Introduction Patients with diabetes are at increased risk of developing foot ulcers. Peripheral neuropathy increases the likelihood of foot injury, and peripheral vascular disease reduces normal healing of minor trauma, allowing development of ulcers. It is estimated that 15 percent of patients with type 1 or type 2 diabetes will develop an ulcer on a foot or ankle within the course of the disease.[1,2] Current standard care for diabetic foot ulcers includes removal of mechanical stress, sharp wound debridement, and use of dressings to maintain a moist wound environment.[2] In addition, infections of the wound site must be managed. Rates of healing with standard care are 24 percent after 12 weeks.[3] A majority of patients with failure of wound healing ultimately require surgery or amputation, and ulcers contribute to 85 percent of lower-extremity amputations for patients with diabetes.[4,5] The development of a new foot ulcer by a 40- to 65-year-old man can generate nearly $28,000 in additional cost of care during the two years after diagnosis.[6] The need to improve the success rate of healing in diabetic ulcers has prompted new therapeutic approaches. Biologic dressings, such as cultured epidermal allografts, can promote healing of a variety of wounds, including burns, venous ulcers, and split-thickness skin graft donor sites.[7–13] Because the allograft cells survive only briefly and do not become a permanent part of the regenerating tissue,[14] the postulated mechanism of action of cultured grafts is stimulation of wound repair through the release of multiple cytokines and matrix components.[15–18] Bilayered cellular matrix (BCM, OrCel™, Ortec International, New York, New York) is a porous collagen sponge containing cocultured allogeneic keratinocytes and fibroblasts harvested from human neonatal foreskin. This device has been approved for use in the United States (US) by the Food and Drug Administration (FDA) for the treatment of split-thickness donor sites of burn patients and in patients with mitten-hand deformities secondary to recessive dystrophic epidermolysis bullosa (RDEB).[19] BCM is not approved by the FDA for use on diabetic foot ulcers. The objective of the current study was to examine the effectiveness and safety of treatment of diabetic neuropathic foot ulcers using BCM plus standard care compared with standard care alone. Methods Study population. Patients included in the study were of either sex, between 18 and 85 years old, and had either type 1 or type 2 diabetes that was controlled (defined as no more than one hospitalization in the previous six months due to hyperglycemia, hypoglycemia, or ketoacidosis). Female patients could be neither pregnant nor nursing. Peripheral neuropathy had to be shown by the absence of protective sensation from the 5.07 (10g) monofilament pressure test. Included patients had adequate peripheral circulation (ABI > 0.7 and great toe pressure > 0.6) and glycosylated hemoglobin HbA1C < 12 percent. Patients with immunocompromising disease or other disease or treatments that would interfere with the study treatment were excluded. To be included, ulcers were Grade 1A as defined by The University of Texas Health Services classification of diabetic wounds. A Grade 1A wound is a superficial ulcer not involving tendon, capsule, or bone, without the presence of infection and ischemia.[21] Included ulcers were located on the plantar surface of the foot, between 1cm2 and 12cm2 in size and present for at least 30 days. Absence of osteomyelitis had to be shown clinically and radiographically (negative probe to bone test, plain film radiographs, and magnetic resonance imaging [MRI] if indicated). If a patient had more than one ulcer, the study ulcer had to be at least 2cm away from any other ulcer and could not have healed more than 30 percent during the two-week screening period. Study design. This study was an open-label, multicenter (eight centers in the US), controlled, randomized, parallel-group pilot study of patients with diabetes with neuropathic foot ulcers. The study protocol and written consent form were approved by an independent Institutional Review Board, and the trial was conducted in accordance with the ethical principals originating from the Declaration of Helsinki and in compliance with US and local regulatory requirements. All patients gave written informed consent before study entry. Patients meeting entrance criteria were enrolled in a two-week screening period, during which protocol-defined standard care treatment was initiated with the study ulcer. Standard care consisted of sharp wound debridement as necessary and a covering of moist saline gauze with a layer of transparent adhesive dressing (OpSite™ Transparent Adhesive Film Dressing, Smith & Nephew Inc., Largo, Florida) and gauze wrap; this dressing was changed twice daily. Sharp debridement of devitalized tissue utilizing a