The Use of New Antimicrobial Gauze Dressings:Effects on the Rate of Epithelialization ofPartial-Thickness Wounds

Introduction For many years, gauze has been used for a wide variety of situations: 1) primary and secondary dressing in conjunction with compression therapy, 2) debridement, 3) secondary dressing for grafts, 4) abdominal incisions, 5) packing of wounds, etc. One of the benefits of gauze is its excellent absorbent property. However, gauze has never been viewed as a barrier to infection. The idea of an antimicrobial gauze dressing that could prevent the entrance of wound pathogens is very attractive. The use of topical antimicrobials in wounds has been controversial, and various investigators recommend they not be used directly in wounds.1–3 Since antimicrobials can be effective at killing pathogenic bacteria, their influence on cells that are vital to the wound healing process is a legitimate concern. Antimicrobials that were incorporated into dressing materials have been shown to be effective in reducing the number of bacteria in wounds.4–6 From the authors’ knowledge, many of these dressings have not been evaluated for their ability to protect wounds from exogenous pathogens; their effect on healing is even less documented. The purpose of this study was to evaluate the effect of a new gauze dressing* impregnated with 0.2% polyhexamethylene biguanide (PHMB) (Avecia, Inc., Wilmington, DE) on the rate of epithelialization of partial-thickness wounds. Polyhexamethylene biguanide is a heterodisperse mixture of polymers whose activity as an antimicrobial agent is well accepted.7–9 This agent’s basic unit can be repeated from two to 30 times with increasing chain length correlating with increasing antimicrobial efficacy. End groups (specifically aminohydrochloride group or a cyanoguanidine or guanidine group) also contribute to activity but less so as molecular weight increases. Lethal action of PHMB concerns interaction at the cytoplasmic membrane to cause nonspecific alterations in membrane permeability. There is a specific association of PHMB with the acidic lipid components of the membrane to cause phase separation and domain formation. Increased size of the polymer increases the magnitude of the membrane perturbation. The authors performed studies in an established in-vivo porcine model.10–14 They believe in-vivo studies, rather than in-vitro experiments that use only keratinocytes or fibroblasts, more closely correspond to the clinical setting. Materials and Methods Experimental animals. Six young Specific Pathogen Free (SPF) pigs (Ken-O-Kaw Farms, Windsor, IL) weighing 40–50kg were used. A porcine model was chosen because of the morphological and functional similarity of pig skin to human skin.15,16 The animals were conditioned for two weeks before the experimental studies were initiated. The animals were fed a nonantibiotic chow ad libitum and housed in the researchers’ facilities (meeting United States Department of Agriculture compliance) with controlled temperatures (19–21 degrees C) and controlled light and dark cycles (12 hours light and 12 hours dark). The University of Miami Animal Care Committee approved the animal protocols used in these studies, and all procedures followed were in compliance with the federal guidelines for the care and use of laboratory animals (NIH Publication No. 86-23, Revised 1985). Wounding technique. For both experimental designs, animals were pre-anesthetized with ketamine HCl (20mg/kg), atropine (0.5mg/kg), and xylazine (0.5mg/kg) IM, followed by mask inhalation of an isoflurane and oxygen combination. Buprenorphine HCl (Buprenex, Reckitt & Collman, Richmond, VA) 0.1mg/kg was administered IM on the first day only. For pain management, fentanyl transdermal system patches (Duragesic, Janssen Pharmaceutica, Titusville, TN) (25mg/hr) were applied to the animals and replaced every three days. The skin on the back and both sides of the animals was clipped with standard animal clippers and prepared for wounding by washing with a nonantibiotic soap and sterile water. Partial-thickness rectangular wounds (7mm x 10mm x 0.3mm deep) were made using a modified electrokeratome (Storz, St. Louis, MO). Hemostasis was achieved using sterile surgical gauze sponges. Study design. One hundred and twenty partial-thickness wounds were made on the paravertebral and thoracic areas of each animal. The wounds were separated by at least 1cm of unwounded skin. The wounds on each animal were divided into three groups of 40 wounds each. The treatment groups included: 1) air-exposed, untreated control; 2) gauze with 0.2% PHMB*; and 3) gauze without the antimicrobial agent, control* to help maintain a moist environment. The experimental dressings were coded by the sponsor to maintain blind study design and were randomly assigned to a treatment area. Treatment 1 (i.e., the air-exposed, untreated control) was always kept on the rear of the animal since the other treatments required bandage wrap to secure them in place. Dressings were replaced daily. (No regional healing