A Prospective, Multicenter, Randomized, Controlled Clinical Trial Comparing a Bioengineered Skin Substitute to a Human Skin Allograft

An estimated 25% of all people with diabetes may experience a foot ulcer in their lifetime, which may lead to serious complications including infection and amputation. A prospective, multicenter, randomized, controlled clinical trial was conducted to compare an in vitro-engineered, human fibroblast-derived dermal skin (HFDS) substitute and a biologically active cryopreserved human skin allograft (HSA) to determine the relative number of diabetic foot ulcers (DFUs) healed (100% epithelialization without any drainage) and the number of grafts required by week 12.   Secondary variables included the proportion of healed patients at weeks 16 and 20, time to healing during the study, and wound size progression. The 23 eligible participants (11 randomized to the HSA, 12 to the HFDS group) were recruited from two hospital-based outpatient wound care centers. Baseline patient (body mass index, age, gender, race, type and duration of diabetes, presence of neuropathy and/or peripheral arterial disease, tobacco use) and wound characteristics (size and duration) were recorded, and follow-up visits occurred every week for up to 20 weeks. Descriptive and multivariate regression analyses were used to compare wound outcomes. At baseline, no statistically significant differences between patients and wounds were observed. At week 12, seven (63.6%) patients in the HSA and four (33.3%) in the HFDS group were healed (P = 0.0498). At the end of the 20-week evaluation period, 90.91% of HSA versus 66.67% of HFDS were healed (P = 0.4282). Among the subset of wounds that healed during the first 12 weeks of treatment, an average of 4.36 (range 2–7) HSA grafts were applied versus 8.92 (range 6–12) in the HFDS subset (P <0.0001, SE 0.77584). Time to healing in the HSA group was significantly shorter (8.9 weeks) than in the HFDS group (12.5 weeks) (log-rank test, P = 0.0323). The results of this study are similar to previous outcomes reported using these treatment modalities and suggest that, after 12 weeks of care, DFUs managed with HSA are approximately twice as likely to heal as DFUs managed with HFDS with approximately half the number of grafts required. Research confirming these results with a larger sample size and in patients with different types of wounds is warranted.

Foot ulcer prevalence among people diagnosed with diabetes is 4% to 10%, and the lifetime incidence has been reported to be as high as 25%.¹ The burden of diabetic foot ulcers (DFUs) is enormous — 29.1 million people or 9.3% of the United States’ population have diabetes.² DFUs represent a substantial public health concern and pose major physical, social, and financial burdens, including a high risk of lower extremity amputation.^1,3,4 Affected individuals experience profoundly compromised physical quality of life, which is worse in persons with unhealed ulcers.³ Patients with DFUs require more frequent emergency department visits, are more commonly admitted to the hospital, and require longer lengths of stay.⁴ Pedal complications associated with diabetes such as ulceration, infection, and gangrene are the leading causes of hospitalization. An estimated 60% to 70% of people with diabetes have mild to severe forms of neuropathy,⁵ which results in impaired sensation in the feet and hands with elevated risk of injury. Severe forms of diabetic neuropathy are a major contributing cause of lower extremity amputations. More than 85% of nontraumatic lower extremity amputations are preceded by an active foot ulcer, and failure of wound healing is the most prevalent cause leading to amputation.⁶ In 2010, approximately 73,000 nontraumatic amputations were performed on people with diabetes.² In 2012, the treatment of diabetes and its complications in the United States generated approximately $176 billion in direct costs²; at least one third of these costs were linked to the treatment of DFUs.⁴ Average medical costs for people with diagnosed diabetes are more than double of what would be in the absence of diabetes.²

The primary goal in treating a DFU is to obtain wound closure in the shortest amount of time. Healing is often a lengthy and frustrating process for people with diabetes, for their families, and for their healthcare providers. Wound healing can be complicated by peripheral neuropathy, arterial insufficiency, infection, renal disease, and impaired inflammatory response.^6-8 Impaired wound healing is associated with morbidity, amputations, mortality, and high healthcare costs.^2,4,7,8 Accelerating the healing process and minimizing these complications become paramount for the treating practitioner.

