Diabetic Foot Ulcers Treated With Porcine Urinary Bladder Extracellular Matrix and Total Contact Cast: Interim Analysis of a Randomized, Controlled Trial

Objective. To evaluate the effectiveness of a porcine urinary bladder-derived extracellular matrix (UBM) for the treatment of recalcitrant, neuropathic diabetic foot ulcers (DFUs).Materials and Methods. This prospective, parallel, randomized, single-center study involving 17 subjects was performed in an outpatient wound care center setting. Each subject with a DFU was randomized (2:1) to receive the urinary bladder matrix (UBM) plus offloading with a total contact cast (TCC) or standard care (nonadherent dressing plus TCC). All DFUs were on the plantar surface of the foot and all were Grade I-A according to the University of Texas Wound Classification System. A traditional TCC was used in all patients and consisted of a minimally padded, well-molded, and rigid (plaster plus fiberglass) construct that maintains contact with the entire plantar surface of the foot and lower leg. The endpoints of the study were wound healing at 12 and 16 weeks and ulcer recurrence at 1 year. Wound evaluations were performed weekly and wound surface area was measured by photo-digital planimetry. Results. In the UBM group, the incidence of wound healing at 12 and 16 weeks was 90% and 100%, respectively, compared with 33% and 83.3% in the control (P = .062). The mean time to healing in the UBM-treated group was 62.4 days compared with 92.8 days in the control group (P = .031). The incidence of ulcer recurrence at 1 year was 10% (1/11) in the UBM-treated group and 50% (3/6) in the control. Conclusion. Results of this interim analysis suggest treatment of DFUs with a UBM could significantly reduce the time to healing and improve the rate of recurrence. 

It is well recognized that the primary factor in the cause of diabetic foot ulcerations (DFUs) is distal symmetrical neuropathy, the most common syndrome of diabetic polyneuropathy.¹ Peripheral neuropathy leads to diminished or absent sensation thus allowing excessive and prolonged stresses and pressures to occur in the diabetic foot, which ultimately results in skin and subcutaneous tissue breakdown.^2-5 Healing the ulcer is often difficult and prolonged due to the chronic wound environment that leads to progressive tissue degradation, neuropathic changes in the skin that decrease the sensitivity to applied pressure, difficulties in offloading based on the anatomical location of the ulcer, and less than optimal circulation (microvascular disease) that impedes the delivery of nutrients required to heal the wound and fight infection.^6-8

An adequate assessment of the patient and the foot ulcer is essential in the design of an effective standardized program for local wound management. A physical examination, detailed history, and diagnostic procedures designed to rule out peripheral arterial disease and osteomyelitis are essential. Clinical assessment of the DFU should guide management principles by helping determine whether the wound must be treated for infection, whether major or minor debridement is needed, appropriate selection of an effective offloading device, how long it will take the ulcer to heal, and what type of dressing should be used as healing progresses. After the ulcer has been debrided of the nonviable tissue, topical agents and dressings may be applied to the wound. Depending on the severity of the ulcer and the effectiveness of offloading, healing may take several weeks or months.^9,10 If the affected foot is effectively offloaded and there are no complications, most neuropathic ulcers heal completely in > 12 weeks.¹¹ However, DFU recurrence is common¹² and with every recurrence there is increased risk of nonhealing, infection, and osteomyelitis.¹³ Diabetic foot ulcer recurrence is caused by a combination of factors, including the quality of the healed tissues (scar), localized perfusion, and effective footwear that reduces friction, shear stress, and distributes pressure appropriately.¹⁴ 

Several extracellular matrix (ECM) materials or collagen-containing wound dressings and tissue-engineered constructs with and without cells have been shown to accelerate the healing of DFUs.^15-18 These ECM materials may have several advantageous features. It has been postulated that their use in chronic wounds leads to a reduction in collagenase and gelatinase (MMP-2, MMP-9) activity.¹⁸ It has been suggested that various ECM wound dressings may bind growth factors, promote angiogenesis, increase fibroblast production, and provide an antibacterial effect to the wound by their natural degradation.^15-17 

