A Guide To Amniotic Membrane Modalities

With the emergence of amniotic membrane in the treatment of chronic wounds in the lower extremity, these authors examine key differences among available products as well as the current research on these treatment options.

Chronic non-healing wounds affect one-fifth of the United States population and may be secondary to a plethora of underlying causes including venous insufficiency, diabetic neuropathy, peripheral vascular disease and pressure.^1-4 Chronic wounds are difficult to treat and are often recalcitrant to healing via standard wound management methods. Billions of dollars are spent annually in the United States for the treatment of chronic wounds.² The financial burden continues to escalate due to an aging population and the rise in healthcare costs. Furthermore, the loss of productivity and diminished quality of life mounts a priceless emotional toll on the individuals affected and the family members who care for them.^2,4-6

Chronic wounds are those that have failed to proceed through an orderly and timely reparative process. With chronic wounds, there is an inability to achieve a 50 percent reduction in wound size over a period of four weeks using the appropriate standard of care.^2,7 Advanced treatment modalities are often indicated for chronic non-healing wounds to facilitate wound closure. Recent advances in wound care technologies, especially the advent of bioengineered alternative tissue, have proven to offer an optimized wound environment but have varying efficacy rates.^8,9

Placenta-derived amniotic membrane modalities are gaining newfound interest. Readily available following cesarean operation without ethical implications, placental tissue contains vital growth factors and extracellular matrix components that have implications in reducing inflammation and scar tissue while providing low immunogenicity without adverse effects.¹⁰ Amniotic membrane as a means of natural human transplantation has been in use since 1910. More recently, however, it has gained value in tissue regeneration for the purposes of wound healing due to an increased availability for use in the clinical office owing to advanced preservation techniques.^1,11–14

A Closer Look At The Wound Healing Properties And Potential Of Amniotic Membrane
Amniotic membrane is one of two layers comprising the human placenta with chorion being the other. Amniotic membrane is the innermost layer closest to the fetus and chorion is the layer adjacent to the uterus of the mother.¹⁰ The amniotic membrane emerges around the umbilical cord to form the epithelial layers of the umbilical cord. The function of the amniotic membrane is to protect and support the fetus during the gestation period. The membrane is a translucent, avascular structure lacking nerves, muscles and lymph vessels, and it modulates metabolic activity by growth factors, cytokines and other biological factors.¹⁵ Amniotic membrane also has antibacterial properties and offers protection from adhesions.

Amniotic membrane is composed of five layers: an epithelial monolayer resting on a thick basement membrane, which is comprised of a compact layer, a fibroblast layer and a spongy layer.¹⁰ The epithelium maintains the cell shape. All of the layers, excluding epithelium, contain collagen, which is known for its scaffold possessing properties for enhancing tissue growth.¹⁶ The basement membrane is a combination of polymeric hyaluronan, glycoproteins, proteoglycans, collagen and sparse mesenchymal cells. The basement membrane not only acts as a barrier but plays a role in the wound healing process by contributing to cell differentiation, proliferation and migration.^10,13,16-19 The spongy layer provides an advantage when processing placental membranes in that one can easily separate the spongy layer from the chorionic membrane. As we will discuss later, having a solely amnion allograft versus an amnion/chorion allograft is a distinguishing factor between certain products that utilize placental membranes in wound healing.

Amniotic membrane is rich in endogenous growth factors, including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta (TGF-b), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).¹³ Growth factors are proteins that bind to receptors on the cell surface with the primary result of activating cellular proliferation and/or differentiation. A type of cytokine acts as a mitogen in stimulating the proliferation and migration of cells. Amniotic membranes also contain antimicrobial proteins including neutrophil gelatinase-associated lipocalin and defensins.¹³

In the complex cascade of wound healing involving inflammation, proliferation and remodeling, a chronic non-healing wound is marked by prolonged inflammation. This creates a disruption in the healing cycle, leading to a decrease in normal proliferation of growth factors as well as an imbalance in protective versus degradative enzymes favoring destruction of the extracellular matrix.^4,13 Amniotic membrane can help facilitate wound healing by providing the necessary growth factors and other biological algorithms that adhere to the extracellular matrix to enhance tissue growth and healing, and ultimately work to overcome the interruption in the series of healing events.^13,20

The use of fresh amniotic membrane tissue was once impractical for office use due to a decreased shelf life and severe risk of disease transmission. The advent of several preservation methods has overcome these limitations. In order to maintain the regenerative properties of amniotic membranes, commercially available amniotic membrane modalities have adopted either the cryopreservation or dehydration preservation method. Depending on the wound care technology, some manufacturers opt to utilize more than one part of the human placenta containing solely amnion, amnion and chorion, or amnion and umbilical cord. Currently, there are more than 25 commercial placenta membrane products available on the market. Most are dehydrated amniotic membranes.

