Acellular Fish Skin Graft Use for Diabetic Lower Extremity Wound Healing: A Retrospective Study of 58 Ulcerations and a Literature Review

Objective. This retrospective study evaluates the efficacy of acellular fish skin graft for the treatment of full-thickness diabetic foot ulcers (DFUs). The primary objective is to calculate the total wound surface area (cm²) healed over a 16-week period. The secondary objective is to provide a systematic review on acellular fish skin grafts. Materials and Methods. There were 51 patients with a total of 58 DFUs treated with an acellular fish skin graft by the principal investigator. The initial wound surface area at first application was compared with the final wound surface area to conclude the percentage of total wound healed over a 16-week treatment period. Results. At 16 weeks, there was a mean reduction of wound surface area by 87.57% and 35 wounds (60.34%) fully healed. The systematic literature review included 10 fish graft articles, 3 of which specifically evaluated lower extremity ulcers. The reviewed studies supported improved wound healing with fish graft application, with benefits noted in dentistry, neurology, and wound care. Conclusions. This retrospective study further supports previous evidence that acellular fish skin graft promotes wound healing in DFUs. In particular, a rapid increase in wound healing was observed during the initial 4 weeks following graft application. This study and review of the literature indicated that fish graft encourages wound healing by enabling the wound to transition from a chronic to an acute stage of healing.

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Clinical Experience Using a dACM Construct for the Management of Wounds

In 2017, the Centers for Disease Control and Prevention estimated 30.3 million people in the United States (9.4% of the population) have diabetes.¹ Due to this large patient population, diabetic wounds have become a growing problem. Lev-Tov et al² acknowledged that 10% to 25% of these patients develop a diabetes-related foot ulceration. As a result, treatment of diabetic foot ulcers (DFUs) creates a large financial burden of about $38.6 billion in the United States annually.³ Therefore, extensive research in the treatment of DFUs is necessary to provide the most beneficial and cost-effective care. Diabetic foot ulcers are costly, dangerous, and the leading cause of nontraumatic lower extremity amputations in the United States.² The 5-year mortality rate after a nontraumatic lower extremity amputation has been studied extensively with results ranging from 56% to 70%.^4-6

In order to prevent limb- and life-threatening sequela from diabetes, it is essential that research be conducted to evaluate the effects of wound care products on wound healing. Rapid wound healing is vital to prevent lower extremity amputations. By utilizing the most effective measures to heal wounds, nontraumatic amputation may be avoided in these high-risk patients.

The patient with diabetes is at high risk for amputation due to a delayed wound healing response. In a healthy patient, wound healing progresses in a predictable fashion through 4 stages: hemostasis, inflammation, proliferation, and remodeling. First, the inflammation stage lasts about 2 to 4 days. During this stage, platelets release factors that acquire neutrophils and monocytes, which then draw in lymphocytes and fibroblasts. Next, at days 3 to 7, the proliferation stage progresses with angiogenesis, collagen formation, and epithelialization by fibroblasts. Finally, the remodeling phase lasts up to 1 year; over that time, newly formed capillaries collapse to allow the vascular density and tissue strength of the wounded area to return to normal. Diabetic wounds do not undergo these 3 typical stages and become stuck in the first stage (ie, inflammatory stage), resulting in chronic inflammation and delayed wound healing.^7,8

Wound care products have been created to encourage a wound to transition from a chronic inflammatory phase into an acute proliferation phase of healing. Numerous xenografts have been developed to assist with the progression of wound healing. In 2013, the US Food and Drug Administration approved Kerecis Omega3 (Kerecis, Isafjordur, Iceland), an acellular fish skin graft, for the treatment of various wounds including diabetic ulcers. Piscine grafts have an advantage over mammalian grafts because they do not risk transmission of disease⁹; therefore, a piscine graft is able to undergo a simpler sterilization process. During mammalian graft sterilization, harsh detergents are used to eliminate any risk of viral transmission, but these detergents also remove fat cells. Since piscine grafts do not undergo the same sterilization process, they are able to retain an omega-3 fat source.⁹ Previous research has supported that omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), reduce inflammatory responses and advance proinflammatory cytokines that promote wound healing.¹⁰ These factors are found largely in piscine skin.¹¹ The aforementioned proprietary piscine graft is an acellular intact fish skin that contains omega-3 polyunsaturated fatty acids, which has been shown to assist the wound in leaving the chronic inflammation state.⁷