The ideal matrix scaffold for wound healing would be amenable to cell infiltration and proliferation, and it would establish milieus for the deposition of fibrous connective tissue and an epithelial layer. A previous study¹⁹ evaluated the cell infiltration of cells cultured in vitro into 3 ECM scaffolds that are currently indicated for wound management: urinary bladder matrix (UBM), small intestinal submucosa (SIS), and liver stroma. While all 3 scaffolds allowed for cell infiltration, only UBM showed bimodal cell growth patterns with cell infiltration on 1 surface of the scaffold and development of a monolayer of cells on the other surface. The bimodal cell growth patterns were due to the presence of an epithelial basement membrane on the surface that did not allow cell infiltration, as the epithelial basement membrane is the substrate upon which epithelial tissue grows.¹⁹ In addition, ECM scaffolds derived from UBM provide a biocompatible material that allows inductive tissue remodeling as well as promotes a normalized wound healing response.^18,19 Based upon this data, the present study was developed to evaluate the potential of UBM to facilitate healing of DFUs as compared to standard of care. This is the interim analysis of a study conducted to test the hypothesis that UBM improves both the rate of healing and scar durability when used in conjunction with a total contact cast (TCC).

Study objective and ethics statement. The purpose of this study was to assess the effectiveness of the UBM (MatriStem Wound Matrix; ACell, Inc, Columbia, MD) plus standard local wound care versus standard wound care alone for the treatment of DFUs as assessed by incidence of complete wound healing, which is defined as complete epithelialization with no drainage or dressings required by 16 weeks. Scar durability was also monitored for the incidence of ulcer recurrence.  

This study was performed in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki (20013), Title 21, Parts 50, 54, 56 of the US Code of Federal Regulations. Study stopping rules included stopping the study at the discretion of the Sponsor (ACell, Inc, Columbia, MD) or Principal Investigator if subject safety was of concern or at the subject’s request. The study protocol, investigators, and consent documents were reviewed and approved by an accredited institutional review board (IRB) (Calvary Hospital IRB, Bronx, NY), and all patients provided written informed consent before participating in the study. This study was registered at ClinicalTrials.gov as NCT02750280.  

Study design and population. This study was a prospective, randomized, controlled, parallel group longitudinal study testing the efficacy of the UBM for the treatment of DFUs. It was conducted in a single clinical center in an outpatient setting. The study was an open-label study comparing 2 treatment regimens: 1) UBM plus standard local wound care; and 2) control arm consisting of standard local wound care alone. Standard local wound care included offloading with a TCC. 

This study was initially designed to enroll about 50 subjects. While an interim analysis was not prospectively planned, one was undertaken to gauge a quantitative treatment effect. The authors selected to perform this analysis after 17 study patients had completed the 20-week treatment period and the 1-year follow-up period. The subjects were randomized (2:1 ratio) into 1 of 2 arms (UBM plus TCC or TCC alone [control]). Eleven subjects were treated with the UBM and 6 with standard local wound care.

The primary study endpoint was incidence of complete wound closure by 16 weeks. Secondary endpoints included incidence of 50% wound healing by week 4, ulcer recurrence assessed posttreatment at week 17, and once monthly up to week 52 (1 year). 

Key safety assessments included signs and symptoms of infection, ability to tolerate local treatment plus TCC, potential dermatological sensitivity to the porcine UBM (as measured by observed and reported discomfort and changes in skin during and after the treatment period), and frequency of adverse events (including unexpected adverse device events or serious adverse events) throughout the duration of the study.

Either male or female subjects aged between 18 and 85 years and diagnosed with diabetes mellitus (type 1 or 2) were eligible for enrollment. The DFU had to be on the plantar surface of the foot with a grade I-A DFU according to the University of Texas Classification System and a history of nonhealing > 2 months. The subject had to have adequate arterial circulation to the foot as determined by an ankle-brachial index (ABI) > 0.75, toe-brachial index (TBI) > 0.65, or toe systolic pressure > 50 mm Hg. Subjects were excluded if the DFU was not a grade I-A, if there were any clinical signs of infection, evidence of osteomyelitis, and if the ulcer was due to a nondiabetic etiology. Patients were also excluded if they had or previously had cancer (other than cutaneous epithelioma) not in remission, were pregnant or lactating women, were receiving oral or parenteral corticosteroids, had other advanced wound therapy (eg, autologous platelet-rich plasma gel, becaplermin, bilayered cell therapy, dermal substitute, ECM) or received topical collagenase, or currently enrolled or participated in another device, drug, or biological trial within the previous 30 days of enrollment 

At the initial visit (day 0), all study subjects had an abbreviated physical exam with medical and surgical histories and list of medications recorded. Laboratory assessments were performed and included radiologic studies (3-view X-ray) of the affected foot, routine bloodwork (metabolic panel [Chem 12], complete blood count plus differential, pregnancy test [urine or serum]), and an ABI using Doppler method. High-resolution ultrasonography was performed to evaluate for osteomyelitis.  