differences have been observed with the partial-thickness model.) Epidermal migration assessment. Beginning on day 3 after wounding (day 0), and on each day thereafter for 7 days, five wounds and the surrounding normal skin from each treatment group were excised using a standard width (22mm) electrokeratome blade set at a depth of 0.5mm (see A in Figure 1). All specimens that were not excised intact were discarded. The excised skin containing the wound site was incubated in 0.5m sodium bromide at 37 degrees C for 24 hours, allowing for a separation of the dermis from the epidermis (see B in Figure 1). After separation, the epidermal sheet was examined macroscopically for defects (see C and D in Figure 1). Defects were defined as holes in the epidermal sheet or as a lack of epidermal continuum in the area of the wound. Epithelialization was considered complete if no defect(s) was present. Any defect(s) in the wound area indicated that healing was incomplete. The mounted samples were preserved for a permanent record (see E in Figure 1). Histological assessment. Three of the six animals selected for the epithelialization study had two excisional biopsies taken from each treatment group on days 3 and 7. The biopsies were obtained through the center of the wounds and included unwounded adjacent skin on both sides. Specimens were processed routinely and stained with hematoxylin and eosin (H&E). One section per block was analyzed. The specimens were evaluated via light microscopy by a dermatopathologist who was unaware from which treatment group the specimens were taken. The specimens were examined for the following elements of wound healing to determine a potential treatment response: 1. Percent of wound epithelialized (%). This measurement is of the length of the wound surface that has been covered with epithelium. 2. Epithelial thickness (cell layers mm). The epithelial thickness may vary from area to area within the biopsy. The thickness of the epithelium in mm was measured on five equal distance points from each other in the biopsy and averaged. 3. White cell infiltrate. Measured by the presence and amount of subepithelial mixed leukocytic infiltrates. Mean score: 1 = absent, 2 = mild, 3 = moderate, 4 = marked, and 5 = exuberant. 4. Scale/crust formation. The amount of scale/crust formation was measured by the following scale: 1 = absence, 2 = minimal, 3 = moderate, 4 = marked, and 5 = exuberant. Results Epidermal migration assessment. After the study was completed, the codes were revealed. The number of wounds healed (completely epithelialized) was divided by the total number of wounds sampled per day and multiplied by 100 (Table 1). The percentage of wounds healed was then plotted against days after wounding (Figure 2). Statistical analysis using Chi square with four tables was performed on all data. On day 5, differences in the rate of epithelialization were seen with wounds treated with gauze without antimicrobial agent (47% completely epithelialized) as compared to those wounds treated with either gauze with antimicrobial agent or left untreated (air exposed) (13% and 0% completely epithelialized, respectively). On days 6 and 7, there were no differences between gauze dressings, and both stimulated complete epithelialization as compared to untreated air exposed. Histological assessment (Table 2). Both gauze dressings (with and without antimicrobial) enhanced the percent of epithelialization and epithelial thickness of wounds as compared to untreated air-exposed wounds. The air-exposed wounds had more inflammatory cells and scale/crust than those treated with either of the gauze dressings. Discussion In this study, the authors found that both gauze dressings accelerated the rate of epidermal migration as compared to untreated control. This was probably due to the fact that the gauze was soaked with saline and covered with an occlusive dressing, which kept the wounds moist. Gauze dressings are used extensively in wound care. The concept of a gauze dressing with an antimicrobial is a novel therapy. Although the use of some antimicrobials may be controversial, the authors have previously documented that they can be effective without inhibiting the wound healing process.17 In a concurrent study, the authors demonstrated the ability of the same gauze with PHMB to act as a barrier to bacteria. During this barrier study, an occlusive environment was maintained. The authors believe that this moisture allowed the antimicrobial to become activated. Interestingly, release of the antimicrobial demonstrated no adverse effects on the rate of epithelialization of partial-thickness wounds. The ability of an antimicrobial gauze dressing to prevent and/or reduce infections while not adversely influencing the wound healing process may have important clinical implications. * Kerlix A.M.D. Gauze Dressing, Kendall, a division of Tyco Healthcare Group LP, Mansfield, MA * BlisterFilm‚ Kendall, a division of Tyco Healthcare Group LP, Mansfield, MA Flex-Wrap, Kendall, a division of Tyco Healthcare Group LP, Mansfield, MA