Two advanced biologically active therapies are available for the treatment of DFUs. Dermagraft® (Organogenesis, Inc, Canton, MA) is an in vitro-engineered human fibroblast-derived dermal substitute (HFDS) manufactured from human fibroblast cells derived from newborn foreskin tissue that are seeded onto a bioabsorbable polyglactin mesh scaffold.⁹ Once formed, the graft is cryopreserved. HFDS is indicated for use in the treatment of full-thickness DFUs >6 weeks duration that extend into the dermis, but without tendon, muscle, joint capsule, or bone exposure.^9,10

TheraSkin® (Soluble Systems, Newport News, VA) is a biologically active human skin allograft (HSA) derived from donated human skin that has been screened via medical and behavior risk assessment in addition to microbiologic and serologic testing in compliance with the requirements of the American Association of Tissue Banks and the US Food and Drug Administration (FDA). The split-thickness skin graft is procured with a dermatome from tissue donors within 24 hours post mortem and is minimally processed and disinfected and cryopreserved at a controlled cooling rate to maintain the cellular and extracellular components. HSA is a human cell, tissue, and cellular- and tissue-based product (HCT/P) as defined by the FDA.¹¹ HSA is used to treat any partial- or full-thickness wound, including DFUs, and can be used over tendon, muscle, joint capsule, or bone.^12,13 In a previous study,¹⁴ it was demonstrated that the cryopreserved HSA contained more Type I, and significantly more Type II and IV collagen than either of the two most common HFDS products. A large retrospective study¹⁵ (N = 188) demonstrated great efficacy with cryopreserved HSA for the treatment of DFUs.

Despite the use of advanced, biologically active wound healing products for the treatment of DFUs, randomized controlled clinical studies comparing these products are rarely published. The intent of this study was to compare the use of two existing biologically active products to inform practitioners on appropriate treatments for their patients with DFUs. The primary objective of this prospective, multicenter, randomized, controlled clinical trial was to study patients with type 1 and type 2 diabetes and foot ulcers to determine the likelihood of healing and the number of grafts applied by week 12 using HFDS versus HSA — ie, to compare wound closure rate and the number of biologics required to achieve closure. The impact of additional cofactors such as body mass index (BMI) and wound size also was considered. Based on this objective, it was hypothesized that a statistically significant different number of wounds would close (100% epithelialization without any drainage) during the first 12 weeks of treatment with either an HSA or HFDS and that the number of grafts required to achieve closure also would differ significantly.

Setting. The study was conducted at two hospital-based wound care centers in three phases. An onsite monitor was present to observe the collection of data throughout the study.

Patient eligibility and randomization. The first phase (Screening) consisted of a series of screening assessments designed to determine study eligibility (see Table 1). Baseline measurements were taken and recorded before study enrollment. Patients who met the study eligibility criteria were randomly assigned to one of two treatment cohorts. Randomization was performed using a series of sealed envelopes that designated the biologically active treatment to be applied. Envelopes were randomized in blocks of six; however, the investigators were unaware of the block size or randomization scheme.

Block randomization is a widely used technique to prevent the investigator from second-guessing the randomization scheme. In this case, with a block of six, three out of every six envelopes would contain assignment to one treatment and three to the other. If the investigator was aware of the block size, he/she would know if three assignments to group one had been made; the remaining three assignments would be to the alternate group. Because the investigator did not know the block size, he/she still could not guess what the next envelope contained. Envelopes were filled by a third party using a randomization table or similar technique. Blocks are necessary to keep the group assignments approximately balanced. Although unlikely, without blocks, it would be possible to assign 20 participants in a row to the same treatment, depending on how the assignments were drawn. Block assignments guarantee that once six subjects have been enrolled, three will be placed in each group.

Patient demographic and wound characteristic variables recorded in the case report forms and included: age, BMI, diabetes type, race, ethnicity, gender, duration of diabetes, presence of insulin dependence, number of diabetic medications the patient currently was taking, wound duration, presence of peripheral artery disease (PAD), presence of peripheral neuropathy, smoking status, baseline wound size, and number of grafts used. Patient anonymity was maintained by assigning study code numbers to each patient at the beginning of the study; these codes were used as identifiers throughout the period of time during and after the participant was enrolled in the study. Codes could only be broken by the principal investigator in the event of an emergency.

Once randomized, patients returned 1 week later to begin treatment. Patients who presented with more than one wound were permitted to participate in the study. In this case, the study wound was the largest wound, and all edges of the study wound had to be at least 2 cm from the nearest adjacent wound edge.

Treatment and evaluation. In the second phase (Treatment), the patient returned to the clinic for initial application of the biologically active product. Patients randomized to the HSA group received a product application every other week; those randomized to the HFDS group were treated every week in accordance with each manufacturer’s product instructions.^9,13 In addition, preparation of the product (ie, thawing) before application was done precisely according to manufacturer instructions. In each case, wounds were prepared in the same way: nonviable tissue and callous were debrided from the wound surface and surrounding wound perimeter, respectively. Wounds were thoroughly cleansed with saline before application of the treatment product. The investigator physician at each site performed wound debridement and applied the biologically active product. All investigators were trained by company representatives for each product in order to ensure correct technique and had attended an investigator meeting to guarantee uniformity in technique across sites.