Wound assessment, preparation of wound site, and evaluations. Only 1 study ulcer was identified for each subject. If the subject had more than 1 ulcer that qualified for the study, the ulcer with the longest history of nonhealing was selected as the target ulcer.  

An initial, extensive debridement of the ulcer was performed to remove all nonviable tissue and wound margins excised to healthy bleeding tissue, so that the wound had a saucer-shape formation. After debridement, the ulcer was photographed with a 3-5 megapixel digital camera for measurement of surface area by photodigital planimetry²⁰ (PictZar; BioVisual Technologies LLC, Elmwood Park, NJ).

Clinical wound assessment and ulcer characteristics were recorded at the initial visit and then weekly during the follow-up visits for 16 weeks. Wound characteristics included signs and symptoms of local wound infection (erythema, edema, induration, and localized pain), undermining, the degree and amount of granulation, wound drainage, and the amount of nonviable tissue.

Test articles. The UBM product is an extracellular connective tissue matrix derived from porcine bladder. It is individually packaged and sterile. The product used, recently rebranded as Cytal Wound Matrix (ACell, Inc, Columbia, MD), is a Food and Drug Administration-cleared device designed for the management of chronic wounds. Standard local wound care consisted of nonadherent (siliconized) medical-grade foam (Mepilex Wound Dressing; Mölnlycke Health Care, Norcross, GA). A TCC was used in all patients and consisted of a minimally padded, well-molded, and rigid (plaster plus fiberglass) construct that maintained contact with the entire plantar surface of the foot and lower leg.  

The group randomized to receive the UBM received an application of the test agent upon initiation, at the visit on day 0, and then once per week during the treatment period for 16 weeks or until healing was achieved. After cleansing and drying the wound, it was traced to obtain a template that was then used to trim the UBM to fit exactly within the margins of the wound. The UBM was then moistened with saline and applied directly onto the wound bed. The UBM was secured to the wound with adhesive skin closure strips, and the secondary dressing was applied. On day 0, the control group received the initial treatment and subsequently once per week for 16 weeks or until healing was achieved. Healing for both groups was defined as complete epithelialization without drainage and no longer needing a dressing. Evaluations for all treatment groups were performed at day 0 and once per week during weeks 1–16. 

All study subjects (in both treatment arms) were off-loaded with a TCC. Recasting was applied at each visit (weekly) for 16 weeks or until healing was achieved.

All subjects whose DFU healed within the 16-week period were followed-up once monthly up to 1 year to evaluate for ulcer recurrence.  

Statistics. Proportion of subjects with complete ulcer closure over the 12-week and 16-week treatment periods were calculated using the nonparametric Cochran-Mantel-Haenszel test. Wound healing rates (percent change from baseline, time to 50% and 100% healing) were compared using the Wilcoxon rank-sum test. Chi-squared test was used for categorical values and Student’s t-test used for continuous variables. The analysis was by intention to treat (ITT), and SPSS for Windows Version 11.5 (SPSS Inc, Chicago, IL) was used for all statistical calculations.

A total of 17 patients provided informed consent and were randomized to treatment; 11 patients were in the UBM group and 6 in the control. All 17 subjects were evaluable for the ITT analysis. Table 1 summarizes the demographics and wound characteristics for both treatment arms. The demographics showed 82% of the subjects were < 70 years old, 83% male, 47% Hispanic, 41% African American, and 2% Caucasian. The mean ulcer size was 12.85 cm² with a mean ulcer age of 5.2 months. There were no statistically significant differences between treatment groups for any of these parameters.   

The mean time in days to reach 50% and 100% healing is shown in Figure 1. The mean time to 50% healing was similar between treatment groups (UBM: 26.7 days; control: 25 days). However, there was a 48.7% difference in the time to 100% wound healing in favor of the UBM-treated group (UBM: 62 days; control: 93 days).  

A significantly greater proportion of ulcers were healed at week 12 in the UBM group than in the control (91%, n = 10 vs. 33%, n = 2; P = .041). The incidence of complete healing was also greater for the UBM group at week 16 (100%, n = 11 vs. 83%, n = 5), a difference of 63% at week 12 and 20% at week 16 (Figure 2). 

The incidence of DFU recurrence is shown in Table 2. A recurring DFU is defined as the development of a new wound on the same anatomical location of the healed DFU. Of the 11 wounds treated with the UBM, only 1 had ulcer recurrence compared with 3 of the 6 subjects in the control group.  