The acellular fish skin graft is harvested from North Atlantic cod and contains collagen, fibrin, proteoglycans, and glycosaminoglycans; therefore, it acts as a skin substitute.¹⁰ In addition, the omega-3 fatty acids within the acellular fish skin graft promote wound healing by functioning as an anti-inflammatory factor. Utilizing this evidence, the primary purpose of this study is to retrospectively review DFU healing with acellular fish skin graft application. To the best of the authors’ knowledge, this is the largest and longest study on DFUs treated with an acellular fish skin graft. The secondary objective is to provide a systematic review on piscine grafts. Ultimately, the present results will be compared with previously published reports on fish graft use for lower extremity wounds.

Retrospective study
A retrospective, nonblinded evaluation of DFU healing with acellular fish skin graft application between January 1, 2014, and December 31, 2017, was performed. 

The St. Vincent Hospital Institutional Review Board (Indianapolis, IN), in collaboration with the American Health Network of Indianapolis, approved collection of deidentified patient information to be stored securely on REDCap (Vanderbilt University, Nashville, TN).¹² Patients were included in the study if they had diabetes and more than 18 years of age, had a history of an acellular fish skin graft application to a full-thickness DFU (distal to the ankle malleoli), and were followed until the wound healed or for 16 weeks. Data were obtained for 51 patients by the principal investigator. Patients were excluded if the wounds were partial thickness or not followed for a full 16 weeks.

Patient charts were reviewed starting with their first presentation of the treated wound. The initial application of the acellular fish skin graft was recorded along with the wound’s progress through 16 weeks post graft application. A deidentified spreadsheet was utilized to record data, which was stored on REDCap. The data sheet included wound location, classification (Wagner grade), duration, and size as well as use of offloading at each visit. Additional information recorded included gender, age, comorbidities, vascular status, and hemoglobin A_1c (HbA_1c). Vascular status was evaluated first by palpation of the dorsalis pedis and posterior tibial pulses. Pulses then were recorded as either palpable or nonpalpable. If nonpalpable, then ankle-brachial index (ABI) results were obtained and evaluated. The ABI results were deemed abnormal if the radiology report specifically stated they were abnormal. Additionally, charts were evaluated for HbA_1c levels to provide an indication of diabetes control.

The included patients had recommended follow-up appointments every 1 to 2 weeks with wound measurements obtained at each visit; however, due to the retrospective nature of the study, adherence was not enforced. Patients were not excluded from the study if their suggested 1- to 2-week appointments were missed; however, they were excluded if they did not follow-up at 16 weeks. The broad inclusion criteria allowed for a realistic study population. Wound measurements (length, width, depth) were obtained and recorded only by the principal investigator.

Wound graft application was completed according to local standard of care. Each target wound was thoroughly debrided of nonviable soft tissue and then measured. Next, a piece of acellular fish skin graft was applied to the wound bed and followed by application of a nonadherent dressing (ADAPTIC TOUCH Non-Adhering Silicone Dressing; KCI, an Acelity Company, San Antonio, TX). Wound healing was defined as complete epithelialization of the wound and deemed as such by the principal investigator. Based on these measurements, the surface area of the wound was calculated from length multiplied by width (cm²).

Wound healing rates were calculated by normalizing the wound area at initial graft application to a value of 100%. The rel. wound area was then calculated at each visit using the Formula to allow for analysis of wound healing rates throughout the study, which was the same method Trinh et al¹³ utilized to evaluate wound healing rates.

