Dermal Nanoemulsion Treatment Reduces Burn Wound Conversion and Improves Skin Healing in a Porcine Model of Thermal Burn Injury

Abstract

Burn wound progression is an inflammation-driven process where an initial partial-thickness thermal burn wound can evolve over time to a full-thickness injury. We have developed an oil-in-water nanoemulsion formulation (NB-201) containing benzalkonium chloride for use in burn wounds that is antimicrobial and potentially inhibits burn wound progression. We used a porcine burn injury model to evaluate the effect of topical nanoemulsion treatment on burn wound conversion and healing. Anesthetized swine received thermal burn wounds using a 25-cm² surface area copper bar heated to 80°C. Three different concentrations of NB-201 (10, 20, or 40% nanoemulsion), silver sulfadiazine cream, or saline were applied to burned skin immediately after injury and on days 1, 2, 4, 7, 10, 14, and 18 postinjury. Digital images and skin biopsies were taken at each dressing change. Skin biopsy samples were stained for histological evaluation and graded. Skin tissue samples were also assayed for mediators of inflammation. Dermal treatment with NB-201 diminished thermal burn wound conversion to a full-thickness injury as determined by both histological and visual evaluation. Comparison of epithelial restoration on day 21 showed that 77.8% of the nanoemulsion-treated wounds had an epidermal injury score of 0 compared to 16.7% of the silver sulfadiazine-treated burns (P = .01). Silver sulfadiazine cream- and saline-treated wounds (controls) converted to full-thickness burns by day 4. Histological evaluation revealed reduced inflammation and evidence of skin injury in NB-201-treated sites compared to control wounds. The nanoemulsion-treated wounds often healed with complete regrowth of epithelium and no loss of hair follicles (NB-201: 4.8 ± 2.1, saline: 0 ± 0, silver sulfadiazine: 0 ± 0 hair follicles per 4-mm biopsy section, P < .05). Production of inflammatory mediators and sequestration of neutrophils were also inhibited by NB-201. Topically applied NB-201 prevented the progression of a partial-thickness burn wound to full-thickness injury and was associated with a concurrent decrease in dermal inflammation.

Burn injury requiring medical attention affects 1.1 million patients annually in the United States.¹ Burn wound healing is a function of the depth of the burn wound and avoidance of burn wound infection.^2,3 The majority of burn wounds presenting to our American Burn Association-verified burn center are initially partial-thickness (65%) or a mixture of partial- and full-thickness (28%).⁴ Burn wound progression is a process by which an initial partial-thickness burn injury can convert to a deep partial-thickness or full-thickness burn wound due to additional tissue damage and/or onset of burn wound infection. The mechanism of burn wound progression is multifactorial and depends on components of the host response, inflammation, erythrocyte aggregation, and tissue ischemia.⁵ A partial-thickness burn wound, which does not progress to full-thickness injury, is capable of self-healing. Conversely, progression of a partial-thickness burn wound to a full-thickness burn wound requires intensive surgical treatment involving operative excision of the dead tissue and skin grafting to restore the patient’s skin integrity.^2,6

Current topical treatments, to minimize burn wound infection, include use of silver sulfadiazine (Silvadene^®) cream, bacitracin ointment, or nanocrystalline silver-impregnated dressings (Acticoat^®, Mepilex^®). These treatments have clinical gaps in coverage and may not be effective against multidrug-resistant organisms (eg, Pseudomonas aeruginosa, Enterobacter sp., Acinetobacter baumannii, Enterococcus sp.) found in human burn wounds. Specific, known problems with silver sulfadiazine are that it does not dependably suppress bacterial growth in large burn wounds, does not penetrate eschar, creates a proteinaceous pseudoeschar, is not antifungal, and can impede burned skin reepithelization.^7–9 Disadvantages of nanocrystalline silver dressings include the inability to penetrate eschar, poor contact with irregular surfaces, and brown/black staining of burn wounds. Treatment of a patient with bacterial- and fungal-infected wounds currently requires rotation of topical agents at each dressing change (more dressing changes, partial coverage due to alternating antibacterial and antifungal agents) or off-label mixing of antifungal agents with the topical antimicrobial (with unproven efficacy).

