross section of the human Amniotic Membrane indicating the biochemical characteristics of the section.8

The basement membrane is a thin layer composed of reticular fibres, closely adherent to the amniotic epithelium, while the adjacent compact layer is more dense and devoid of cells consisting of a complex reticular network.3,4 It has low immunogenic potential and contains bioactive factors that have been shown to be beneficial in wound treatment, such as: collagen, cell-adhesion bio-active factors (fibronectin, laminins, proteoglycans and glycosaminoglycans) and growth factors.3,7

The following fibroblastic layer is thickest of the five layers consisting of fibroblasts embedded in a loose reticular network.

The outer spongy layer forms the interface between amniotic and chorionic membranes, consisting of wavy reticulum bundles bathed in mucin.

Amniotic membrane tissue shows little to no HLA-A, B, C antigens or β2 microglobulins and is therefore immunologically inert.3

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Characteristics9–13

The following therapeutic benefits are derived from the biological factors and cytokines:

Anti-inflammatory effects

  • Amniotic membrane suppress the pro-inflammatory cytokines IL-1α and IL-1β11,14
  • Amniotic membrane produce natural metalloprotease (MMP’s) inhibitors11,15

Analgesic properties (absence of pain)

  • Rapid Pain Relief [1,25] due to efficient covering of the nerve endings

Anti-microbial

  • The amniotic membrane serves as a physical barrier against the external environment with close adherence of the membrane to the wound surface.
  • Amniotic membrane produce β-defensins, secretory leukocyte proteinase inhibitor (SLPI) and elafin16,17.

Anti-angiogenic (Prevents the formation of new blood vessels)

  • Hao et al, used reverse transcriptase polymerase chain reaction to identify anti-angiogenic chemicals, such as Thrombospondin-1 and endostatin, expressed by the amniotic epithelial cells. [3,11]
  • In addition tissue inhibitors such as metalloproteinase, TIMPs-1,2,3,4 has also been isolated from the amniotic epithelium as well as the potent anti-angiogenic factor PEDF (Pigment epithelium-derived factor). [26]

Anti-scarring and Anti-adhesive activity

  • Amniotic membrane also reduces protease activity via the secretion of TIMP’s (tissue inhibitors of metalloproteinases) and therefore has an anti-fibrotic effect.
  • TGF-ß is down regulated which is responsible for the activation of fibroblasts and prevent the adhesion of injured surfaces to each other 18–21.

Protection against loss of fluids and proteins

  • Intact collagen matrix provides structure for cellular migration and proliferation.
  • Contains collagen types IV, V and VII which promote cellular differentiation and adhesion.

Non-immunogenic and low antigenicity

  • Due to absence of viable epithelium cells.
  • Low or lack of expression of Histocompatibility (HLA Class II) antigens A, B, C DR or beta2 microglobulin 22–24.

Promotion of Epithelialisation

  • The basement membrane side of the amnion is a good substrate for re-epithelialisation and the maintenance of epithelial cell polarity.
  • Expression of growth factors such as KGF, b-FGF, HGF and TGF-ß that promotes epithelialisation, cell proliferation and differentiation.
  • Provides a new collagen rich basement membrane.
  • Amnion facilitate the migration of epithelial cell and promotes their differentiation.

Mode of Action

The amniotic membrane is used for its high concentrations of cytokines and growth factors. The use of the amniotic membrane to cover inflamed or exposed areas favourably influences the wound healing process, as well as, reducing the levels of pain and discomfort of the patient.3

AmnioMatrix [c][d]™ conforms easily to the wound surface and can be either glued or sutured to the wound surface. The membrane is hydrophilic and naturally absorbs the surrounding fluids. As part of the healing process, AmnioMatrix [c][d]™ resorbs into the wound.27General literature suggests that the membranes completely resorb into the wound in about 14 – 21 days.28

