EpiFix Membrane for Non-Healing Ulcers

Stephan J. LaPointe, DPM, PhDYusuf Opakunle, DPM

INTRODUCTION

Non-healing ulcers are arguably one of the most frustrating pathologies faced by the podiatric physician, and they can be even more frustrating to the patient (1). A non-healing ulcer can be defined as a wound that shows no significant improvement after 4 weeks or does not epithelize and heal after 8 weeks. Wound healing occurs in 4 stages. It is important to understand the physiologic processes going on in each stage of wound healing to appreciate the role wound grafts play in non-healing ulcers (2). The first stage is hemostasis, which occurs immediately after injury to the skin and lasts for 24 hours. Platelets aggregate to the site of injury clotting the bleeding vessels. Inflammation, the second stage lasts from the first day to 3-4 days. During this phase, blood vessels dilate bringing neutrophils, monocytes (macrophages) to phagocytize micro-organisms to prevent infection. Several growth factors are also present at this stage. The inflammation stage presents clinically as warmth, swelling, and erythema and the patient may experience pain and discomfort. A podiatric physician must be able to distinguish the inflammatory process from infection at this stage. The third is the proliferation stage, which lasts from 4 to 21 days depending on the size of the wound and overall health status of the patient. Cell migration and division occur at this stage as more macrophages, lymphocytes, neurocytes, fibroblasts, and keratinocytes play various roles leading to wound contraction and epithelization. Attention should be given to this stage of wound healing because non-healing ulcers usually experience suppression of cell migration and division, thereby leading to an increase in inflammatory cells. There is a marked decrease in response to growth factors by cells. This results in a non-healing ulcer trapped in what is called a chronic inflammatory state. The final stage of wound healing is the remodeling or maturation stage, which occurs from day 21 and can last up to 2 years. Fibroblasts are building tensile strength by depositing and arranging collagen fibers. Careful attention and protection should be given to the skin at this stage because wound dehiscence and breakdown can occur.

Any factor that interferes with any of the stages of wound healing can lead to a non-healing ulcer. However, most non-healing ulcers are secondary to uncontrolled diabetes mellitus, end-stage renal disease, neuropathy, peripheral vascular disease, pressure deformity, venous stasis,

infection, or a combination thereof. It is not uncommon for a non-healing ulcer to be multifactorial, therefore it is the duty of the podiatric physician to investigate and address all underlying problems that could impede wound healing. When wounds do not respond to traditional wound care management and the underlying complicating factors have been addressed, alternate therapy should be considered. There are several skin graft substitutes as well as skin allografts available. One of the skin graft substitutes available is EpiFix (MiMedx Group).

EPIFIX

EpiFix is a skin graft substitute made from human amniotic membrane (HAM). It was introduced to the market as a tissue allograft in 2006. The allograft is obtained from donated placenta (that would otherwise be discarded) after childbirth and is preserved by dehydration via a proprietary PURION process. Dehydration has the advantage over cryopreservation used by other skin graft substitutes because no freezer is required for storage; therefore it can easily be stored on the shelf for up to 5 years (1). Furthermore, dehydrated HAM has been shown to be more effective at delivering growth factors to a healing wound (3). The amniotic membrane is the innermost layer of the placenta and surrounds the developing fetus. It is a single layer of epithelia cells that are closely attached to the thick basement membrane and is adjacent to the amniotic fluids, which provide nutrients (4). HAM was first used by Davis in 1910 (5) on 550 patients who underwent skin transplantation at Johns Hopkins Hospital.

Amniotic membranes facilitate migration of epithelia cells, differentiation, and promotion of goblet cells that all aid in and promote wound healing (6). A study by Maan et al showed that dehydrated HAM recruited more progenitor cells compared with controls (7). HAM has an anti-inflammatory effect by stimulating the production of activin. It also has an anti-scaring effect, reducing the transformation of growth factors-beta signaling and myofibroblast differentiation of normal fibroblasts. Koob et al demonstrated in a study that approximately 36 cytokines involved in wound healing were identified in HAM (8). Additionally, Koob et al identified angiogenic factors in dehydrated HAM using enzyme-linked immunosorbent assays (9).