Novel oil-in-water nanoemulsions are formulated from nontoxic materials, are well designed for the treatment of burn wounds, and are proven to exhibit broad antibacterial and antifungal properties.^10–13 These oil-in-water emulsions are made using high shear conditions to produce droplets that physically fuse with the lipid layer in microbial outer membranes, leading to membrane destabilization and lysis of pathogens. We have developed a formulation (NB-201) for use in burn wounds that reduces the potential for the introduction of opportunistic multidrug-resistant organisms and inhibits burn wound progression.^14,15

We hypothesize that topical application of an antimicrobial nanoemulsion will minimize microbial growth and reduce burn wound progression in a porcine model of partial-thickness thermal burn injury. Concomitant reduction of neutrophil infiltration and modulation of pro-inflammatory responses may impact the development of burn wound progression in the early postinjury phase. Here we examined the efficacy of topically applied NB-201 nanoemulsion formulations on abatement of burn wound progression, promotion of wound healing, antimicrobial efficacy, and burn wound inflammation.

METHODS

Reagents

Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Animal Ethics and Care

All experiments were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All animal work was reviewed and protocols approved by the University of Michigan Institutional Animal Care & Use Committee (IACUC). Seven commercially obtained 3-month-old Yorkshire-Landrace female swine weighing between 20 and 30 kg (Michigan State University Swine Teaching and Resource Center; Lansing, MI) were acclimated for at least 7 days before the start of the experiments. Three pigs were utilized in burn model development and four pigs were used in the described experiments testing the nanoemulsion. Animals were housed individually in enriched cages with water provided ad libitum. Animals were fed a standard porcine diet (Lab Diet 5801; PMI Nutrition, IN) in accordance with University of Michigan Unit for Laboratory Animal Medicine guidelines.

Anesthesia Induction

Animals were fasted overnight prior to anesthesia. Anesthesia was induced with intramuscular injection of 2.5 to 3 mg/kg tiletamine/zolazepam (Telazol; Zoetis Inc., Kalamazoo, MI) and 2.2 mg/kg xylazine and anesthesia was maintained with isoflurane administered via face mask with oxygen flow of 2 liters per minute. Animals were placed in sternal recumbency for the duration of the procedure. Heart rate, respiratory rate, rectal temperature, and capillary oxygen saturation were monitored at regular intervals. Additional warming support was provided as necessary with a circulating water blanket.

Postprocedure analgesia was provided with injectable buprenorphine and a Butrans transdermal system (Purdue Pharma L.P., Stamford, CT) for systemic delivery of 5 µg per hour buprenorphine for 7 days. A single loading dose of 0.01 mg/kg buprenorphine was administered intramuscularly immediately following burn trauma and again on days 7, 10, 14, and 18 after punch biopsy specimen collection. A Butrans transdermal patch system was applied following the loading dose of injectable buprenorphine and was maintained for 1 week. Swine were monitored twice daily for evidence of discomfort. If necessary, additional analgesic doses were administered under veterinary supervision.

Swine Burn Model

Dorsal hair was removed using depilatory cream (Nair; Church & Dwight Inc., Princeton, NJ) and any remaining hair was clipped. The skin was thoroughly washed with water and then sterile prepped with 2% chlorhexidine gluconate and 70% isopropyl alcohol solution (Chloraprep; Cardinal Health, Dublin, OH). The protocol for creation of progressive partial-thickness burns was adapted from Singer et al.¹⁶ The estimated total BSA burned is 5% and this burn model results in highly reproducible damage to the upper 30 to 50% of the dermis and is described as follows.^16,17 Multiple square 5 × 5 cm copper blocks (weight 530 g) with an attached positioning rod were heated in an 80°C water bath for 30 minutes prior to application to the skin. One block was applied to each of five paralumbar sites on the right side of the body for a duration of 20 seconds per site. On the left side of the body, the exposure time was increased to 30 seconds to increase the severity of the initial partial-thickness burn wound for two of the four pigs and five additional paralumbar burn wounds were created. The blocks were returned to temperature for at least 10 minutes in the water bath prior to being used again. Pressure was supplied by gravity.

