Electroceutical treatment of infected chronic wounds in a dog and a cat

Abstract

Objectives:

To describe the use of an innovative printed electroceutical dressing (PED) to treat non-healing, infected chronic wounds in one dog and one cat, and report outcomes.

Animals:

A four-year-old, female spayed Mastiff and a one-year-old, female spayed domestic shorthair (DSH) cat.

Study design:

Short case series.

Methods:

Both cases had chronic wounds (duration: approximately 1 year for the dog and 6 3/4 months for the cat) that remained open and infected despite various wound management strategies. Both animals were treated with the PED. Observations from the records regarding wound size, antimicrobial susceptibility and time to healing were recorded.

Results:

After 10 days of PED treatment in the dog and 17 days of PED treatment in the cat, the wounds had decreased in size by approximately 4.2 times in the dog and 2.5 times in the cat. Culture of punch biopsies yielded negative results. Wounds were clinically healed at 67 days in the dog and 47 days in the cat. No further treatment of the wounds was required beyond that point.

Conclusion:

Application of a PED led to closure of two chronic wounds and resolution of their persistent infection.

Clinical Significance:

PEDs may provide a new treatment modality to mitigate infection and promote healing of chronic wounds.

Introduction

Chronic wounds in humans are defined as wounds that fail to heal in 4-6 weeks^1,2. Moreover, for animals such as dogs, chronic skin wounds are defined as full-thickness defects that fail to heal despite appropriate antibiotic therapy and bandaging³. Often, chronic wounds show significant bacterial infection^4,5 with recalcitrance towards standard antibiotic treatment^6,7. Chronic wound management and treatment presents a major healthcare and financial burden in the United States. Domestic and livestock animal wound management costs are predicted to be $1.1 billion in 2021 with a 7% expected increase per year⁸, and the costs are even higher for human patients ⁹.

In addition to an economic burden, chronic wounds pose significant scientific, technological, and clinical challenges^9,10. In human chronic wounds, delayed healing has been attributed to the presence of bacterial biofilms; however, the role of biofilm infection in veterinary patients is ill-defined^11-13. The bacteria in biofilms have lower metabolic needs than planktonic bacteria¹⁴, which are non-adherent cells considered to be free-living. In contrast, biofilms are bacterial communities encapsulated in extracellular polymeric substances ¹⁵. Biofilm characteristics allow the bacteria to exhibit avoidant or resistant behavior to state-of-the-art treatments with antimicrobials and host immune defenses¹⁶. Moreover, in both veterinary and human patients, molecular diagnostic methods have identified a broad spectrum of microorganisms suggesting that chronic wound infections are often polymicrobial^10,17,18.

Current clinical practice for bacterial clearance of chronic wounds uses mechanical debridement or a combination of debridement with existing antimicrobials, skin grafts or skin flaps, and negative pressure dressings^19-22. However, using multiple or combinatorial treatments can be costly and prolongs treatment duration. Moreover, for chronic infections, debridement has been noted to be only modestly effective^23,24.

Bacteria were recently found to rely on electrostatic interactions for adhesion to surfaces²⁵, thereby motivating the evaluation of electrical stimulation as a possible bacterial inhibitory agent. Electroceuticals, which generate open circuit potentials due to the presence of dissimilar metals in contact with a conducting fluid, have become an emerging treatment modality as electrical stimulation wound dressings. Electroceuticals, such as Arthrex® and Procellera®, have become commercially available with US Food and Drug Administration approval²⁶. These dressings use the generated electric fields from the open circuit potential to help disinfect but have no current flow²⁶. In contrast, the PED described here uses direct current (DC) with a battery pack as a source of electric potential. DC has been shown to have bacterial inhibitory effects against gram-positive and gram-negative bacteria^27-29. In vitro studies in the presence of electrical stimulation have shown that bacterial inhibition occurs due to the generation of hypochlorous acid (HOCl)^30-32, which is a known antimicrobial agent³³. In addition to having beneficial antimicrobial effects, some studies have suggested that electrical stimulation may promote wound healing by stimulating angiogenesis and keratinocyte proliferation and migration^34-36.