scalpel, tissue nipper, and forceps was performed in the clinic by the investigator. All patients were provided with a pressure relief walker (DH Pressure Relief Walker®, Centec Orthopaedics, Camarillo, California) and encouraged to limit mobility. Activity levels were monitored by reviewing a patient diary at each weekly clinic visit. At a second visit after 14 days, those patients who continued to meet eligibility criteria were individually assigned to the BCM treatment group or to the control group according to a computer-generated randomization code (Figure 1). Patients in the control treatment group continued with standard therapy for up to 12 weeks or until the ulcer was 100-percent reepithelialized with no drainage or need for protective dressings. Patients in the BCM treatment group received standard therapy plus an application of fresh BCM, which was then covered with a nonadherent dressing and gauze wrap (Adaptic®, Johnson & Johnson Wound Management Worldwide, Somerville, New Jersey). The gauze wrap was changed every 48 to 72 hours as required. Patients in the BCM treatment group received standard therapy plus a new BCM application at each weekly visit for up to six total applications until the ulcer was 100-percent epithelialized (complete healing). If healing had not occurred after six applications, standard care was given to the ulcer for an additional six weeks or until complete healing. Patients were evaluated weekly for up to 12 weeks until the study ulcer was 100-percent reepithelialized. At each visit, vital signs were taken, ulcer debridement was performed if needed, and the ulcer was assessed. Healing was measured by photography and sterile tracing for planimetry. The investigator also measured wound dimensions and assessed wound drainage, infection, and degree of healing. Measurements Efficacy. The primary measure for ulcer healing was planimetric analysis. Measurements were also made by photographic analysis and direct investigator assessment. Data from all patients who were randomized for the study (intent-to-treat population) were included in the analysis. For planimetric analysis, the investigator made sterile wound tracings of the open, unepithelialized regions of each ulcer at each visit. Calculation of the perimeter and area of the unhealed regions was made from the tracings by digital scanning and computerized planimetric analysis at a central facility. Personnel performing the measurements were blinded to treatment group assignments. Percentage wound closure over time was calculated based on planimetric measurements. Ulcers were photographed using a uniform protocol with identical equipment, film, and training at each site. Film was processed at a central facility and read in randomized order by two separate wound care experts who were blinded to specific protocol, patient, and visit date. Wounds were classified as unepithelialized, completely epithelialized, or “cannot evaluate” (if precluded by a technical reason). Investigator-assessed wound healing was defined as 100-percent epithelization with no drainage or need for absorbent dressing. Efficacy was measured as the proportion of ulcers in each treatment group that showed complete healing at the end of active treatment, the mean rate of wound closure from visit to visit for each treatment group, and the cumulative proportion of patients in each treatment group showing complete wound closure over time. Safety. Incidence of adverse events, withdrawals due to adverse event, incidence of infection at wound site, and vital signs were monitored. Data analysis. For the continuous variable of wound size, the t-test was used to evaluate the rate of wound closure. Time to healing was evaluated from the Cox Regression Model or Log-Rank Test. For comparison of proportion healed, the Chi-square was used. Results Patient demographics and baseline characteristics. The two treatment groups showed similar demographics (Table 1). Descriptive statistics of baseline ulcer characteristics are shown in Table 2. Ulcers were stratified into two categories based on wound area: ulcers of less than or equal to 6cm2 and those > 6cm2. Approximately 70 percent of the ulcers were < 6cm2 in size. Proportion of ulcers healed at endpoint of study. Within the 12-week study period, 35 percent of all ulcers in the BCM treatment group were completely healed, compared with 20 percent of ulcers in the control treatment group (Figure 2). Examining the stratum of ulcers with baseline size less than or equal to 6cm2 showed that 47 percent of ulcers in the BCM treatment group were completely healed (7/15), compared with 23 percent (3/13) of those in the control treatment group. Ulcers with a baseline area of greater than or equal to 6cm2 showed that none of the ulcers in the BCM group were completely healed (0/5), while 14.3 percent of the wounds receiving standard care alone healed by the 12 week