Accordingly, let us take a closer look at some of the more commonly used placenta membrane modalities and the clinical evidence available for these products. All products are currently designated by the U.S. Food and Drug Administration (FDA) as human cell, tissue and cellular and tissue-based products.

Key Insights On Dehydrated Human Amniotic Membrane Allograft
Dehydrated human amniotic membrane allograft (AmnioExcel, Derma Sciences) has an intact extracellular matrix that is intended to advance soft tissue repair, replacement and reconstruction.¹⁷ The proprietary dehydration process eliminates the need for thawing or any other preparation as AmnioExcel is ready to use from the packaging, which allows product storage at room temperature with a shelf life of five years.^17,21 One may apply the allograft to the wound on either side. The hydrophilic nature of this dehydrated amniotic membrane allograft causes it to adhere immediately to the moist wound bed, eliminating any need to fix the allograft to the wound.²¹

A recently published open-label, randomized, parallel group trial compared the wound healing efficacy of the dehydrated amniotic membrane allograft plus standard of care to standard of care alone in treating chronic diabetic foot ulcers (DFUs).²¹ Five of the 15 patients treated with dehydrated amniotic membrane allograft plus standard of care achieved complete wound closure at or before six weeks. In contrast, none of the 14 patients in the standard of care group achieved wound closure in the six-week period. While the sample size was small, the data indicates that dehydrated human amniotic membrane allograft may help patients achieve complete wound closure earlier than the standard of care alone.

Lintzeris and colleagues conducted a retrospective review of cases using dehydrated amniotic membrane allograft in patients with complex, non-healing wounds.¹⁷ While the authors’ results suggest that dehydrated amniotic membrane allograft may potentially provide healing in slow healing or non-healing wounds, the drawback to this small retrospective case series is that we cannot generalize its results to other patient populations. Randomized controlled studies are needed to truly assess the efficacy of dehydrated human amniotic membrane allografts in wound healing.

Examining The Potential Of Human Viable Wound Matrix
Human viable wound matrix (Grafix, Osiris Therapeutics) is designed to preserve the native components of the human placental membrane in a cryopreserved allograft.^8,22 The maintenance of the native structure of human tissue and its living components have demonstrated angiogenic, anti-inflammatory and antioxidant effects in wound repair models and in vitro studies.^8,22-23 One can store Grafix at -80ºC before use and the product has a two-year shelf life.⁸ Normal preparation of human viable wound matrix includes thawing followed by a wash with saline solution. Human viable wound matrix conforms well and adheres to the wound, and therefore does not require fixation.  

In a prospective, single-blinded, randomized controlled multicenter trial, Lavery and colleagues evaluated the efficacy of weekly applications of human viable wound matrix in treating diabetic foot ulcers (DFUs) in comparison with standard wound care therapy.²² Ninety-seven patients were enrolled in the trial. Fifty patients were treated with human viable wound matrix plus standard of care and 47 study participants received standard of care alone. The results revealed that treatment of DFUs with the combination of human viable wound matrix and standard of care showed a statistical improvement in wound closure (62 percent) in comparison to standard of care alone (21.3 percent).

The median healing time for patients receiving human viable wound matrix was shorter (42 days) in comparison with the control group (about 70 days).²² Moreover, in the study, Lavery and colleagues employed a unique study design element in which a third party of wound care experts confirmed the wound closure assessment to further remove potential bias and increase the reliability of the results.⁸
In another study, Regulski and colleagues conducted a large, retrospective single center study evaluating the effect of human viable wound matrix on chronic wounds that had failed to heal.²⁴ This study consisted of 66 patients with 67 chronic wounds and demonstrated a 76.1 percent (51 of 67 wounds) wound closure rate for all types of wounds after 12 weeks of care. The types of wounds in this study included DFUs, venous leg ulcers and other chronic wounds. The average time to closure in these wounds was 5.8 ± 2.5 weeks with an average 3.2 applications for patients who healed. However, the absence of a control group is a limitation of the study by Regulski and colleagues due to its retrospective nature.⁸ According to the authors, human viable wound matrix is a safe and more effective therapy for treating DFUs than standard wound therapy.²²

Assessing The Evidence On Dehydrated Human Amnion/Chorion Membrane Allograft
Dehydrated human amnion/chorion membrane or dHACM (EpiFix, MiMedx Group) is a laminated product containing two layers, amnion and chorion, from donated placenta following a scheduled cesarean section.²⁵ Chorion is the layer of the human placenta adjacent to the uterus of the mother and is about four times thicker than the amnion.¹⁰ Chorion is comprised of three layers: a reticular layer that forms the majority of the chorion’s thickness, basement membrane and a trophoblast layer.²⁶ The trophoblast layer adheres to the maternal decidua and the reticular layer is the one in contact with the spongy layer of the amnion.