Systematic review
The authors conducted a literature search on PubMed using the following terms: piscine acellular dermal graft, acellular fish skin, and fish skin graft. Articles were restricted to only those published in the English language before December 31, 2017. No additional restrictions were applied. The reference lists from the identified studies then were evaluated to locate additional articles. Researchers reviewed the articles for appropriateness; unanimous agreement was a requirement for final inclusion. The studies were graded according to the American College of Foot and Ankle Surgeons’ Levels of Clinical Evidence Guidelines (LOE). Data obtained from the literature were compiled in a table to include the following: author, publication date, LOE, methods, number of participants, results, and conclusions.

Retrospective review
The study included a total of 51 patients with 58 DFUs. The participants were comprised of 13 (25.49%) women and 38 (74.51%) men, with a mean age of 66 years (range, 45–88 years). Patient demographics are listed in Table 1. The mean number of comorbidities per patient was 4.73 (Figure 1). Hemoglobin A_1c data were available for 50 (98.04%) of the 51 patients. The overall mean HbA_1c was 7.45%, and the mean HbA_1c for healed patients was 7.34% and 7.6% for nonhealed wounds.

Ten patients (19.61%) had an abnormal ABI result. Although ABI results were abnormal, 6 patients (60%) fully healed their wounds. Furthermore, a total of 8 abnormal ABI patients (80%) had > 97% of wound surface area healed.

The mean initial wound size was 3.02 cm² (range, 0.04 cm²–15.0 cm²). The locations of the wounds were as follows: 17 (29.31%) toe, 14 (24.14%) forefoot, 17 (29.31%) midfoot, 5 (8.62%) heel, and 5 (8.62%) dorsal foot (Figure 2). Wounds were classified based on Wagner grades: 43 (74.14%) grade 1, 11 (18.97%) grade 2, and 4 (6.90%) grade 3 (Figure 3). The mean ulcer duration at initial graft application was 18 weeks (range, 1–156 weeks).

The total number of feet (right and left) with wounds was 54; however, only 34 had documentation indicating the type of offloading used. The 34 documented cases were evaluated based on offloading used (Figure 4). A total of 19 feet (55.88%) healed using a form of offloading. Of the 19 healed feet, 15 (78.95%) used a modified controlled ankle movement boot or postoperative shoe (Figure 4).

At 16 weeks, there was a mean reduction of wound surface area by 87.57%, and 35 of 58 (60.34%) wounds fully healed (Figure 5). By 16 weeks, > 90% reduction in surface area was achieved in 43 DFUs (74.14%) and > 75% reduction was seen in 49 (84.48%). There were only 2 wounds that saw no reduction in surface area at 16 weeks; both wounds healed eventually, with 1 healed at 24 weeks after 2 additional applications of acellular fish skin graft. The other healed at 36 weeks with only 3 additional wound debridements and no additional acellular fish skin graft applications; it should be noted that this wound was the oldest and had been present for more than 3 years.

The mean number of acellular fish skin graft applications per patient was 4.9. The mean time to achieve wound healing in the 35 fully healed wounds was 10 weeks. The mean number of grafts used on fully healed patients was 4.5 (Table 2).

Systematic review
A total of 10 articles met inclusion criteria for the literature review, either from initial PubMed review or from additional search of the literature. The current literature indicates that fish graft has benefits in many fields of medicine, including neurology, dentistry, and wound care (Table 3^7,9,14-18).

Three studies evaluated fish skin grafts for strictly lower extremity wounds (Table 4^10,13,19). Cyrek et al¹⁹ presented a case study of an 86-year-old patient with a chronic lower extremity venous ulcer in which the ulcer healed within 14 weeks with fish graft applications. Trinh et al¹³ completed a larger case study with 7 complicated wounds (bone exposed). Five forefoot wounds took 13 to 41 weeks to heal. It was noted that wound area decreased significantly by 50% in the first third of treatment time. Yang et al¹⁰ evaluated wound healing in 18 patients with chronic lower extremity ulcers over 5 weeks. Similar to Trinh et al,¹³ they¹⁰ found wound surface area and wound depth had decreased by 40% and 48% at 5 weeks, respectively. Also at 5 weeks, Yang et al¹⁰ noted 3 of 18 wounds (16.67%) healed.

