Wound healing is a challenging task for any podiatric physician, especially for our high-risk patients with diabetes. Uncontrolled diabetes has consequences for all aspects of the body but is especially detrimental to wound healing. Patients with diabetes have a 15 to 25 percent lifetime risk of developing foot ulcers and their annual treatment costs are estimated to be about $30,000.1
The normal physiology of wound healing encompasses three phases. The inflammatory phase occurs right after the initial insult. Platelets adhere to form a hemostatic plug and release alpha granules containing platelet-derived growth factor (PDGF). The alpha granules act to recruit and activate pro-inflammatory cells such as fibroblasts, macrophages, neutrophils and monocytes. These in turn secrete transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), endothelial growth factor (EGF) and vascular endothelial growth factor (VEGF). Platelets also release TGF-β themselves and platelet-derived angiogenesis factor (PDAF).
In the epithelialization phase, epidermal cells proliferate and migrate, depositing basement membrane components. Neovascularization causes the formation of granulation tissue. Fibroblasts are key to orchestrating the reorganization of the extracellular matrix. Growth factors and VEGF support wound healing and angiogenesis.
During the remodeling phase, fibroblasts assist in wound contraction and matrix metalloproteinases (MMPs) continuously remodel collagen until wound re-epithelialization has occurred.
There are numerous bioengineered alternative tissues (BATs) available to the podiatric physician but there are basic principles of wound care that physicians must follow prior to recruiting BATs.2 These principles include:
• removal of non-vital tissue via enzymatic, mechanical, biologic or surgical debridement, including hydrosurgical debridement;
• maintenance of moist wound healing environment;
• avoidance of excessive cytotoxic therapies (povidone-iodine or silver agents);
• assessment of micro- and macrovascular disease;
• offloading pressure sites; and
• aggressive treatment of infection.
A chronic non-healing wound, as defined by Medicare and based on the research of Sheehan and colleagues, is a wound that does not heal by at least 50 percent after receiving basic wound care for 30 days.3-5 This definition is necessary to establish an endpoint at which more expensive biological therapies may be warranted.2
Remember that chronic wounds are those in which there is an imbalance of the phases of wound healing. Although MMPs are present and important at every stage of wound healing, chronic diabetic wounds contain higher levels of MMPs in comparison to acute wounds, and this increase plays an inhibitory role at the wound base. Fibroblasts produce tissue inhibitors of metalloproteinases (TIMPs) that regulate MMPs. Interestingly, research has shown that diabetic wounds have decreased levels of TIMPs, suggesting even less regulation of MMPs.6,7
A Pertinent Overview On Bioengineered Alternative Tissues
Physicians have adopted the term bioengineered alternative tissue recently mainly because it is more descriptive than previous terms. The old terms, such as “tissue-engineered skin,” “biologic skin substitutes,” etc., caused much confusion.
Bioengineered alternative tissues are not autogenous skin and we should not treat them as such. All BATs are not created equal. It is imperative that one understands the capabilities and limits of these BATs in order to prevent misuse.
Just like in bone healing with reference to osteoinductive and osteoconductive properties of bone fillers, the terms “dermoinductive” and “dermoconductive” can apply to BATs.8
Dermoinductive products derive from living cell cultures containing human living keratinocytes, fibroblasts or both. These products recruit and activate tissue within the wound bed. Products include Apligraf (Organogenesis), Dermagraft (Shire Regenerative Medicine), Epicel (Genzyme) and Laserskin/Vivoderm (Fida Advanced Biopolymers). Apligraf and Dermagraft are FDA-approved for diabetic foot ulcers.
Dermoconductive products do not contain living cells and serve as a scaffold matrix allowing the infiltration of cells in an organized fashion. Products include Integra (Integra Life Sciences), Oasis (Smith and Nephew), Graftjacket (KCI), GammaGraft (Promethean LifeSciences), EZ-Derm (Molnlycke Health Care), Alloderm (LifeCell), Biobrane (Smith and Nephew), TransCyte (Smith and Nephew), Primatrix (TEI Biosciences) and MatriStem (Acell). Oasis and MatriStem are FDA approved for diabetic foot ulcers.
How Human Amniotic Membrane Is Emerging As A Viable Treatment
Recently, much attention has focused on human amniotic membrane for the treatment of chronic wounds. However, researchers have actually used human amniotic membrane for wound care treatment since the early 1900s. In 1910, Davis published a review of cases from Johns Hopkins Hospital.9 In 1913, Stern reported on amniotic membranes to treat ulcerated skin surfaces due to burns and Sabella used fetal membranes in skin grafting.10,11 Human amniotic membrane is currently in use in the field of ophthalmology promoting corneal epithelialization but has also been in use for burn injuries and ulcers, mandibular vestibuloplasty, dural defect repair, intra-abdominal surgery and gynecological reconstructive surgery.12
Amniotic membrane is the innermost layer of the placenta. Amniotic membrane is composed of a thin epithelial layer, a thick basement membrane and avascular stroma.