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A prospective randomized study by Zelen et al (10) compared the healing efficacy of diabetic foot ulcer treated with dehydrated HAM (n = 13) with standard of care (n = 12). This was a single-center clinical trial, which examined diabetic patients who had a diabetic foot ulcer of ≥4 week’s duration, adequate blood flow, and no infection. Wounds were evaluated for size and rate of complete healing at 4 weeks and 6 weeks. The overall rate of healing for the dehydrated HAM at 4 weeks and 6 weeks was 77%, and 92%, respectively compared with standard of care, which had a rate of healing at 4 and 6 weeks of 0% and 8%, respectively. These results showed that dehydrated HAM with standard of care is more efficient for wound healing.

Another prospective randomized study by Zelen et al a year later (11) compared weekly versus biweekly application of dehydrated HAM in the management of diabetic foot ulcers. Patients with more than 4 weeks of non-infected ulcers were included in the study. Patients were randomized to receive weekly or biweekly application of dehydrated HAM. The result showed that ulcers treated with weekly application healed faster than wounds treated with biweekly application. Additional research by Massee et al in vitro (12) demonstrated that dehydrated HAM promotes wound healing by increasing proliferation and migration of adipose-derived stem cells, mesenchymal stem cells, and hematopoietic stem cells. A similar in vitro study confirmed that diabetic adipose stem cells responded to dehydrated HAM therapy (6).

CASE REPORT ONE

A 25-year-old man who was profoundly neuropathic, had diabetes mellitus, and had a history of methamphetamine use was discharged from our practice in April of 2012 for non-compliance. The patient had been receiving care in our practice since the age of 19 years, when he presented with fractures of all five metatarsals of the left foot. He later had a fifth digit ulcer that eventually required amputation. After being discharged from our practice in 2012, he underwent a trans-metatarsal amputation of the right foot per vascular surgery.

In May, 2016 the patient called our office pleading for us to reconsider treating him since “they” wanted to amputate his left leg. The patient was examined on May 5th. A partial fifth ray amputation, an ulcer over the base of the fifth metatarsal, and cellulitis were noted (Figure 1 and Figure 2). We recommended hospital admission, debridement and biopsy, but the patient refused because he had to travel to Florida on personal business.

On May 11th, the patient returned and we resected the entire fifth metatarsal base in an attempt to remove infected bone and primarily close the wound. A biopsy of the fourth metatarsal head was performed. Debridement of the necrotic tissue prevented primary closure. He was directly admitted

to the hospital for treatment with intravenous antibiotics. The fourth metatarsal head biopsy sample was positive for osteomyelitis and the patient was transferred to a long-term skilled nursing facility on the same campus.

On May 18th the fourth metatarsal was resected, and the proximal margin submitted for evaluation for osteomyelitis, which returned as positive. We recommended a vascular consult for a below-knee amputation, but the patient declined, so resection of the entire fourth metatarsal was performed on June 2nd. The wound dehisced with significant gapping, possibly due to the patient being

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Figure 1. Initial presentation of Case 1. Note the cellulitis and osteomyelitis of the remaining fifth metatarsal base.

Figure 2. Radiographic anteroposterior view at initial presentation.

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noncompliant even though he was in a skilled nursing facility (Figure 3).

On June 22nd, the patient was taken again to surgery, the wound was debrided down to bleeding margins, and Dermaclose was applied in an attempt to close the wound (Figure 4). The cuboid was biopsied at that time and was negative. Dermaclose was removed on June 28th with another attempt at wound closure. Patient was discharged home on June 29th. He presented to our office noncompliant with nonweightbearing (Figure 5) on July 11th. A week later

on July 18th all the sutures had pulled through, and wound margins were fibrotic and did not appear viable. We once again advised the patient on remaining nonweightbearing and told him that we were running out of options. Another procedure was scheduled, which included Epifix graft. On July 20th we debrided the wound, applied Epifix graft and closed the wound over the graft. Six weeks later on August 8th the patient presented with a healed ulcer of the lateral border of the left foot (Figure 6). A new ulcer developed over the lateral fifth transmetatarsal amputation on the

Figure 3. Wound dehiscence after the fourth metatarsal resection.