endpoint (1/7). Rate of reepithelization. The rate of wound closure was calculated based on planimetrically measured total epithelialized area at each visit. The mean rate of wound closure per day was higher for the BCM treatment group (1.8 ± 2.5% per day) than for the control treatment group (1.1 ± 1.9% per day) over the 12-week treatment period (p = 0.0087). The mean rate of wound closure per day in BCM-treated wounds was 2.2 ± 2.8 percent per day (Table 3) compared to 1.1 ± 1.9 percent per day in those wounds receiving standard care alone (p = .001). Figure 3 illustrates the rates of closure of wounds that were of less than or equal to 6cm2 at baseline. The mean rate of wound closure of the BCM-treated group was significantly higher than that of the standard treatment group during the initial three weeks of treatment, gradually declining to approximately 1.4 percent per day by week 5. The mean rate of healing of wounds in the standard-care treatment group showed an approximately steady rate of 1.1 percent per day during the 12 weeks of treatment. Safety. There were no treatment-related adverse events in either treatment group. In the BCM treatment group, two patients had infections associated with the study ulcer compared with four patients in the standard treatment group. None of the infections in the BCM treatment group were serious compared with one serious infection in the standard treatment group. Two patients in the BCM treatment group were withdrawn prior to the end of treatment. One patient had an adverse event unrelated to the study treatment, and the second was withdrawn due to treatment failure. The baseline size of the ulcer (17.6cm2) was larger than allowed by the protocol and may have contributed to the treatment failure. Conclusions Standard state-of-the-art care of diabetic foot ulcers requires intense medical attention, wound debridement, control of infection, offloading of pressure, and monitoring circulatory adequacy.[2] However, standard care is not always successful4 and is highly expensive.[6] Diabetic peripheral neuropathy, both sensory and autonomic, and peripheral vascular disease contribute to the abnormal healing of these chronic ulcers,[2] producing the need for interventions that will augment the healing process. The BCM treatment described in this study augments the wound healing process in patients with diabetes, increasing the speed of wound closure and the success rate for complete healing. In this 12-week trial, initial treatment with up to six weeks of BCM followed by standard moist saline care produced a greater percent of patients with complete healing of chronic diabetic foot ulcers than did treatment with standard care alone. The mean rate of reepithelization (wound closure) averaged over the duration of the study was significantly higher with BCM-treatment than with standard care alone. Treatment with BCM produced high initial rates of wound closure that gradually slowed during the first three weeks, while the rate of wound closure with standard treatment remained low and relatively constant over the 12 weeks of observation. Recently, Margolis, et al.,[21] showed that wound size, duration, and grade directly impact the likelihood of healing. Our results reflect these findings in that those patients with larger wounds (of lgreater than or equal to 6cm2) responded poorly compared to those with smaller wounds regardless of treatment arm. In fact, only one of the 12 wounds in the group with baseline size greater than or equal to 6cm2 healed after 12 weeks; this patient received standard care without BCM. Additionally, there was no statistically significant difference in the rate of wound closure in the class of wounds with baseline size greater than or equal to 6cm2, while statistical significance was demonstrated in both the less than or equal to 6cm2 group and the entire study population. Growth factors and cytokines secreted by the BCM graft are proposed to stimulate wound repair by the patient’s own cells. The observed accelerated rate of reepithelization of chronic diabetic foot ulcers with BCM treatment is consistent with the hypothesis that the BCM graft cells exert a stimulatory effect on wound repair. In summary, this study has demonstrated pilot evidence of BCM’s effectiveness in the treatment of neuropathic diabetic foot ulcers and will serve as baseline to construct an adequately powered larger pivotal trial. Acknowledgments We acknowledge the following investigators for their contributions to this clinical trial: John Capotorto, MD, Staten Island University Medical Center, Staten Island, New York; Russell Caprioli, DPM, Long Island Jewish Medical Center, New Hyde Park, New York; Michael DellaCorte, DPM, St. Joseph’s Hospital, Flushing, New York; Jill Babbitt, MD, Wound Care Consultants, Milwaukee, Wisconsin; and David P. Steed, DPM, Lehigh Valley Podiatry Associates, Allentown, Pennsylvania.