In 1979, Trelford and colleagues found that amniotic fluid promoted epithelial healing, reduced inflammation, increased comfort and decreased the severity of insufficient vascularization.13 In 2004, Zhang and coworkers noted that mesenchymal stem cells in human placenta are able to differentiate into osteogenic, adipogenic and chondrogenic lineages and demonstrated an ability to suppress T-cell proliferation.14 Solomon and colleagues in 2005 showed that amniotic membrane transplantation promotes re-epithelialization, decreases inflammation and fibrosis, and modulates angiogenesis.15
Amniotic membrane (or amnion) is therefore of particular interest because it can provide cells with multipotency. One can easily obtain amnion from the human placenta after a Caesarean section and the controversies that surround human embryonic stem cells do not apply in the procurement of amniotic membranes since placentas are discarded after childbirth. Serological testing occurs prior to C-sections and with the mother’s/donor’s consent.
Amniotic membrane contains amnion epithelial cells derived from embryonic ectoderm and amnion mesenchymal cells derived from embryonic mesoderm. Amnion and mesenchymal cells lack immunogenicity as the placenta is an organ that is immune privileged and therefore can be part of any immunocompromised patient either from human immunodeficiency virus or post-transplant.15
Amniotic membrane comes in two forms: dehydrated human amniotic membrane and cryopreserved human amniotic membrane. Dehydrated human amniotic membrane is pliable at room temperature, has a shelf life of five years, comes in different sizes and contains chorion, a part of the membrane that separates the fetus from the placenta. The cryopreserved form has a two-year shelf life and does not contain chorion. To date, there is no research available on the benefits or disadvantages to having chorion in the amniotic membrane.
However, with the graft form of amniotic membrane, controversy exists as to which side should face the wound, the amniotic or chorionic side. In a study of sheep, Trelford and colleagues showed an immunological response when the chorionic side faced the wound.16 Such a response suggested maternal decidual fragments may inadvertently accompany the chorion. Robson and coworkers studied “vascular invasion” and recommended not separating the chorionic surface from the amnion in order to see vascularization.17
Lim and coworkers compared the biological and ultra-structural properties of the dehydrated human amniotic membrane and cryopreserved human amniotic membrane cellular components, and biochemical composition with respect to ocular surface disease that required resurfacing with human amniotic membrane.20 They found that although there are significant differences in composition and ultrastructure between the two forms, they do not appear to compromise cell survival in vivo.
Zelen and colleagues compared healing characteristics (wound reduction and rates of complete healing) of dehydrated human amniotic membrane (EpiFix, MiMedx) versus standard of care in indolent neurotrophic diabetic ulcers.5 Researchers applied EpiFix every two weeks until complete healing and did not exceed 10 weeks. Ninety-two percent of chronic diabetic foot ulcers healed over a six-week period in comparison to only 8 percent with standard of care alone.
Werber and colleagues studied 20 patients with foot ulcers due to diabetes and arterial insufficiency that had been recalcitrant to treatment for over a year.19 These wounds displayed undermining and/or sinus tract or tunneling. The study used the cryopreserved allograft form AmnioMatrix (Applied Biologics), a mixture of amniotic membrane and amniotic fluid, which is intended for treatment of wounds. At 12 weeks, 18 of 20 patients experienced 100 percent wound closure and the undermining/tunneling of the wound was the first to respond to AmnioMatrix. Reductions in wound volume and area followed.
How To Apply Amniotic Membrane
As always, sharp debridement and appropriate preparation of the wound bed are absolutely necessary. Amniotic membrane comes in liquid form, in powder form (that one can mix with saline for injection) and as a graft. When using the injectable form, make the injection in the periwound area about 0.5 cm from the wound edge and directly into the superficial fascia and subcutaneous tissue of the wound at the 12, 3, 6 and 9 o’clock positions of the wound. The needle should be parallel to the wound margin at each location and one should deposit equal amounts at each site at about 10 to 15 mm in depth.
After application, apply a non-adhesive dressing followed by a dry, sterile dressing. The graft form comes in different sizes that one can cut to fit the wound. Graft orientation is important. The epithelial layer should be on top and dressed with non-adherent dressing. Do not disturb the graft form for at least two weeks.