The necrotic epidermis was gently removed by abrading each burn wound with dry cotton gauze. The first topical treatment and dressing occurred 30 minutes after creation of the burn wounds. An occlusive dressing of sterile Telfa (Kendall Co., Mansfield, MA) and Tegaderm HP (3M HealthCare, St Paul, MN) was applied to prevent wound contamination. A clean laparotomy pad was placed over the Tegaderm. The thorax was covered with self-adherent wrap (MediChoice, Buford, GA) and the ends secured with heavyweight stretch tape (BSN Medical, Inc., Charlotte, NC). A cloth jacket (MWI Veterinary Supply Inc., Rochester Hills, MI) was placed over the dressing to prevent fecal contamination. Pigs 1–3 were used in burn wound model and experimental assay development. No results are reported for these animals. Pigs 4–7 were used for the experiments reported. A total of four pigs with 10 burn wounds yielded N = 8 separate burn wounds per treatment group. One pig underwent burn creation and treatment with no interval biopsies to allow for optimal burn wound photograph documentation at each dressing change. Pig 4 and 7 underwent 20-second burn creation on the right side and 30-second exposure on the left side. For pigs 5 and 6 all of the burn wounds were the result of 20-second exposure to the 80°C copper block.

Topical Burn Wound Treatment and Evaluation

Each pig was anesthetized during all subsequent dressing changes. The burn sites were treated with saline, silver sulfadiazine cream (1% silver sulfadiazine, Pfizer), or NB-201 (10, 20, or 40% nanoemulsion). NB-201 nanoemulsion formulations were obtained from BlueWillow Biologics Inc. (Ann Arbor, MI). NB-201 was applied topically to the burn wound surface using a Fine Mist Sprayer (Mistette MK 140-T; MeadWestvaco Calmar GmbH, Germany) from a 6-ml U-Save Type 1 glass vial (Neville & More, West Sussex, United Kingdom). The treatment was directly applied to the wound (step 1) and wounds were covered with saturated treatment (step 2). Step 1), 5 ml of topical NB-201 was delivered in a uniform manner to each burn with pauses for absorption between each spray application. Step 2), for the NB-201- or saline-treated groups, a 6 × 6 cm Telfa square was soaked with 5 ml of NB-201 or saline. For Silvadene-treated wounds, 1 g of silver sulfadiazine cream was applied evenly over a 6 × 6 cm Telfa square which was then applied to the burned skin. Dressing changes were performed on days 1, 2, 4, 7, 10, 14, and 18 after burn injury. Full-thickness skin biopsies were obtained with a 4-mm-diameter punch biopsy device on days 4, 7, 10, 14, 18, and 21 after burn injury. Digital pictures were taken at the time of each dressing change to monitor burn wound progression and subsequent healing. Animals were euthanized at 21 days postburn.

Nanoemulsion Formulation

The oil-in-water NB-201 nanoemulsions were prepared by emulsification of a composition of: a cationic surfactant (benzalkonium chloride [BAC]), a nonionic surfactant (Tween 20), ethanol humectant (glycerol), a chelating agent (EDTA), an oil (highly refined soybean oil), and water using proprietary manufacturing methodology. These formulations are composed of pharmaceutically approved ingredients that are included on the Food and Drug Administration (FDA) Inactive Ingredient List for Approved Drugs. The final concentrations of BAC in the compositions were: 10% NB-201 is 0.2% (w/v) BAC, 20% NB-201 is 0.4% (w/v) BAC, and 40% NB-201 is 0.8% (w/v) BAC. The surfactants, both cationic and nonionic, reside at the interface between the oil and water phases. The hydrophobic tail of the surfactant distributes in the oil core and its polar head group resides in the water phase as shown in Figure 1.

Figure 1.

Schematic of NB-201 nanoemulsion droplet.