Once applied, both study products were covered with either Mepitel (Mölnlycke Health Care US, LLC, Norcross, GA) or Polymem (Ferris Manufacturing Corporation, Fort Worth, TX), according to the investigator’s preference. All wounds were offloaded with ½-inch felt as part of an aperture type of device. All participants were given either a healing sandal fabricated from a surgical shoe (Darco, International, Inc, Huntington, WV) or a fixed ankle boot (DH Off-Loading Walker; Ossur, Foot Hill Ranch, CA) if the patient required more protection. The decision to treat with either a surgical shoe or fixed ankle boot was based on the clinician’s assessment of the wound and the patient’s need for more stability. One participant in each group received the fixed ankle boot, and one in the HFDS group was treated with a total contact cast due to the inability to tolerate either a healing shoe or fixed ankle boot.

Treatment evaluations included investigator assessment and documentation on data collection sheets of wound closure and wound size. Only fully epithelialized wounds were considered healed. Because the grafts have a different physical appearance, it was not possible to disguise the type of graft used at the time of evaluation. At each visit, wounds were photographed and the margins of the wounds were traced along the inner edge of the margins. This was done after removal of the calloused perimeter, when present. Wound area was determined by measuring the length and width of the wound from the tracing and inserting these measurements into the equation for an ellipse. This is the same technique as described by Gilman.¹⁶ Adverse events, new medications, and any changes in the medical history were recorded. Any evidence of infection, as well as associated treatments, also was documented. No additional biologically active materials could be used and no vascular interventions were permitted during the course of the study. Patients who received either of these types of treatments during the course of the study were excluded from this analysis. The treatment phase continued for 12 weeks. However, if the wound closed before 12 weeks, one follow-up visit was performed to confirm wound closure. The time to closure was measured based on the visit where closure was first observed, not on the confirmation visit.

Follow-up. Patients with an unhealed wound at week 12 continued on to the third phase (Follow-Up) for treatment and evaluation for up to eight additional weeks. No additional biologically active products were used in either treatment group after the week 12 visit. Patients who still had ulcers were treated with saline-moistened gauze and debridement as needed, per the investigators. Persons whose ulcers were recorded as healed (100% closure as determined by the investigator) at the last scheduled treatment visit returned for one confirmatory visit. Participants with incomplete wound closure continued to be assessed until completion of week 20, at which time any subsequent treatment was provided outside of the scope of the study.

Data and statistical analysis. All data were transferred from the data collection sheets to a digital spreadsheet for analysis by an experienced, professional research monitor. The correctness of the data and any missing data points were noted. Each investigator confirmed the correct transfer and interpretation of their data. Unless specified otherwise, SAS Version 9.0 (SAS Institute Inc, Cary, NC) or higher was utilized to perform all statistical analyses. Continuous variables were summarized using general descriptive statistics, and categorical variables were summarized by percentages for proportions. Change in wound size per wound measurements was based on a comparison to the baseline area.

Data were analyzed using Student’s t-test and chi-square analysis to determine the equivalency of the two cohorts. Continuous covariates were assessed for normality and dichotomized or broken into quartiles if normality could not be justified (eg, BMI, wound duration, and baseline wound size were broken into quartiles for the analysis). Normality assessment included the Shapiro-Wilks test for normality, stem leaf plot, box plot, and normal probability plot. Any variable containing >20% missing data was excluded from further analysis. Any categorical or binary variable identified to cause a possible nonconvergence problem from pure results was collapsed if nominal or treated as a continuous variable if ordinal.

Multivariate logistic regression was used to evaluate the association of biologically active therapy agent and the proportion of healed wounds after 12 weeks from initial allograft application in order to control for identified confounders. Hypothesis testing was performed at the 5% significance level, with two-sided P values rounded to two decimal places. All confidence intervals had 95% coverage.

A Kaplan-Meier curve also was generated to demonstrate the cumulative percentage of healing in both groups.

Ethical considerations. Written informed consent was obtained from all study participants. This clinical research study was approved through the Western Institutional Review Board, Puyallup, WA.