Figure 4. Appearance after Dermaclose application for wound dehiscence.

Figure 5. Appearance after removal of the Dermaclose device and before the sutures were removed.

Figure 6. Six weeks after application of Epifix and primary closure of wound at approximately 3 months after initial presentation.

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opposite limb. This was healed via modified shoe gear and off weighting. Then we prescribed bilateral custom shoes and double upright braces. In February 2017 the patient subsequently developed an ulcer of the lateral malleolus of the left ipsilateral limb and a grade 0 lesion over the prior incision site (Figure 7).

After almost 1 year of trying to heal the ulcer over the lateral malleolus, an Achilles tendon lengthening was performed on August 30, 2017 to provide the patient with a more braceable foot. Although he was non-compliant with nonweightbearing, the patient healed the tendon lengthening and the ulcer. Although this patient would likely benefit from a tibialis anterior tendon transfer, it has not been recommended at this time, due to his non-compliance (Figure 8 and Figure 9).

CASE REPORT TWO

A 67-year-old man with diabetic neuropathy presented to the office on December 15th, 2016 as a new patient for routine diabetic foot care. As part of his care, a lower extremity arterial flow (LEAF) study was performed. The ankle brachial index (ABI) was 0.94 and 0.84 in the right and left foot, respectively, and the hallux digital pressures were 100 mm Hg and 84 mm Hg on the right and left, respectively. On May 22nd, the patient presented with a grade 1 ulcer of the medial first metatarsal head measuring 6 x 6 mm, and cellulitis that had been present for over a week (Figure 10). The patient’s primary care physician had prescribed Keflex. Wound cultures were obtained and debridement of the ulcer was performed. Three days later (May 25th) the wound measured 18 x 15 mm, and the results of the culture showed pan-sensitivity to Streptococcus agalactaie. Two weeks later (June 1st) the wound measured 10 x 10 mm, and the cellulitis continued to resolve. The patient was scheduled to be seen in the office every 2 weeks, but after missing 2 appointmets, presented to the office with a larger wound, now measuring 10 x 17 mm. Regranex was prescribed, and the wound initially responded. However, on September 21st the wound measured 15 x 15 mm and cellulitis had recurred. We referred to vascular although the LEAF study was not significant. The patient was seen the same day and had the LEAF study repeated with reported ABI of 1.07 on the right, and 1.12 on the left. Digital

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Figure 7. Lateral malleolus ulcer and grade 0 lesion of the incision site.

Figure 8. Radiograph after fourth ray resection and healed ulcers.

Figure 9. Appearance after Achilles lengthening with all ulcers healed 20 months after initial presentation.

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pressures were 120 mm Hg on the right and 105 on the left, with markedly diminished dorsal pedis waveforms noted. On October 4th arthrectomy and angioplasty of the popliteal, pereonal, and tibialis posterior vessels were preformed. Three weeks later on October 24th, the ulcer measured 12 x 12 mm, and a white fibrotic base and joint capsule were noted in the wound. We then received approval for five Epifix applications (Figure 11). On December 11th after 4 weekly-Epifix applications, the wound was almost completely resolved (Figure 12); however, the patient subsequently lost to follow-up.

CASE REPORT THREE

A 53-year-old woman with diabetes mellitus underwent a Baxter’s nerve release on October 4th, 2016. At the first postoperative visit (5 days after surgery), the incision was well-coapted, there was no erythema, and only mild edema was noted. On her second postoperative visit, the edema was significant and the sutures had started to pull through (Figure 13). The patient reported that she had been working on the farm and going horseback riding. Delayed closure in

the office was discussed, but the patient preferred steri-strips because she did not want to receive an injection.

Two weeks later, the wound measured 35 mm x 8 mm. We were concerned with the integrity of the underlying neurovascular bundle, and again discussed closure in the office with the patient, but chose to schedule surgery. At the preoperative visit, the wound measured 32 mm x 10 mm. On November 15th, Epifix was applied with an attempt at delayed closure. However, there was significant tension on the vertical mattress sutures and the wound margins could not be opposed. The patient then presented to the office for the first postoperative visit. At this point, although the wound was deep, the margins appeared to be fairly healthy (Figure 14). We ordered a wound vacuum and after two weeks the wound had filled in. The wound continued to respond well and was resolved at six weeks (Figure 15).