Quantitation of Bacterial Wound Infection

Four-millimeter full-thickness punch biopsies were mechanically homogenized in 1 ml of sterile saline solution. This homogenate was then further diluted with 9 ml of sterile saline solution. Serial dilutions were performed, and skin homogenates were plated in triplicate on blood agar plates (Becton Dickinson). Culture plates were incubated for 24 hours at 37°C and colony forming units (CFUs) were counted.¹⁸ Speciation of bacterial colonies was performed by the Michigan Medicine Clinical Burn Laboratory using standard clinical bacteria identification practices. In our clinical lab, a positive quantitative wound culture result is considered to be growth of bacteria at greater than 1 × 10⁵ CFUs per g of tissue.^14,18

Quantitation of Soluble Mediators by Enzyme-Linked Immunosorbent Assay

Four-millimeter full-thickness skin tissue punch biopsies were mechanically homogenized in 1 ml of sterile phosphate-buffered saline (1×), pH 7.4, containing 0.01% (w/v) Triton X (Roche) and complete protease inhibitors cocktail (Complete X, Roche, Indianapolis, IN). This homogenate was then centrifuged at 3000g for 5 minutes at 4°C and used for sandwich enzyme-linked immunosorbent assay (ELISA). Pig interleukin 1-beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α) were measured by ELISA using DuoSets from R&D Systems Inc. (Minneapolis, MN). Pig myeloperoxidase ELISA kit was from TZS ELISA (Ellison Park, MA). Results were expressed as picograms per milliliter (pg/ml).

Detection of Neutrophils (Myeloperoxidase Activity and Cell Count)

One hundred milligrams of skin tissue was mechanically homogenized in 1 ml ice-cold potassium phosphate buffer consisting of 115 mM monobasic potassium phosphate. Homogenates were centrifuged at 3000g for 10 minutes at 4°C, supernatants were removed, and the pellets resuspended in 1 ml C-TAB buffer consisting of dibasic potassium phosphate, cetyltrimethylammonium bromide, and acetic acid. The samples were sonicated (Branson Sonifier 250, Danbury, CT) on ice for 40 seconds, centrifuged at 3000g for 10 minutes at 4°C, and the supernatant collected. Supernatants were incubated in 60°C water bath for 2 hours (Shaker Bath, 2568; Forma Scientific, Marietta, OH). Samples were stored at −80°C until assayed.

Twenty-microliter standards (Calbiochem, Gibbstown, NJ) or samples were added to a 96-well immunosorbent microplates (NUNC, Rochester, NY), followed by the addition of 155 µl of 20 mM TMB/DMF consisting of 3,3′,5,5′-tetramethylbenzidine/N,N-dimethylformamide in 115 mM potassium phosphate buffer (Fischer Scientific, Pittsburgh, PA) to each well. The samples were mixed well, after which 20 µl of 3 mM H₂O₂ was added to each well. The reaction was stopped by adding 50 µl/well of 0.061 mg/ml Catalase (Roche, Indianapolis, IN). The plates were read using a microplate reader at 620 nm. Myeloperoxidase levels were calculated using a linear standard curve and adjusted for prior dilution. The final concentrations were expressed as µg/ml.

Histology and Cell Counts

Fresh 4-mm full-thickness skin tissue punch biopsies were fixed in 10% buffered formalin and embedded in paraffin. Sections 4 µm thick were sliced and affixed to slides, deparaffinized and stained with hematoxylin and eosin to assess morphological changes. The number of neutrophils were counted per slide based on visual identification. Each skin histology sample was scored by two independent and double-masked dermal pathologists using the following scoring system (Table 1). The two scores for each burn wound/treatment at each time point were averaged. A total histology score was computed by summing the scores in each category. Individual and total scores were averaged across treatments for the 20- and 30-second burn wounds.

Table 1.

Histological scoring system

Epidermis	Dermal necrosis	Deep granulation tissue	Immature granulation tissue
0 = Complete	0 = None	0 = None	0 = None
1 = Acute necrosis	1 = ≤100 µm	1 = ≤500 µm	1 = ≤100 µm
2 = Separation	2 = 100–300 µm	2 = 500–1000 µm	2 = 100–300 µm
3 = Absent	3 = 300–500 µm	3 = 1000–3000 µm	3 = 300–500 µm
	4 = ≥500 µm	4 = 3000–5000 µm	4 = ≥500 µm
		5 = >5000 µm
Necrotic inflammation	Superficial dermal inflammation	Perivascular inflammation	Total
0 = None	0 = None	0 = None	Sum of all scores
1 = ≤100 µm	1 = Focal	1 = Mild multifocal	Minimum = 0
2 = 100–300 µm	2 = Locally extensive	2 = Moderate multifocal	Maximum = 31
3 = 300–500 µm	3 = Diffuse	3 = Mild diffuse
4 = ≥500 µm	4 = Focal necrotizing	4 = Moderate diffuse
	5 = Locally extensive necrotizing	5 = Severe
	6 = Diffuse necrotizing