From this analysis, based on the magnitude of the effect (effect size r = 0.35), a conventional level of alpha (0.05) and a 90% probability of detecting an effect, the minimum sample size would be N = 67.

The results of this interim analysis suggest that treating DFUs with UBM in conjunction with TCC could significantly reduce the time to healing and improve the rate of recurrence as compared with standard local wound care or TCC alone. The potential reduction in healing time for DFUs treated with UBM is observed despite no difference in the time to 50% wound closure. This suggests the wound environment that develops in the presence of the UBM is different than the wound environment of management with standard local wound care alone.

The study design in this trial incorporates the use of TCC, the “gold standard” for the treatment of DFUs. The design is consistent with real-life medical treatment for DFUs allowing for treatment of the ulcer while eliminating the issue of compliance associated with removable offloading controlled ankle motion walkers, footwear, or orthotics. It also demonstrated that UBM can be utilized in a safe and efficacious manner when applied once weekly under a TCC.  

 A preclinical study²¹ has shown a rapid cell infiltration into UBM, followed by a robust angiogenic response and new host tissue deposition in the presence of UBM materials in a variety of body systems. Another study²² also suggests the immune response to UBM is different than the immune response in classical wound healing. The immune response to UBM typically includes a prominent infiltration of macrophages that express a M2-like phenotype as opposed to M1 macrophages. M2 macrophages tend to be associated with tissue accommodation and remodeling, while M1 macrophages tend to be associated with scar tissue formation.²² It is possible that the modulated immune response and robust vascular supply enable the body to increase the rate and improve quality of new tissue deposition, which could explain the potential reduction in time to healing and rate of recurrence when wounds were treated with UBM.  Further studies are required to confirm these potential mechanisms.

The results of this prospective study of wound healing in chronic DFUs are consistent with the results of several published articles describing the use of UBM in wound healing.  Kimmel et al²³ described the successful healing of 3 chronic DFUs after application of UBM devices. A larger case series¹⁵ described the successful healing of 34 consecutive acute and chronic wounds in a 12-month period, including healing many wounds that had previously stalled after treatment with other advanced wound care therapies. The use of UBM has also been described in a case series where it was found to facilitate definitive soft tissue reconstruction by establishing a neovascularized soft tissue base acceptable for second-stage wound and skin coverage options within traumatic and combat-related wounds.²⁴ A recent study²⁵ describing results from a multicenter, randomized clinical trial showed that UBM had comparable wound healing characteristics, lower cost, and improved quality of life scores when compared to Dermagraft (Organogenesis Inc, Canton, MA). When considered along with the current results, there is a growing body of literature supporting the efficacy of UBM for managing hard-to-heal wounds.

The data from this preliminary analysis showed wounds managed with UBM healed more rapidly and with fewer risks of recurrence than the control-treated wounds, which is consistent with earlier reports using bioengineered skin and other cellular and acellular ECM materials.^16,17,26,27 Adverse events encountered were consistent with those observed generally in the treatment of DFUs with TCCs and were evenly distributed between treatment groups. The most common adverse events were local wound infection (n = 6), dermatitis (n = 4), and discomfort associated with a TCC (n = 1). There were 3 serious adverse events (cellulitis, n = 1; urinary tract infection, n = 1; congestive heart failure, n = 1), and all were unrelated to the test agent.

When interpreting the results, it is important to keep in mind that this is the report of an interim analysis and only involves 17 study subjects. These results merely indicate that continuation of the present study is warranted and provides a more reliable power calculation, which indicates the inclusion of 65 to 70 subjects would be needed to achieve the statistical goal related to the primary endpoint. 

Results of this interim analysis suggest that treating DFUs with a UBM together with adequate offloading with a TCC could significantly reduce the time to healing. In addition, the results also suggest UBM may improve the rate of DFU recurrence.

From the Center for Curative and Palliative Wound Care, Calvary Hospital, Bronx, NY; Department of Medicine, New York Medical College, Valhalla, NY; ACell, Inc, Columbia, MD; and Skin of All Color Dermatology and Aesthetic Medicine, LLC, East Windsor, NJ 

Address correspondence to:
Oscar M. Alvarez, PhD
Program Director, Vascular and Wound Care Center  
University Hospital, Rutgers NJMS 
150 Bergen Street (D-106) 
Newark, NJ 07101
alvareom@uhnj.org

Disclosure: The funding and product for this study were provided by ACell, Inc (Columbia, MD).