Figure 10. Initial appearance of patient in Case 2 with new ulcer and cellulitis.

Figure 11. Appearance prior to first application of Epifix.

Figure 12. Appearance prior to application of fifth Epifix graft.

Figure 13. Appearance of patient in Case 3 at second postoperative visit for Baxter’s nerve release.

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Diabetic foot ulcers are difficult to treat. We need to use all the available tools to resolve the ulcers before the extremity is threatened. These tools include mechanical protection, vascular optimization, proper wound care, treatment of infections, and at times biopsy for suspicious appearing and non-healing wounds. We have many tertiary products on the market including amniotic membranes. Presented here were 3 cases that all resolved well. Two of the cases involved long-standing ulcers that were unlikely to resolve with traditional wound care. The third case most likely would have resolved, but due to its location prompted us to expedite the application of the graft. Epifix is relatively cost effective, has a long shelf life and does not preclude other treatment options such as a split-thickness skin graft. It is an additional option that should be considered in a patient with a difficult, non-healing ulcer.

REFERENCES1. Sheikh ES, Sheikh ES, Fetterolf DE. Use of dehydrated human

amniotic membrane allografts to promote healing in patients withrefractory non healing wounds. Int Wound J 2013;11:711-7.

2. Martin P, Leibovich SJ. Inflammatory cells during wound repair: thegood, the bad and the ugly. Trends Cell Biol 2005;15:599-607.

3. Koob TJ, Lim JJ, Zabek N, Massee M. Cytokines in single layeramnion allografts compared to multilayer amnion/chorionallografts for wound healing. J Biom Mater Res B Appl Biomater2014;103:1133-40.

4. Gude NM, Roberts CT, Kalionis B, King RG. Growth and functionof the normal human placenta. Thromb Rese 2004;114:397-407.

5. Davis JW. Skin transplantation with a review of 550 cases at theJohns Hopkins Hospital. Johns Hopkins Med J 1910;15:307-96.

6. Massee M, Chinn K, Lim JJ, Godwin L, Young CS, Koob TJ. TypeI and II diabetic adipose-derived stem cells respond in vitro todehydrated human amnion/chorion membrane allograft treatmentby increasing proliferation, migration, and altering cytokinesecretion. Adv Wound Care (New Rochelle) 2016;5:43-54.

7. Maan ZN, Rennert RC, Koob TJ, Januszyk M, Li WW, GurtnerGC. Cell recruitment by amnion chorion grafts promotesneovascularization. J Surg Res 2015;193:953-62.

8. Koob TJ, Lim JJ, Massee M, Zabek N, Denoziere G. Properties ofdehydrated human amnion/chorion composite grafts: Implicationsfor wound repair and soft tissue regeneration. J Biomed Mater ResB Appl Biomater 2014;102:1353-62.

9. Koob TJ, Lim JJ, Massee M, Zabek N Rennert R, Gurtner G,et al Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair andregeneration. Vasc Cell 2014;6:10.

10. Zelen CM, Serena TE, Denoziere G, Fetterolf DE. A prospectiverandomized comparative parallel study of amniotic membranewound graft in the management of diabetic foot ulcers. Int WoundJ 2013;10:502-7.

11. Zelen CM, Serena TE. Snyder RJ. A prospective, randomizedcomparative study of weekly versus biweekly application ofdehydrated human amnion/chorion membrane allograft in themanagement of diabetic foot ulcers. Int Wound J 2014;11:122-8.

12. Massee M, Chinn K, Lei J, Lim JJ, Young CS, Koob TJ. Dehydratedhuman amnion/chorion membrane regulates stem cell activityinvitro. J Biom Mater Res B Appl Biomater 2015;104:1495-503.

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Figure 14. First postoperative visit after Epifix application and attempt at delayed primary closure.

Figure 15. Appearance at six weeks after Epifix and wound vacuum.