The current standard of care for DFUs includes glucose control, adequate extremity perfusion, debridement of nonviable tissue, offloading, infection control, local wound care, and patient education.² Despite an extensive and thorough standard care regimen, about 30% of DFUs heal at 20 weeks.³ In order to expedite healing, physicians have added biologics into the treatment regimen of DFUs. Unlike other biologics on the market (eg, human, porcine, ovine, bovine, equine), fish graft is able to retain its natural fat source of omega-3 polyunsaturated fatty acids, EPA and DHA. These lipids have been shown to reduce inflammatory responses and advance proinflammatory cytokines in wounds; therefore, enabling the DFU to transition from a chronic inflammatory state into an acute wound.¹⁰ Baldursson et al¹⁴ compared acellular fish skin graft with porcine grafts in a double-blind, randomized, controlled clinical trial. The results¹⁴ indicated 77.5% of wounds treated with fish graft healed compared with 65% of wounds healed at 3.5 weeks with porcine graft. Those results¹⁴ support faster wound healing with fish graft compared with porcine grafts.

In comparison with amnion/chorion membrane products, the acellular fish skin graft structure is porous rather than dense. This porous structure facilitates 3D wound cell ingrowth.¹⁵ Additional prospective controlled studies are necessary to compare wound healing with acellular fish skin graft to healing with other allografts.

Although the quantity of current research is limited on strictly lower extremity ulcerations treated with fish graft, the results and conclusions agree that improved wound healing is noted. At 4 weeks, studies have reported a decrease in wound surface area by 40% to 50%.^10,13 The current results are similar and indicate a 64% reduction in wound area at 4 weeks. The findings of this retrospective study support current evidence that fish graft encourages the progression of healing from a chronic to an acute wound.

To the best of the authors’ knowledge, this is the largest and longest retrospective study on acellular fish graft for DFUs.

In the current study, broad inclusion criteria was utilized to represent a high-risk, complicated, and realistic patient group; however, this may have adversely affected the results. Patients were included even if they were noncompliant with the preferred treatment plan. All patients were instructed to limit weightbearing using an offloading device, but many patients refused offloading devices and did not limit their weightbearing. Also, patients were included even if scheduled appointments were missed. An additional study is necessary to evaluate acellular fish skin graft use in patients with strict inclusion criteria that mandates offloading and weekly graft applications, which may influence and possibly improve results. It is important to note that despite the broad inclusion criterion, the present study shows favorable outcomes with acellular fish skin graft for DFU healing. 

The results of this 3-year retrospective study support increased wound healing rates with the use of acellular fish skin graft for treatment of DFUs. Furthermore, rapid wound surface area reduction during the initial 4 weeks following graft application corroborates previous research^10,13 that acellular fish skin graft enables the DFU to transition from a chronic inflammatory stage to an acute wound environment for healing. 

Authors: Shannon Michael, DPM^1,2; Christopher Winters, DPM³; and Maliha Khan, DPM¹

Affiliations: ¹St. Vincent Hospital, Indianapolis, IN; ²Foot & Ankle Clinics of Arizona, Chandler, AZ; and ³American Health Network, St. Vincent Hospital

Correspondence: Shannon Michael, DPM, Foot & Ankle Clinics of Arizona, 1831 East Queen Creek Road, Chandler, AZ 85286; srmichael527@gmail.com 

Disclosure: Dr. Winters is a member of the Kerecis (Isafjordur, Iceland) Speaker’s Bureau. No financial support was received for this study. During the time of this study, Dr. Michael was a resident with St. Vincent Hospital.