There are no current reports of using DC dressings clinically in cats. However, we have previously reported proof of concept for the feasibility of clinical PED use in dog wounds³¹. The previous report included images of the dog wound but did not provide detailed results for the clinical case. The primary focus was on developing the PED as a dressing with the design, fabrication, systems engineering, in vitro disinfection, and electrochemical characterization of the PED³¹. One of the key findings reported was a proposal for a new physical biomarker, which was the electrical impedance of the wound, to monitor wound healing remotely³¹ with the PED. The objectives of the current report were: (1) to describe the clinical findings, specifically, the culture results and change in wound size in a dog and a cat that were treated with an innovative electroceutical dressing and (2) to report the outcome of these cases post treatment.

Materials and Methods

Printed Electroceutical Dressing

The printed electroceutical dressing (PED) fabrication and its safe use has been previously reported^31,37. Briefly, the fabric-based PED was constructed by screen-printing medically compatible Ag/AgCl ink (Creative Materials #113-09) onto a habotai silk substrate (Figure 1A)³¹. The PED was powered by a 6 V battery with maximum current limited to 600 μA through a 10 kΩ ballast resistor. Medical tape encapsulates all electrical components while providing an electrical and fluid isolation layer, leaving the printed electrodes and battery pack accessible for use. The larger dressings (7.5 cm x 7.5 cm) presented an interdigitated electrode layout that maximizes the anode area (Figure 1B, 1C) for highest bacterial clearance^30,31. For cat wounds, a smaller PED (1 cm x 2 cm) was used with the simplified electrode geometry (Figure 1D, 1E) to match previously reported in vitro bactericidal effects^30,32.

Figure 1.

Overview of PED design. (A) The 7.5 cm x 7.5 cm PED with the electrodes in an interdigitated design. (B) and (C) The 7.5 cm x 7.5 cm PED used on the dog is connected to a 6V battery pack with circuit connections concealed with medical tape. (D) The 1 cm x 2 cm used on the cat PED has simplified electrodes with equally sized anode and cathode. (E) The 1 cm x 2 cm PED is connected to a 6V battery and the circuit connections sealed with medical tape. Both PEDs have a ballast resistor (10 kΩ) limiting the current to 0.6 mA^31,37.

The laboratory fabricated PEDs needed to be secured to the wound for treatment and the electrodes require a moist environment for proper functioning. Therefore, for PED treatment, a hydrogel was first applied to the wound bed to ensure the PED electrodes were moistened when applied. The PED was then affixed with bandages so that the electrodes lay within the wound bed.

PED Application in a Dog

Before PED application, the dog was sedated with intravenous butorphanol tartrate (0.2 mg/kg) and dexmedetomidine HCl (4 mcg/kg). The peri-wound skin was clipped and cleaned with a chlorhexidine scrub (2%) then benzalkonium chloride (Zephiran) was used to remove the chlorhexidine scrub. A 6 mm punch biopsy of the center of the granulation tissue was cultured and revealed Staphylococcus pseudintermedius and Streptococcus canis with antimicrobial insensitivity to ampicillin and penicillin. The purpose of the biopsy was to conduct a deep-tissue culture for detection and identification of infection. Hydrogel (Skintegrity, Medline Industries, Inc) was copiously applied to the granulation bed. A 7.5 cm x 7.5 cm PED (Figures 1B, 1C) was applied to the wound and secured to the limb with a soft padded bandage. Four PEDs were applied (initial dressing plus three dressing changes) over a 10-day treatment period with the first PED application classified as day 0.

PED Application in a Cat

Prior to applying each PED, the cat was sedated with methadone HCl (0.2 mg/kg), dexmedetomidine HCl (10 mcg/kg), and ketamine HCl (5 mg/kg) given intramuscularly. The peri-wound skin was prepped as previously described for the dog and affixed with a tie over bandage to secure the PED to the cat chronic wound.

A total of five PEDs over 17 days were used for the treatment course determined by wound evaluations with dressing changes every 3-6 days.

Wound Area Measurements

Before each PED application, a digital photograph of the wound was captured with a ruler for reference. Each wound image captured was imported into MATLAB to determine the wound area using MATLAB’s image processing toolbox (Figure 2). The pictured ruler provided a size reference for image processing determining the wound area by pixel count. Wound area calculations were repeated three times with the reported average area +/− the associated root mean square variance.

Figure 2.

Process flow diagram showing the calculation of wound area. The blue areas are “masks” for use with MATLAB to quantify wound area. In each marked area, pixels were counted to determine wound size. Three measurements using independent masks are averaged and referenced to a ruler to determine the wound size by pixel count.