Epidermal necrosis was characterized by partial separation to complete necrosis or loss of the epidermis, with features of hypereosinophilia, loss of architecture, and loss of cellular detail. Dermal necrosis was characterized by hypereosinophilia, loss of collagen detail, and nuclear pyknosis and karyorrhexis. Necrotic inflammation was reported when there was cellular debris admixed with degenerate neutrophils, predominantly appearing in a band bordering zones of dermal necrosis. Superficial and deep dermal inflammation were characterized by the presence of various inflammatory cells in the superficial and deep dermal layers, respectively. Deep dermal granulation tissue was reported as proliferation of capillaries and fibroblasts between collagen bundles within the dermis or zones subjacent to dermal necrosis.

Hair Follicle Assessment

The number of hair follicles were counted per slide. Each slide represented a cross section of each 4-mm full-thickness skin tissue punch biopsy. Skin sections were stained with hematoxylin and eosin.

Statistical Methods

All statistical analysis was performed using GraphPad Prism software, version 6.0 (GraphPad Software, La Jolla, CA). Results are presented as mean values ± the standard deviation (SD) unless otherwise noted. Continuous variables were analyzed using a one-way or two-way analysis of variance and Tukey’s Newman–Keuls or Sidak’s multiple comparison. Statistical significance was defined as a P value < .05.

RESULTS

NB-201 Reduces Burn Wound Progression in a Partial-Thickness Thermal Burn Wound

Digital photographs were taken to document macroscopic healing. The burn wounds treated with saline or silver sulfadiazine progressed to full-thickness burns by day 4 with evidence successive eschar formation and contraction (Figure 2A). Macroscopic healing was achieved by day 21 postburn in the NB-201-treated burn wounds. Twenty and 40% NB-201-treated wounds were histologically healed on day 21 and appeared similar to unburned skin with the exception of some residual hypercellularity (Figure 2B). Silver sulfadiazine-treated wounds were not healed by day 21 and significant leukocytic infiltration was noted. The visual and histopathologic appearance of wounds induced by exposure to 80°C heated copper bar for 20 seconds vs 30 seconds was not different (data not shown). Both 20-second and 30-second exposure times resulted in similar initial partial-thickness wounds which progressed to full-thickness wounds by day 4–7 in saline- and silver sulfadiazine-treated burns.

Figure 2.

Visual burn wound healing. (A) Burn wound sites were created by application of a copper bar heated to 80°C in a water bath for 20 seconds. Photographs of burn wounds by topical treatment (saline, silver sulfadiazine, 10% NB-201, 20% NB-201, 40% NB-201 taken on day 1, 4, 10, and 21 postburn injury). (B) Representative hematoxylin and eosin-stained cross-sectional histology samples for normal unburned skin, 40% NB-201-treated skin, and silver sulfadiazine-treated skin sampled on day 21 (20× magnification). Burn wound sites were created by application of a copper bar heated to 80°C in a water bath for 20 seconds.

NB-201 Inhibits Production of Inflammatory Mediators in Wounds

Soluble dermal inflammatory mediator production was quantified from punch biopsies obtained from wounds on days 4 and 21. In 20-second exposure burn wounds, treatment with NB-201 significantly reduced wound levels of IL-1β, IL-6, and IL-8 compared to saline- and silver sulfadiazine-treated burn wounds (Figure 3A). Similar results were found in the 30-second exposure burn wounds for IL-1β on day 4 (Figure 3B). In the 30-second wounds there were also significant differences for IL-6 at 21 days. IL-8 exhibited differences for silver sulfadiazine vs NB-201 at the higher treatment concentrations of NB-201. Significantly less TNF-α was present at day 21 in the wounds treated with NB-201 compared to silver sulfadiazine in the 30-second exposure burns. The values presented as normal represent unburned skin. Although we saw some variation in the levels of soluble mediators generated between the 20-second and 30-second exposure burns there was minimal visual or histological difference documented by day 21.