Click here for References
  1. Perepelkin N, Hayward K, Mokeona T et al.; Cryopreserved amniotic membrane as a transplant allograft: viability and post-transplant outcome; Cell Tissue Bank [DOI 10.1007/s10561-015-9530-9] published online: 11 September 2015.
  2. Rahman I, Said DG, Maharajan VS, Dua HS. Amniotic membrane in ophthalmology: indications and limitations. Eye. 2009;23(10):1954–61.
  3. Dua HS, Gomes JA., King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Survey of Ophthalmology. 2004 Jan;49(1):51–77.
  4. Niknejad H, Peirovi H, Jorjani M, Ahmadiani A, Ghanavi J, Seifalian AM. Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater. 2008;15:88–99.
  5. Tyszkiewicz JT, Uhrynowska-Tyszkiewicz IA, Kaminski A, Dziedzic-Goclawska A. Amnion allografts prepared in the Central Tissue Bank in Warsaw. Annals of transplantation: quarterly of the Polish Transplantation Society. 1999;4(3-4):85.
  6. Bourne G. The Fœtal Membranes. Postgrad Med J. 1962 Apr;38(438):193–201.
  7. Jiang A, Li C, Gao Y, Zhang M, Hu J, Kuang W, et al. In vivo and in vitro inhibitory effect of amniotic extraction on neovascularization. Cornea. 2006;25:S36–S40.
  8. Science of Amniotic Tissue [Internet]. [cited 2013 Jun 20]. Available from: http://www.afcellmedical.comimagesamnioticfluid_chart_large.jpg
  9. Ahn J-I, Jang I-K, Lee D-H, Seo Y-K, Yoon H-H, Shin Y-H, et al. A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium. Biotechnology and Bioprocess Engineering. 2005;10(3):262–9.
  10. Baradaran-Rafii A, Aghayan H-R, Arjmand B, Javadi M-A. Amniotic membrane transplantation. Journal of Ophthalmic & Vision Research. 2008;2(1):58–75.
  11. Hao Y, Ma DH-K, Hwang DG, Kim W-S, Zhang F. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea. 2000;19(3):348–52.
  12. Shimmura S, Shimazaki J, Ohashi Y, Tsubota K. Antiinflammatory effects of amniotic membrane transplantation in ocular surface disorders. Cornea. 2001;20(4):408–13.
  13. Riau AK, Beuerman RW, Lim LS, Mehta JS. Preservation, sterilization and de-epithelialization of human amniotic membrane for use in ocular surface reconstruction. Biomaterials. 2010 Jan;31(2):216–25.
  14. Solomon A. Suppression of interleukin 1alpha and interleukin 1beta in human limbal epithelial cells cultured on the amniotic membrane stromal matrix. British Journal of Ophthalmology. 2001 Apr 1;85(4):444–9.
  15. Kim JS, Kim JC, Na BK, Jeong JM, Song CY. Amniotic membrane patching promotes healing and inhibits proteinase activity on wound healing following acute corneal alkali burn. Exp. Eye Res. 2000 Mar;70(3):329–37.
  16. King AE, Paltoo A, Kelly RW, Sallenave J-M, Bocking AD, Challis JRG. Expression of Natural Antimicrobials by Human Placenta and Fetal Membranes. Placenta. 2007 Feb;28(2–3):161–9.
  17. Buhimschi IA, Jabr M, Buhimschi CS, Petkova AP, Weiner CP, Saed GM. The novel antimicrobial peptide β3-defensin is produced by the amnion: A possible role of the fetal membranes in innate immunity of the amniotic cavity. American Journal of Obstetrics & Gynecology. 2004 Nov;191(5):1678–87.
  18. Tseng SCG, Li D-Q, Ma X. Suppression of transforming growth factor‐beta isoforms, TGF‐β receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. Journal of Cellular Physiology. 1999 Jun 1;179(3):325–35.
  19. Lee S-B, Li D-Q, Tan DT, Meller D, Tseng SC. Suppression of TGF-ß signaling in both normal conjunctival fibroblasts and pterygial body fibroblasts by amniotic membrane. Current eye research. 2000;20(4):325–34.
  20. Adzick NS, Longaker MT. Scarless fetal healing. Therapeutic implications. Annals of surgery. 1992;215(1):3.
  21. Cuttle L, Nataatmadja M, Fraser JF, Kempf M, Kimble RM, Hayes MT. Collagen in the scarless fetal skin wound: Detection with Picrosirius-polarization. Wound repair and regeneration. 2005;13(2):198–204.
  22. Aagaard-Tillery KM, Silver R, Dalton J. Immunology of normal pregnancy. Seminars in Fetal and Neonatal Medicine. 2006 Oct;11(5):279–95.
  23. Chen EH, Tofe AJ. A literature review of the safety and biocompatibility of amnion tissue. J Impl Adv Clin Dent. 2010;2(3):67–75.
  24. Bailo M, Soncini M, Vertua E, Signoroni PB, Sanzone S, Lombardi G, et al. Engraftment Potential of Human Amnion and Chorion Cells Derived from Term Placenta. Transplantation. 2004 Nov;78(10):1439–48.
  25. Mermet I, Pottier N, Sainthillier JM, Malugani C, Cairey-Remonnay S, Maddens S, et al. Use of amniotic membrane transplantation in the treatment of venous leg ulcers. Wound repair and regeneration. 2007;15(4):459–64.
  26. Liu J, Sheha H, Fu Y, Liang L, Tseng SC. Update on amniotic membrane transplantation. Expert Rev Ophthalmol. 2010 Oct;5(5):645–61.
  27. Tao H, Fan H. Implantation of amniotic membrane to reduce postlaminectomy epidural adhesions. European Spine Journal. 2009 Apr 30;18(8):1202–12.
  28. Kesting MR, Loeffelbein DJ, Steinstraesser L, Muecke T, Demtroeder C, Sommerer F, et al. Cryopreserved Human Amniotic Membrane for Soft Tissue Repair in Rats. Annals of Plastic Surgery. 2008 Jun;60(6):684–91.

