One-third of diabetic foot ulcers (DFUs) fail standard of care treatment (which involves debridement, offloading, moist wound therapy and bioburden control), and enter a state of chronically impaired healing.1 Along with abnormally expressed growth factors and cytokines in DFUs, there is impaired extracellular matrix remodeling and deregulation of protease levels that researchers widely believe result in chronic inflammation, a principal factor with delayed healing.2-5
Also crucial to wound healing and compromise in DFUs are fibroblasts, mesenchymal stem cells, adipose-derived stem cells and hematopoietic stem cells that regulate and coordinate the healing process, perform cellular recruitment, and secrete growth factors and cytokines.2,6-8 Emerging therapies in wound care therefore target the extracellular matrix, growth factors, cytokines and stem cells necessary to promote the healing of DFUs.8-10
Placental tissues are a potential exogenous source of the critical wound healing factors. For nearly 120 years, clinicians have used amniotic membranes to treat wounds.11 Placental membranes are composed primarily of amnion and chorion membranes. These membranes are made of structural collagen and extracellular matrix, regenerative molecules (including the growth factors and cytokines inhibited in DFUs), and biologically active cells, all of which interact with one another to regulate the healing processes.12,13 The amnion membrane is made up of five layers: the epithelium, the basement membrane, the compact layer, the fibroblast layer and the intermediate layer.14 Chorion membrane has a reticular layer—which contributes to the placental tissue being three to four times thicker than amnion—a basement membrane and a trophoblast layer.14-18
The use of amnion/chorion allografts on chronic DFUs is more effective for jump-starting the healing process and closing the wound than amnion allografts alone due to the collaborative biomechanical properties of the combined graft, the abundance of pro-healing growth factors and proteins, and the preservation technique that retains all biological functions.
Understanding The Biological Properties And Mechanism Of Action Of Amnion/Chorion Allografts
The mechanism of action for amnion/chorion allografts merits further investigation. However, studies on amnion/chorion allografts have revealed that these grafts contain more than 50 growth factors, proteins and tissue inhibitors of metalloproteinases (TIMPs).6,7,9,14 These molecules include chemokines and proteins that assist with leukocyte migration, immunomodulatory cytokines and mitogenic growth factors as well as cytokines for tissue growth.6
Pro-healing growth factors identified in amnion/chorion allografts include platelet-derived growth factor-AA (PDGF-AA), platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-b (TGFb), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PGF) and granulocyte colony-stimulating factor.7 Researchers have also identified in these tissues anti-inflammatory interleukins (ILs) including IL-1ra, IL-5 and IL-19, as well as TIMP-1, TIMP-2, and TIMP-4, which facilitate the regulation of matrix metalloproteinase activity. In addition, the amnion/chorion allografts are rich in extracellular matrix components including collagens (types I, III, IV, V, VI), elastin, laminin and fibronectin that provide both structural and signaling cues.6,7
More than 25 products derived from placental tissue are commercially available but the majority of the products are single-layer.19 Two amnion/chorion allografts currently available are the Purion processed dehydrated amnion/chorion membrane (dHACM) (EpiFix®, MiMedx) with electron-beam sterilization and aseptically processed dehydrated human amnion and chorion allograft (dHACA) (AmnioBand, Musculoskeletal Transplant Foundation).
Given that single layer amnion membranes are three to four times thinner than their chorion counterparts, it is easy to understand that they have less matrix structure as well as fewer biomechanical properties, growth factors and cytokines that are beneficial to wound healing.9,20 In comparison to an amnion allograft, researchers have reported that amnion/chorion allografts have four to five times more cytokines and proteins that are crucial to the delivery of growth factors and mediation of inflammation in the wound.20
The growth factors and cytokines inherent to amnion/chorion allografts facilitate the recruitment of local stem cells to the wound. Studies have demonstrated that in normal mesenchymal stem cells, adipose-derived stem cells and hematopoietic stem cells, the application of soluble extracts of dHACM in vitro stimulated cellular migration and proliferation, and modulated cytokine secretion.6 Adipose-derived stem cells in patients with diabetes exposed in vitro to eluted soluble growth factors and cytokines from dHACM also showed an increased wound healing response with researchers observing recruitment and proliferation of adipose-derived stem cells as well as an altered expression of cytokines.2 Studies have thus clearly demonstrated that amnion/chorion allografts have at least one soluble factor that can increase the cellular response by attracting local stem cells to wounds, modulating their activities and stimulating the natural healing process.7
In summary, the abundant, preserved growth factors, chemokines, cytokines and other wound healing factors in the amnion/chorion allografts comprise a biological matrix and natural barrier that performs the following wound healing activities:
• Induces cellular proliferation and migration2,6,7,9,14
• Modulates the cellular response and coordination of the healing processes2,6,7
• Modulates inflammation7,14
• Promotes angiogenesis and neovascularization9
• Inhibits matrix metalloproteinases and promotes tissue regeneration7
Why Amnion/Chorion Allografts Are Superior To Amnion Allografts
Clinical studies of the treatment of DFUs with amnion/chorion allografts have further demonstrated the beneficial impact of these activities and functions.