Figure 3.

Dermal expression of burn-induced inflammatory mediators at day 4 and day 21. Four-millimeter full-thickness punch biopsies of skin were homogenized and assayed using ELISA for level of IL-1β, IL-6, IL-8, and TNF-α. N = 8 separate burn wounds per treatment group. Statistics: two-way ANOVA with Sidak’s multiple comparisons test. Displayed as mean ± SD. (A) Burn wound created by application of a copper bar heated to 80°C in a water bath for 20 seconds. (B) Burn wound created by application of a copper bar heated to 80°C in a water bath for 30 seconds.

NB-201 Suppresses the Growth of Bacteria in Burn Wounds

Quantitative burn wound culture testing revealed significant growth of bacteria within the saline- and silver sulfadiazine-treated burn wounds in tissue biopsies obtained on day 21 postburn (Figure 4). Isolates included Staphylococcus aureus, coagulase negative Staphylococcus spp., and enteric gram-negative rods. (Corynebacterium spp.). Bacterial growth was not found in any of the NB-201-treated burn wounds.

Figure 4.

Quantitative skin biopsy bacterial growth in burn wounds on day 21 postinjury. N = 8 separate burn wounds per treatment group. Results expressed as median number of colony forming units/g of tissue (bar). Statistics: ordinary one-way ANOVA with Tukey’s multiple comparisons test. *P < .05 saline vs NB-201, 10 or 20 or 40%. ^#P < .05 silver sulfadiazine vs NB-201, 10 or 20 or 40%.

Histopathologic Examination

We analyzed hematoxylin and eosin-stained sections of burn wound skin for presence of injury to epidermis, dermal necrosis, necrotic inflammation, perivascular inflammation, superficial dermal inflammation, immature granulation tissue, and deep granulation tissue (scoring system present in Table 1). A total histopathologic score was calculated by summing individual scores for all of the above parameters. Results for individual dermal injury assessments and total histology score are displayed in Figure 5. Significant differences were found between the saline- and silver sulfadiazine-treated wounds when compared to the NB-201-treated wounds for the 20 and 40% nanoemulsion formulations.

Figure 5.

Pathologic scoring of skin samples stained with hematoxylin and eosin and analyzed by two independent/masked pathologists. Scores determined by pathologists were averaged and plotted. Statistics: three pigs, six burn wounds per treatment group, two-way ANOVA with Sidak’s multiple comparisons test. Displayed as mean ± SD, *P < .05. Burn wound created by application of a copper bar heated to 80°C in a water bath for 20 seconds.

Comparison of epithelial restoration by day 21 showed that 77.8% (n = 18) of the nanoemulsion- (10, 20, and 40%) treated wounds had an epidermal injury score of 0 compared to 16.7% (n = 6) of the silver sulfadiazine-treated burns (P = .01, Fisher’s exact test). The same result was observed for the nanoemulsion-treated burns in comparison to the saline-treated control burns 16.7% (n = 6, P = .01, Fisher’s exact test).

NB-201 Inhibits Neutrophil Sequestration (Myeloperoxidase Assay)

Neutrophil sequestration associated with burn wound inflammation was significantly reduced by treatment with NB-201 compared to silver sulfadiazine and saline controls on days 4 (P < .0001) and 21 (P < .0001, Figure 6A). Representative slides of histopathologic sections demonstrated epithelial loss (day 4) with regrowth of epithelium in burned skin treated with NB-201 vs continued loss of epithelium in the saline control or silver sulfadiazine-treated wounds on day 21 (Figure 6B).

Figure 6.

Evidence of neutrophil sequestration into the burn wound after thermal injury and topical treatment at day 4 and day 21 postinjury. (A) Myeloperoxidase assay. (B) Histopathologic neutrophil count. (C) Representative photomicrographs of slides from histopathologic examination (10× magnification, inset 60× magnification). N = 8 wounds per treatment group. Statistics: two-way ANOVA with Sidak’s multiple comparisons test. Displayed as mean ± SD.