Baseline demographics and characteristics. Patient baseline demographics and wound characteristics did not differ significantly between the 23 patients randomized to the HFDS (12 patients) or HSA (11 patients) treatment groups (see Table 2). No statistically significant difference was noted in age, gender, race, BMI, percentage of type 1 versus type 2 diabetes, duration of diabetes, number of medications taken, neuropathy, presence of peripheral vascular disease, percentage of subjects who were smokers, wound duration, PAD, and baseline wound size.

Wound healing. At week 12, seven (63.6%) wounds in the HSA versus four (33.3%) in the HFDS subset healed. Primary analysis revealed patients treated with HSA had greater odds of complete wound healing by 12 weeks (P = 0.0498) (see Table 3). Considering the sample size, confounders such as participant age, gender, BMI, wound size, wound duration before treatment, and type of diabetes were statistically indistinguishable (see Table 4). The overall model fit was better than intercept alone (likelihood ratio test = 17.62, P = 0.0015; intercept alone likelihood ratio test = 2.1443, P = 0.1431).

The percent healed after 16 and 20 weeks was greater in the HSA group but did not maintain statistical significance due to the small number of wounds that went on to close beyond the 12-week mark (see Table 3). However, when utilizing multivariate linear regression to assess the percentage in wound size reduction over the 20-week period, a statistically significant difference favoring HSA was noted (P = 0.0277) (see Table 5).

An insignificant number of adverse events were observed in both groups. In the HSA group, one patient developed erythema surrounding the ulcer site. X-rays performed on the day of observation were negative for any signs of bone infection, and the erythema resolved without the use of antibiotics. In the HFDS group, two patients developed maceration around the wound, which resolved after 1 week, and one patient required an increase in medication for hypertension, which was determined to be unrelated to the study treatment.

The average time to healing in the HSA group was 8.9 (range 5–20) weeks compared to 12.5 (range 7–20) weeks for HFDS. Furthermore, the HSA treatment had a significantly shorter time to healing compared to HFDS (log-rank test, P = 0.0323) (see Table 6). Kaplan-Meier analysis of percentage of wounds healed showed divergence of percentage of wounds healed starting at week 4 (see Figure 1).

Among the healed wound subsets, an average of 4.36 (range 2–7) HSA grafts were applied versus 8.92 (range 6–12) HFDS grafts (P <0.0001, SE 0.77584). This is consistent with manufacturer’s recommendations that HSA should be applied every other week; HFDS is applied weekly. Wounds that did not close continued to receive grafts every 2 weeks in the HSA group and every week in the HFDS group. Among patients who did not heal, the average number of HSA grafts applied was six, and the average number of HFDS grafts was 12 during the 12 initial weeks of treatment. No grafts were applied after week 12 in either group. Therefore, among those wounds that did not close, those patients received twice as many HFDS grafts, as compared to the HSA group.

The data presented in this study are consistent with previously published studies using the same biologically active products. In Landsman et al’s¹⁵ retrospective clinical study, 188 consecutive patients with DFUs and venous leg ulcers (VLUs) were treated with HSA. Multivariate logistic regression was used to evaluate the relationship between baseline wound size and the proportion of wounds healed after 12 and 20 weeks from initial HSA application. The authors found by week 12, DFUs closed 60.38% of the time and VLUs closed 60.77% of the time. After 20 weeks, the number of closed DFUs increased to 74.1% and the number of VLUs increased to 74.6%. In this study, the wounds were stratified by size, and the authors found that wounds that are smaller initially tended to close more quickly. The analysis demonstrated that the primary factor in wound closure was the use of HSA, and other factors such as neuropathy, glucose control, age of the wound, and age of the patient appear to have no statistically significant impact on the outcomes. This is consistent with the current study.

DiDominico et al’s¹⁷ prospective, comparative study of 28 patients with DFUs treated with either HSA or a human fibroblast and keratinocyte-derived dermal skin (HFKDS) substitute (Apligraf®, Organogenesis, Inc, Canton, MA) evaluated the percentage of wounds that achieved complete closure and the rate of wound closure. At 12 weeks, 66.7% of the HSA patients healed, compared to 41.3% of the HFKDS patients. The average time to wound closure for patients who received HSA was 5 weeks (SD 3.43) versus 6.86 weeks (SD 4.12) for patients who received HFKDS.

Marston et al’s¹⁰ prospective, randomized trial of HFDS demonstrated that patients with chronic DFUs of >6 weeks duration experienced a significant clinical benefit when treated with HFDS versus conventional therapy (saline-moistened gauze) alone. Complete wound closure by week 12 was achieved in 39 of 130 (30.0%) of the HFDS patients compared with 21 of 115 (18.3%) control patients (P = 0.023).