Results

PED Application in a Dog

A four-year-old, female spayed Mastiff presented with a chronic wound on the distal antebrachium that was caused by a bite wound with marked tissue infection. Prior to the use of the PED, one year of treatments included open wound management, negative pressure wound therapy, and skin grafting with 50% graft failure. Prior to the skin graft, a punch biopsy was taken for antimicrobial susceptibility that showed antimicrobial insensitivity to several antibiotics (e.g., amoxicillin, ampicillin, cefazolin, cefpodoxime, doxycycline, trimethoprim/sulfamethoxazole, and chloramphenicol). Following these treatments, the patient was transferred to the primary care veterinarian for open wound management. One year after the initial wounding event, a 4.2 cm² full-thickness wound was on the distomedial aspect of the right antebrachium with pale white unhealthy granulation tissue within the wound bed and peripheral fibrous tissue. The wound had minimal exudation.

At this time, a layer of hydrogel and the PED were applied to the wound. The PED was changed two days following initial application, and the wound was observed to decrease from 4.2 cm² to 3.2 cm². At the next dressing change four days later, wound area decreased from 3.2 cm² to 2.4 cm². After the last PED was removed on day 10, the wound area decreased to 1.0 cm² (Figure 3). The root mean square variance in the wound area for each measurement was ±0.08 cm².

Figure 3.

A summary of treatment prior and post-PED for the dog. Punch biopsy detected no infection after the last PED. The wound fully healed 67 days after initial PED treatment. The root mean square variance in wound area for each measurement was ±0.08 cm². OSU stands for The Ohio State University.

A punch biopsy of the tissue was performed and submitted for culture and antimicrobial susceptibility after the last dressing change. No bacterial growth occurred after this last PED treatment as labeled by the green-box in Fig. 3. Over the 10 days of treatment there was a ~4.2X reduction in the wound area. The dog was discharged for continued care with the primary care veterinarian for completion of second intention healing. The exact details of second intention healing are unknown (due to delayed communication by the owner until day 67) except knowing the wound was covered with a non-adherent dressing (Telfa) and a soft padded bandage. No antibiotics were prescribed. A re-check was performed at the clinic 67 days later and showed complete healing of the wound (Figure 3; day 67 image).

PED Application in a Cat

A one-year-old, female-spayed domestic shorthair (DSH) cat presented with a full thickness wound extending from the left flank to the lateral stifle. Open wound management was performed until a healthy bed of granulation tissue was present as determined by the attending clinician. Five months after the initial injury, an advancement flap was used to close the defect. Three days following surgery, partial dehiscence of the wound occurred, and a wound culture detected Pseudomonas aeruginosa. The cat was treated with pradofloxacin (5.5mg/kg PO q24h) with continued open wound management until a healthy bed of granulation tissue was re-established. Six months following the initial wounding, an interpolation flap was used to close the defect, and dehiscence of the distal 40% of the interpolation flap occurred 12 days post-operatively.

The wound remained static despite treatment. A wound biopsy was cultured and showed an eosinophilic granuloma complex (EGC) seven months following the initial wounding. The EGC was managed with prednisone (2mg/kg PO q24h) for the following month with open wound management. After that time, another biopsy revealed resolution of the EGC. Prednisone was tapered and discontinued over three weeks. The wound remained static (2.4 cm² wound area) despite open wound management, eight months following the initial injury.

After resolution of the EGC, another biopsy of the wound was cultured and showed Staphylococcus epidermidis. The cat was not on systemic antibiotics and no new antibiotics were initiated. At this time, hydrogel was applied to the wound bed and the smaller PED (Figures 1D, 1E) was applied. A tie over bandage was used to affix the PED to the wound.

The first PED change occurred after six days, and the wound area decreased from 2.4 cm² to 1.7 cm². Four days later, a dressing change revealed the wound at 1.6 cm². The next PED was applied three days later, showing the wound size of 1.3 cm². The last PED change was four days later, and the wound area decreased to 0.9 cm² (Figure 4). The root mean square variance in the wound area for each measurement was ±0.03 cm². A 2 mm punch biopsy was submitted for culture on day 17 and there was no bacterial growth. Over the 17 days, wound area decreased by ~2.5X. Open wound management was continued for another 6 weeks with hydrophilic foam (Covidien) as the primary layer until complete re-epithelialization of the wound was observed (Figure 4; day 47 image).