Surgical Application

Ophthalmic indications – used as a substrate to replace the damaged ocular tissue or as a biological dressing in10:

Indications for Corneal Surface Reconstruction
  • Persistent Epithelial Defects.
  • Non-healing Stromal Ulcers.
  • Partial Limbal Stem Cell Deficiency.
  • Total Limbal Stem Cell Deficiency.
  • Bullous Keratopathy.
  • Band Keratopathy.
  • Mooren’s Ulcer
Indications for Conjunctival Surface Reconstruction
  • Pterygium surgery.
  • Chemical Burns.
  • Cicatrizing Conjunctivitis.
  • Ocular Surface Squamous Neoplasia (OSSN).
  • Leaking Blebs.
  • Filtering surgery.
  • Symblepharon release.
  • Fornix Reconstruction.
  • Socket Reconstruction.
  • Entropion correction.
  • Scleral Melt.
Internal Soft Tissue Healing and Anti-Adhesion Barrier
Periodontal Surgery – wound cover for gingival repair
Ear, Nose and Throat
  • Tympanoplasty 29–31
  • Nasal septum repair.
Indications for Conjunctival Surface Reconstruction
  • Pterygium surgery.
  • Chemical Burns.
  • Cicatrizing Conjunctivitis.
  • Ocular Surface Squamous Neoplasia (OSSN).
  • Leaking Blebs.
  • Filtering surgery.
  • Symblepharon release.
  • Fornix Reconstruction.
  • Socket Reconstruction.
  • Entropion correction.
  • Scleral Melt.
Click here for References

10. Baradaran-Rafii A, Aghayan H-R, Arjmand B, Javadi M-A. Amniotic membrane transplantation. Journal of Ophthalmic & Vision Research. 2008;2(1):58–75.

29. Fouad T, Rifaat M, Buhaibeh Q. Utilization of amniotic membrane graft for repair of the tympanic membrane perforation. Egypt J Ear Nose Throat Allied Sci. 2010;11:31.

30. Shojaku H, Takakura H, Okabe M, Fujisaka M, Watanabe Y, Nikaido T. Effect of hyperdry amniotic membrane patches attached over the bony surface of mastoid cavities in canal wall down tympanoplasty. The Laryngoscope. 2011;n/a–n/a.

31. Myringoplasty U. Amniotic Membrane to Temporalis Fascia Graft. Med J Malaysia. 2005;60(5):585.

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Training Videos

Video to illustrate the use of cryopreserved amniotic membrane as a graft in conjunctival surgery.

Video to illustrate Eye Socket Reconstruction Deepening of the Inferior Fornix

Video to illustrate the use of dehydrated amniotic membrane as a graft in Pterygium surgery.

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AmnioMatrix Products

  • AmnioMatrix (c) - Cryopreserved Amniotic Membrane
  • AmnioMatrix (d) - Denuded Dehydrated Amniotic Membrane

AmnioMatrix (c) is a processed, cryopreserved amniotic membrane tissue graft. The processing and preservation methods used by Next Biosciences retains the vital cytokines and growth factors of the amniotic membrane. This has been shown to be of particular value in ophthalmic surgery.