Harding et al’s¹⁸ open-label, prospective randomized trial of HFDS in patients with VLUs showed that 34% of 186 patients were healed by week 12 compared with 31% of 180 patients in the control group. For ulcers <12 months’ duration, 52% of 94 patients in the HFDS group versus 37% of 97 patients in the control group healed by week 12.

Although this was a prospective, randomized study, the sample size is small. This, of course, brings into question the power of a study to detect statistically significant relationships. In the current study, a statistically significant association was observed regarding the primary outcome. Subject randomization resulted in group balance based on measured demographic covariates (seen in Table 2). The near significance of one covariate (insulin dependent, %) may be indicative of inadequate power due to the small sample size, but this seems unlikely given statistical significance was achieved with regard to primary and secondary outcomes. The authors believe the near significance of that single covariate is likely attributable to the multiplicity problem inherent to univariate testing; future publications will seek to explore the relevance of this covariate. Nonetheless, randomization successfully achieved similarities between the two cohorts and minimized the impact of any secondary influences, and the power of this study is 0.80 based on the closure rate of the wounds in each group, making the difference in closure rates statistically significant. No significant differences were noted in the baseline wound size or other characteristics.

Other limitations are that two of the patients used a fixed ankle boot and one patient had a total contact cast. Thus, a slight inconsistency existed in the offloading techniques used due to the clinical needs of the patients.

Healing DFUs in a timely and cost-effective manner is an essential component of limb preservation and amputation prevention and should be considered in the selection of advanced biologically active products. Patients in the HSA treatment group had greater odds of complete wound healing by 12 weeks (P = 0.0498). Of those who healed, study participants who received HSA treatment also had a significantly shorter time to healing (mean 8.9 weeks) compared to persons receiving HFDS (mean 12.5 weeks) (P = 0.0323) and required significantly fewer graft applications to do so (average 4.36 in HSA versus 8.92 in the HFDS group). No adverse ulcer-related events were observed with either treatment group. The authors suggest HSA may be a highly effective, alternative to HFDS for the treatment of DFUs. Additional studies may include larger sample sizes and a variety of other wound types, including deeper and more complex wounds. Although HSA costs less and requires fewer grafts on average, a more detailed cost analysis would be beneficial as well in order to help direct a clinician toward one treatment or the other.

Data collection was performed by two of the investigators, and data were analyzed by independent analysts. This manuscript was authored by the physicians who participated in this study and was reviewed by the sponsors before publication. No data, findings, or statements were altered as a result of that review. The authors thank the Bon Secours Wound Care Clinic at Mary Immaculate Hospital, Newport News, VA, and Washington Hospital Wound Center, Washington, PA for their participation. The authors also recognize the contributions of Cynthia A. Dowd, RN, CWON; Susan R. Russell, RN; and the following investigators: Matthew Hopson, DPM; Brendan McConnell, DPM; Brandon Crim, DPM; and Nicholas Lowery, DPM.

Dr. Sanders and Dr. Arnold Landsman serve on the Scientific Advisory Board of Soluble Systems, Newport News, VA, and are paid consultants. Dr. Adam Landsman serves on the Scientific Advisory Board of Soluble Systems and on the Board of Directors of LifeNet Health, Virginia Beach, VA. Soluble Systems, LLC, and LifeNet Health supported this research investigation.

Dr. Sanders is a Clinical Professor (Adjunct), Department of Podiatric Medicine, Temple University, School of Podiatric Medicine, Philadelphia, PA. Dr. Adam Landsman is Assistant Professor of Surgery, Harvard Medical School; and Chief Podiatric Surgery, Cambridge Health Alliance, Cambridge, MA. Dr. Arnold Landsman is a Consultant to Soluble Systems, Newport News, VA; and former Chief, Podiatric Surgery, Inova Mt. Vernon Hospital, Alexandria, VA. Dr. Keller is a clinician, Colonial Foot Care, Hampton, VA. Dr. J. Cook is a clinical instructor, Harvard Medical School. Dr. E. Cook is a clinical instructor, Harvard Medical School; and Director of Podiatric Residency Training, Mt. Auburn Hospital, Harvard Medical School. Dr. Hopson is a clinician, TPMG Orthopedics and Sports Medicine, Williamsburg, VA.

Please address correspondence to: Adam S. Landsman, DPM, PhD, Harvard Medical School, Podiatric Surgery-Cambridge Hospital, 1493 Cambridge Street, Cambridge, MA 02445; email: adamlandsman@aol.com or alandsman@cha.harvard.edu.