Figure 4.

A summary of treatment prior and post-PED for the cat. Five PEDs were applied over the treatment. Punch biopsy detected no infection after the last PED. The wound fully healed 47 days after initial PED treatment. The root mean square variance in wound area for each measurement was ±0.03 cm². OSU stands for The Ohio State University.

Discussion

The veterinary cases presented here suggest that PED electroceutical dressings show promise for the treatment of chronic wounds. PED treatment cleared infection and promoted healing in a dog and a cat. The results reported here are also considered important for human chronic wounds³⁸. For example, wound healing in dogs has been studied as translational model for both veterinary and human applications³⁹. Prior to the clinical use described here, efficacy of the PED for infection mitigation had been reported in vitro^30-32 and for safe operation in humans³⁷. The two cases presented here showed efficacy in eradicating persistent infections over a 10-day PED treatment course for the dog and a 17-day treatment course for the cat. Infection removal in both animals correlates to past in vitro results where 3-5 log reduction in bacterial CFU counts for 24-hour Pseudomonas aeruginosa and Staphylococcus aureus lawn biofilms treated with electroceuticals were reported^30-32. The PED, in vitro, generates reactive oxygen species, primarily HOCl³⁰, to mitigate bacterial infection. In both the dog and the cat, no observations of host cytotoxicity were recorded, which visually can be observed by abnormal discoloration of the wound or surrounding tissue. Other researchers have evaluated cytotoxicity in excised porcine dermal tissue subjected to electroceutical treatment by reviewing results from a blinded histopathological assessment, which did not show differences between untreated and treated dermal explants⁴⁰. In vitro, the observed bacterial clearance occurs over the anode, motivating the enlarged anode area on the PED (Figure 1B). Less substantial inhibitory effects may take place over the cathode due to the electric potential favoring the production of H₂O₂⁴¹; however, the bacteria may be able to recover from the oxidative stress from H₂O₂-mediated disinfection⁴².

Past in vitro results have reported eradication of antibiotic tolerant variants that may arise in cultures³². The bacterial cultures from the dog tissue biopsies showed insensitivity to several antibiotics (e.g., amoxicillin, ampicillin, cefazolin, cefpodoxime, doxycycline, trimethoprim/sulfamethoxazole, and chloramphenical) prior to the skin-graft surgery. Moreover, the wound after the skin graft failed with persistent infection (Staphylococcus pseudintermedius and Streptococcus canis) remained resistant to ampicillin and penicillin. The PED treatment resolved infection and demonstrated a continued reduction (~4.2X) in wound area over 10 days. Similarly, PED treatment was beneficial in a cat chronic wound. Prior to PED treatment, the wound showed the presence of Staphylococcus epidermidis following two skin-flap surgeries with dehiscence post-operatively. Despite the clearance of EGC within the wound after the second flap, Staphylococcus epidermidis persisted, and the wound remained static. However, after 17 days of PED treatment there was no detectable infection and the wound area decreased by 2.5X.

After various standard, established wound care treatments failed to resolve the chronic wounds on the dog and the cat, the PED proved to be an effective treatment in eliminating infections with subsequent, complete wound healing. While the results reported here did show that PED treatment was effective in promoting healing in chronic wounds in both cases, there were some limitations. Notably, dosing of electrical stimulation parameters and treatment course was not consistent between the two cases. Therefore, the results here are limited to key observations from each case.

Past work evaluating safe PED use in humans has also been reported. Previously, a pilot feasibility study to determine physically observable effects of PED application on host tissue response for safe use was evaluated. The pilot testing on a small cohort (N = 8) of patients receiving a lower extremity amputation with at least one open wound, showed that with engineered regulation of current flow to the open wound, the PED can be used with little to no visually observable adverse effects on chronic human skin wounds³⁷. We note that in the results reported here for both the dog and the cat, during the treatment course, no complications, adverse events, or side effects were observed.

In summary, the application of a PED led to closure of chronic wounds in two veterinary patients, one dog and one cat. In both cases, long-standing infection was resolved with a concomitant reduction in wound area, suggesting efficacy of PED treatment. The results suggest that PEDs may provide a new treatment modality to mitigate infection and promote healing of chronic wounds. Further testing is needed to validate the clinical findings and to define the underlying mechanism(s) of action of the PED.