We now have more of an understanding of the biological functions and activities of amnion/chorion allografts that benefit wound healing, and the clinical outcomes further support their effectiveness. Healing rates of DFUs from clinical studies, including multiple randomized controlled trials, demonstrate the superior beneficial effect of amnion/chorion allografts over single-layer amnion allografts.
In a recent randomized, controlled trial, DiDomenico and colleagues evaluated the efficacy of dHACA on DFUs and found that 85 percent of wounds healed at 12 weeks with a mean time to heal of 36 days.21 Following treatment with dHACM, greater than 90 percent of DFUs reportedly healed at six weeks and at 12 weeks with a mean time to heal of 23.6 days.12,22-24 Along with clinical efficacy, there is an economic aspect to consider while using these amnion/chorion allografts. Studies have found the cost to closure at 12 weeks for dHACA was $1,400 and dHACM was $2,798, highlighting not only clinical efficacy but economical benefits as well.21,24
In contrast, a single-layer, cryopreserved amnion membrane (Grafix®, Osiris Therapeutics) demonstrated lower DFU healing rates (62 percent) in a randomized controlled trial at 12 weeks.25 Researchers reported even lower healing rates for a single-layer dehydrated amnion allograft (AmnioExcel, Derma Sciences) at six weeks (33 to 45.5 percent).26 Therefore, studies demonstrate the superior clinical effectiveness of amnion/chorion allografts over amnion allografts with drastically higher healing rates for DFUs.
With many options available in the treatment of DFUs, it is important to consider prospective published data and review the clinical and economical data when making decisions. Currently, there are published prospective clinical studies for two commercially available amnion/chorion products that demonstrate the healing of DFUs and show economical value. These studies report significantly superior healing rates with level I evidence that supports amnion/chorion products as the better choices over amnion allografts alone when it comes to DFU care.
Dr. Zelen is the Medical Director of the Professional Education and Research Institute of Roanoke, Va. He is a Fellow of the American College of Foot and Ankle Surgeons.
Dr. Serena is Medical Director and CEO of Serena Group in Hingham, Mass.
- Fife CE, Horn SD, Smout RJ, Barrett RS, Thomson B. A predictive model for diabetic foot ulcer outcome: the Wound Healing Index. Adv Wound Care (New Rochelle). 2016; 5(7):279-287.
- Massee M, Chinn K, Lim JJ, Godwin L, Young CS, Koob TJ. Type I and II diabetic adipose-derived stem cells respond in vitro to dehydrated human amnion/chorion membrane allograft treatment by increasing proliferation, migration, and altering cytokine secretion. Adv Wound Care (New Rochelle). 2016; 5(2):43-54.
- Lauer G, Sollberg S, Cole M, et al. Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds. J Invest Dermatol. 2000; 115(1):12-18.
- Jude EB, Blakytny R, Bulmer J, Boulton AJ, Ferguson MW. Transforming growth factor-beta 1, 2, 3 and receptor type I and II in diabetic foot ulcers. Diabet Med. 2002; 19(6):440-447.
- Yager DR, Nwomeh BC. The proteolytic environment of chronic wounds. Wound Repair Regen. 1999; 7(6):433-441.
- Massee M, Chinn K, Lei J, Lim JJ, Young CS, Koob TJ. Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro. J Biomed Mater Res B Appl Biomater. 2016; 104(7):1495-1503.
- Koob TJ, Rennert R, Zabek N, et al. Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing. Int Wound J. 2013; 10(5):493-500.
- Maxson S, Lopez EA, Yoo D, Danilkovitch-Miagkova A, Leroux MA. Concise review: role of mesenchymal stem cells in wound repair. Stem Cells Transl Med. 2012; 1(2):142-149.