Current research shows that cryopreserved human amniotic membrane has potential in orthopaedic wound covering, and soft tissue barrier to prevent adhesion after primary surgical repair10AmnioMatrix (c) is used in a surgical setting and is attached to the surgical site with sutures or tissue glue.2

Amnion_02

Processing

The amniotic membrane is mechanically separated from the placenta in a clean room environment. The membrane is decontaminated in an antibiotic solution and then either stored on a nitrocellulose membrane carrier, orientated with the stromal side of the membrane on the nitrocellulose paper for AmnioMatrix (c). It is then double packaged in an inner polyethylene pouch and an outer aluminium foil pouch. The packaged membrane is sealed and stored frozen at – 80°C.

The outer pouch can be torn open across the tear notch, allowing the clear inner-pouch to be fished out with sterile forceps to be introduced to the sterile field. Using sterile scissors the pouch can be cut open just under the seal and the graft removed with sterile smooth forceps.

AmnioMatrix (c) is distributed on dry ice (-80 °C). Samples can be used immediately after delivery or can be stored for up to 3 months in a standard home freezer (-20 °C). Once thawed this tissue product must be used immediately.

Storage Location Temperature Use after Receipt
Unopened Insulated Shipping Container
(containing dry ice)
Frozen (-80 °C) Within the expiration date of the dry ice, printed on outer surface of the shipping container
Standard Freezer
(home or general use)
Frozen (-20 °C) Within 3 months of placing the device in the freezer or until expiration date printed on outer product packaging, whichever comes first
Next Biosciences
(-80°C Freezer)
Frozen (- 80 °C) Until the expiration date printed on outer product packaging (shelf-life is 3 years from date of manufacture)

AmnioMatrix (d) is a dehydrated, gamma-sterilized amniotic membrane tissue graft. It is denuded of the cuboidal epithelial layer, so no living cells are exposed to the patient. AmnioMatrix (d) has a five year shelf life that can be stored at room temperature in a clean dry area. AmnioMatrix (d) is used in a surgical setting and is attached to the surgical site with sutures or tissue glue.2

AmnioMatrix (d) is packaged on a polyester net with the epithelial side orientated onto the net. The allografts are packaged aseptically in an inner polyethylene pouch and sealed with an outer peel pouch. The outer pouch can be peeled open using normal aseptic technique, introducing the inner-pouch onto the sterile field.

No special transport conditions are needed for AmnioMatrix (d). The tissue must be protected from excessive heat and moisture.

Fig. 04 Right: Denuded Dehydrated Human Amniotic Membrane

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Preferred storage conditions for AmnioMatrix (d)

Location Temperature Use after Receipt
Unopened Room Temperature (10 °C – 28 °C) Within the expiration date printed on the product label. Usually 5 years since the day of production.


 

Sources of Placentas

Amniotic membranes are obtained from mothers with uncomplicated pregnancies giving birth via caesarean section. All placentas are procured by the Netcare Transplant unit. Full informed consent is obtained from the potential donors prior to the delivery according to the statues of the Declaration of Helsinki.
All donors are screened for infectious diseases such as HIV, Hepatitis B & C, HTLV and Syphilis. Microbial cultures are performed during processing of the tissue to ensure the sterility of the tissue. All serology and bacteriology is reviewed by the Medical Director of Next Biosciences prior to the release of the tissue for use.

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History of the use of the Human Amniotic Membrane

The human placental membranes were first used in the early 1900’s as a skin substitute: in 1910 by Davis and again in 1913 by Sabella. These procedures included the amniotic and chorionic membranes.

The amniotic membrane was first used on its own in ophthalmology in 1940, when De Rotth suggested it for the use in the treatment of an ocular burn wound.2 The treatment was successful, but the use of amniotic membrane then went out of favour until the early 1990’s, when improved processing and storage techniques were developed. The use of amniotic membrane in ophthalmic surgery for various ocular disorders increased and since then, thousands of ocular surgical procedures with amniotic membrane have been performed, and more than 700 peer reviewed articles have been published.

Amniotic membrane has also been used in various reconstructive surgical procedures as a biological dressing, which can aid in healing, and as a foundation material for the re-growth of soft tissue. These procedures include: biological bandages for burns and non-healing ulcers, repair of abdominal hernia closure of the pericardium and as a barrier to the prevention of surgical adhesions.3,4

Recent literature has also indicated the successful use of the amniotic membrane in the treatment of subcutaneous wounds such as diabetic and venous ulcers and partial thickness burn wounds.

The amniotic membrane contains a myriad of growth factors, cytokines and extracellular matrix proteins that have important functions in the wound healing process.

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