In a study published in 2014, Koob and colleagues evaluated the benefits of a single layer amnion allograft in comparison to bilayer amnion/chorion allografts for wound healing.²⁷ The authors noted that the amnion and chorion contained a similar array of growth factors, cytokines and regulatory factors as each layer contributes certain biological algorithms in vitro. The chorion layer, being about four times thicker than the amnion layer, contains substantially more growth factors per square centimeter than the amnion. The resulting bilayer amnion/chorion graft subsequently contains fivefold more growth factors than a single layer of amnion. While the increase in growth factors could in theory suggest increased efficacy, there are currently no studies or reports that compare the clinical wound healing efficacy of a single layer amnion allograft versus a bilayer amnion/chorion allograft.

Dehydrated human amnion/chorion membrane has a shelf life up to five years and requires no refrigeration. It adheres well to the moist wound bed, eliminating the need to fixate the allograft to the wound.
Five studies by Zelen and coworkers in recent years have supported the clinical efficacy of dHACM.^25,28-31 In their latest study, a prospective, randomized, controlled parallel group multicenter clinical trial, Zelen and colleagues evaluated healing outcomes as well as cost at 12 weeks in 100 patients with chronic DFUs.³⁰ The patients received either a bioengineered skin substitute (Apligraf, Organogenesis), dHACM or standard wound care. Wound closure rates were highest among the dHACM group and lowest among the group who had standard wound care. Healing times decreased by almost 50 percent for the dHACM subset of patients (23.6 days) in comparison to standard wound care (57.4 days). The median graft cost per wound was reduced more than 75 percent when clinicians used dHACM.

According to the authors, the study was limited because patients had follow-up for only one week after healing and patients were allowed to withdraw from the study after six weeks if their wound had not reduced in size by at least 50 percent.³⁰ The authors were therefore unable to compare the rates of healing at 12 weeks or the wound recurrence rates in this study. In addition, this study includes a variety of lower extremity diabetic ulcers, both plantar and dorsal. According to the authors, the sample size was insufficient to stratify by wound location nor was it possible to perform any meaningful subgroup analysis to determine factors influencing outcomes or speed of healing.

A few case series studies also support the use of dHACM in the treatment of chronic non-healing wounds of various etiologies.^32,33 Forbes and colleagues reported on dHACM treatment in a population of patients with wounds of various etiologies such as venous leg ulcers, crush injury, arterial insufficiency, immunologic skin disease/scleroderma and snake bite.³² Sheikh and coworkers demonstrated successful healing of refractory, non-healing wounds with dHACM in patients with comorbidities.³³

What You Should Know About Cryopreserved Human Umbilical Cord And Amniotic Membrane
Neox (Amniox Medical) is a wound covering made from cryopreserved amniotic membrane and human umbilical cord. It has been in extensive use in ophthalmology as it helps minimize postoperative inflammation, pain and adhesion formation.^12,34 Clinicians can safely refrigerate the product for up to two years and it does require fixation onto the wound bed by either sutures or staples. While the umbilical cord has been a favorable source of mesenchymal stem cells, there are currently no large clinical studies assessing the benefits of umbilical cord/amniotic membrane allograft in comparison to other single or bilayer amniotic membrane derived modalities.³⁵

There are a few published retrospective case series addressing the use of cryopreserved human umbilical cord and amniotic membrane for wound treatment. Raphael recently published a retrospective chart review looking at the efficacy of cryopreserved umbilical cord/amniotic membrane tissue for the treatment of DFUs.³⁶ In the study, Raphael assessed the treatment of 32 wounds in 29 patients with a mean wound size of 10.6 ± 2.15 cm² at a single healthcare center. Raphael found that 28 of the 32 wounds achieved complete wound closure in an average of 13.8 ± 1.95 weeks with the use of less than two applications. Prospective randomized controlled studies are needed to truly assess the efficacy of this modality in the healing of chronic ulcerations.

In Conclusion
Chronic wounds present a challenge for both the patient and the clinician. An ideal treatment for a non-healing wound would entail complete wound closure in a timely manner while saving healthcare costs. Recent advances in tissue preservation techniques have resulted in commercially available amniotic membrane products for use in the clinical setting. The decision to choose a specific product is multifactorial and new research has shown the benefits of amniotic membrane derived allografts. Although there is limited clinical research regarding the wound healing efficacy of most amniotic membrane-based products, current and future studies will help elucidate the value of amniotic membrane for the treatment of chronic wounds.

Ms. Nanrhe is a second-year student at the Dr. William M. Scholl College of Podiatric Medicine at Rosalind Franklin University of Medicine and Science in Chicago. She is a National Institutes of Health (NIH) T35 funded research scholar and currently serves as an executive board member of the Illinois Podiatric Medical Student Association.

Dr. Wu is the Associate Dean of Research, a Professor of Surgery at the Dr. William M. Scholl College of Podiatric Medicine and a Professor of Stem Cell and Regenerative Medicine at the School of Graduate Medical Sciences at the Rosalind Franklin University of Medicine and Science. She is also the Director of the Center for Lower Extremity Ambulatory Research (CLEAR) in Chicago.

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