- Koob TJ, Lim JJ, Massee M, et al. Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration. Vasc Cell. 2014; 6:10.
- Wieman TJ, Smiell JM, Su Y. Efficacy and safety of a topical gel formulation of recombinant human platelet-derived growth factor-BB (becaplermin) in patients with chronic neuropathic diabetic ulcers. A phase III randomized placebo-controlled double-blind study. Diabetes Care. 1998; 21(5):822-827.
- Davis J. Skin transplantation with a review of 550 cases at the Johns Hopkins Hospital. Johns Hopkins Hospital Report. 1910; 15:307-310.
- Zelen CM, Serena TE, Denoziere G, Fetterolf DE. A prospective randomised comparative parallel study of amniotic membrane wound graft in the management of diabetic foot ulcers. Int Wound J. 2013; 10(5):502-507.
- Schultz GS, Davidson JM, Kirsner RS, Bornstein P, Herman IM. Dynamic reciprocity in the wound microenvironment. Wound Repair Regen. 2011; 19(2):134-148.
- Koob TJ, Lim JJ, Massee M, Zabek N, Denozière G. Properties of dehydrated human amnion/chorion composite grafts: Implications for wound repair and soft tissue regeneration. J Biomed Mater Res B Appl Biomater. 2014; 102(6):1353-1362.
- Bourne G. The foetal membranes. A review of the anatomy of normal amnion and chorion and some aspects of their function. Postgrad Med J. 1962; 38:193-201.
- Parry S, Strauss JF. Premature rupture of the fetal membranes. New Engl J Med. 1998; 338(10):663-670.
- Oyen ML, Cook RF, Calvin SE. Mechanical failure of human fetal membrane tissues. J Mater Sci Mater Med. 2004; 15(6):651-658.
- Chua WK, Oyen ML. Do we know the strength of the chorioamnion? A critical review and analysis. Eur J Obstet Gyn Reprod Biol. 2009; 144(Suppl 1):S128-133.
- Gibbons GW. Grafix®, a cryopreserved placental membrane, for the treatment of chronic/stalled wounds. Adv Wound Care (New Rochelle). 2015; 4(9):534-544.
- Koob TJ, Lim JJ, Zabek N, Massee M. Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing. J Biomed Mater Res B Appl Biomater. 2015; 103(5):1133-1140.
- DiDomenico LA, Orgill DP, Galiano RD, et al. Aseptically processed placental membrane improves healing of diabetic foot ulcerations: prospective, randomized clinical trial. Plast Reconstr Surg Glob Open. 2016; 4(10):e1095.
- Zelen CM, Gould L, Serena TE, Carter MJ, Keller J, Li WW. A prospective, randomised, controlled, multi-centre comparative effectiveness study of healing using dehydrated human amnion/chorion membrane allograft, bioengineered skin substitute or standard of care for treatment of chronic lower extremity diabetic ulcers. Int Wound J. 2015; 12(6):724-732.
- Zelen CM, Serena TE, Snyder RJ. A prospective, randomised comparative study of weekly versus biweekly application of dehydrated human amnion/chorion membrane allograft in the management of diabetic foot ulcers. Int Wound J. 2014; 11(2):122-128.
- Zelen CM, Serena TE, Gould L, et al. Treatment of chronic diabetic lower extremity ulcers with advanced therapies: a prospective, randomised, controlled, multi-centre comparative study examining clinical efficacy and cost. Int Wound J. 2016; 13(2):272-282.
- Lavery LA, Fulmer J, Shebetka KA, et al; Grafix Diabetic Foot Ulcer Study Group. The efficacy and safety of Grafix® for the treatment of chronic diabetic foot ulcers: results of a multi-centre, controlled, randomised, blinded, clinical trial. Int Wound J. 2014; 11(5): 554-560.
- Snyder RJ, Shimozaki K, Tallis A, et al. A prospective, randomized, multicenter, controlled evaluation of the use of dehydrated amniotic membrane allograft compared to standard of care for the closure of chronic diabetic foot ulcer. Wounds. 2016; 28(3):70-77.
For further reading, see “Amniotic Membrane: Can It Facilitate Healing?” in the April 2015 issue of Podiatry Today, “Amniotic Membrane: Does It Have Promise For Diabetic Foot Ulcers?” in the June 2013 issue or “Amniotic Membrane: Can It Have An Impact For DFUs?” in the March 2014 issue.