One should follow any form of application with supportive therapies such as offloading and compression if necessary.5,19
Amniotic membrane has numerous indications and very few contraindications with infection being the main contraindication. Wounds require adequate vascularity to heal regardless of any treatment plan as well as proper offloading. Amniotic membrane is no exception. The fact that amniotic membrane is non-immunogenic and anti-inflammatory with decreased pain and scarring, provides a matrix for cell colonization and acts as a natural barrier makes human allograft ideal for wound treatment in many types of patient. Currently, amniotic membrane is still undergoing research and more robust studies need to be performed. However, amniotic membrane is definitely another tool in the foot and ankle surgeon’s armamentarium when clinicians use it appropriately along with good local wound care.
Dr. Perez is a third-year resident in Podiatric Medicine and Surgery at the New York College of Podiatric Medicine/Metropolitan Hospital/Lincoln Hospital. She is a Fellow of the New York Academy of Medicine.
Dr. Khan is an Associate Professor within the Division of Medical Sciences at the New York College of Podiatric Medicine.
1. Sheikh ES, Sheikh ES, Fetterolf DE. Use of dehydrated human amniotic membrane allografts to promote healing in patients with refractory non healing wounds. International Wound Journal. 2013; epub ahead of print.
2. Forbes J, Fetterolf DE. Dehydrated amniotic membrane allografts for the treatment of chronic wounds: a case series. J Wound Care. 2012; 21(6):290-6.
3. Warriner R (ed). Wound Treatment Protocol. Diversified Clinical Services Clinical Practice Guidelines. Diversified Clinical Services, 2009.
4. Snyder R. Wound percent area reduction and making decisions about utilizing advanced therapies. Podiatry Manage. 2010; 39(3):197-201.
5. Zelen CM, Serena TE, Denoziere G, Fetterolf DE. A prospective randomized comparative parallel study of amniotic membrane wound graft in the management of diabetic foot ulcers. Int Wound J. 2013; 10(5):502-7.
6. Tengrove NJ, Stacey MC, MacAuley S, et al. Analysis of acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair Regen 1999; 7(6):442-452.
7. Lobmann R, Ambrosch A, Schultz G, et. al. Expression of matrix metalloproteinases and their inhibitors in the wounds of diabetic and non-diabetic patients. Diabetologia. 2002; 45(7):1011-1016.
8. Southerland JT, Boberg JS, Downey MS, et al (eds). McGlamry’s Comprehensive Textbook of Foot and Ankle Surgery, fourth edition, chapter 69. Lippincott, Williams and Wilkins, Philadelphia, 2013.
9. Davis JW. Skin transplantation with a review of 550 cases at the Johns Hopkins Hospital. Johns Hopkins Med J. 1910; 15:307-96.
10. Stern M. The grafting of preserved amniotic membrane to burned and ulcerated surfaces, substituting skin grafts. JAMA. 1923; 60:973.
11. Sabella N. Use of fetal membranes in skin grafting. Med Records NY. 1913; 83:478-80.
12. Nordack PH, Miettinen S, Kääriäinen M, et al Amniotic membrane reduces wound size in early stages of the healing process. J Wound Care. 2012; 21(4):190-197.
13. Trelford JD, Trelford-Sauder M. The amnioin in surgery, past and present. Am J Obstet Gynecol. 1979; 134(7):833-845.
14. Zhang Y, Li C, Jiang S, et al. Human placenta-derived
mesenchymal progenitor cells support culture expansion of long-term culture-
initiating cells from cord blood CD34+ cells. Exp Hematol. 2004; 32(7):657–664.
15. Solomon A, Wajngarten M, Alviano F, et al. Suppression of inflammatory and fibrotic responses in an in-vitro model of allergic inflammation by the amniotic membrane stromal matrix. Clin Exp Allergy. 2005; 35(7):941-948.
16. Trelford JD, Anderson DG, Hanson FW, et al. Consideration of the amnion as an autograft and as an allograft in sheep. A preliminary report. J Med. 1972; 3(4):231.
17. Robson MC, Chandrasekharam V. Use of human and bovine amnion as a biological dressing. Arch Surg 1981; 116(7):891.
18. Niknejad H, Pierovi H, Jorjani M, et al. Properties of the amniotic membrane for potential use in tissue engineering. Eur Cells Mater. 2008; 15:88-99
19. Werber B, Martin E. A prospective study of 20 foot and ankle wounds treated with cryopreserved amniotic membrane and fluid allograft. J Foot Ankle Surg. 2013; 52(5):615-21.
20. Lim LS, Poh RW, Riau AK, et al. Biological and ultrastructural properties of Acelegraft, a freeze-dried γ-irradiated human amniotic membrane. Arch Ophthalmol. 2010; 128(10):1303-1310.
For further reading, see “Amniotic Membrane: Does It Have Promise For Diabetic Foot Ulcers?” in the June 2013 issue of Podiatry Today.