NB-201 Preserves Hair Follicles Presence Following Burns

The number of intact hair follicles present per cross-sectional biopsy sample was counted in specimens obtained on day 21 postburn. We documented loss of hair follicles in the skin after silver sulfadiazine or saline treatment (Figure 7). Burn wounds treated with silver sulfadiazine (0 ± 0 hair follicles per 4-mm biopsy section) or saline (0 ± 0 hair follicles) had complete loss of hair follicles. NB-201-treated burn wounds demonstrated 3–6 intact hair follicles per slide and had a similar count to normal unburned skin (4.8 ± 2.1 vs 5.0 ± 0.8, P = not significant).

Figure 7.

Hair follicle preservation in burned skin. NB-201 treatment resulted in hair follicle preservation as compared to hair follicle loss in controls on day 21 postburn. Cross-sectional skin histological samples were stained with H&E and viable hair follicles were counted. N = 4 separate burn wounds per treatment group. Statistics: ordinary one-way ANOVA with Tukey’s multiple comparisons test. Displayed as mean ± SD.

DISCUSSION

Dermal treatment of partial-thickness thermal burns with NB-201 mitigated progression to full-thickness burn injury. Conversely, silver sulfadiazine- or saline- (control) treated burns progressed to full-thickness burns. Secondary tissue loss due to burn wound progression was found to be coupled with sustained inflammation after burn injury as evidenced by dermal IL-1β, IL-6, and IL-8 elevation and histopathologic evaluation.^19–21 Prolonged inflammation, neutrophilia, and continued tissue ischemia in the stasis zone are known to promote conversion of a partial-thickness wound to a full-thickness injury.²² Reduction of inflammation within the burn wound by treatment with NB-201 was coupled with a decreased loss of hair follicles and normal healing as observed visually and on histopathology.

Progression of burn injury is a well-known clinical phenomenon, but the definitive mechanism by which it occurs and treatments to halt progression remain elusive. Shupp et al has summarized three local mediator processes that influence burn wound progression.²² They are rapid dermal infiltration of inflammatory cells, impaired vascular perfusion, and oxidative stress. Following thermal injury, the burn wound undergoes an extended inflammatory response during which neutrophils release cytotoxic mediators and reactive oxidative species. A prolonged inflammatory response after burn injury can have detrimental effects on both a local and systemic basis.^23–28 Source control of dermal inflammation is a potential therapeutic approach toward reduction of burn wound progression. Topical application of SB 202190, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK), can reduce the overall level of inflammation within the dermis.²³ Diminishing the level of dermal inflammation correlated with reduced neutrophil sequestration, microvascular damage, and burn-induced epithelial apoptosis in hair follicle cells. In patients without concomitant inhalational injury, the burn wound generated pro-inflammatory cytokines in the dermis can trigger a systemic inflammatory response and cause further sequestration of neutrophils into the site of dermal inflammation.^29–31

Similar to SB 202190, NB-201 appears to act by diminishing the local dermal inflammatory response and added advantage of being substantially antimicrobial.^14,15 The mechanism of the anti-inflammatory action of the nanoemulsion seems to be distinct from its antimicrobial activity because the anti-inflammatory effect was also present in sterile wounds in rodents and has now been demonstrated in these porcine burn experiments in which bacteria was not inoculated into the wound. Our porcine data confirm the results found in rodents, as topical NB-201 significantly inhibited dermal production of IL-1β, IL-6, and IL-8 when compared to silver sulfadiazine- or saline-treated burn wounds. In addition, the NB-201-treated wounds demonstrated reduced neutrophil infiltration, reduced levels of necrotic inflammation, preservation of hair follicles, and retained proliferation of epithelial cells lining the hair follicle.

Cell death in burn wounds occurs due to apoptosis or direct necrosis.^32,33 Within the zone of stasis in deep partial-thickness burn wounds apoptosis has been identified in up to 44 to 49% of the dermal cells and the manifestation of apoptotic cells can persist for up to 2 weeks after injury.³⁴ Apoptotic dermal cells are found in 19% of healthy skin, 5.6% of superficial partial-thickness skin, and 0% of full-thickness burns.^34,35 However, a definitive correlation between the rate of apoptosis and deep partial-thickness burns conversion to full-thickness has been elusive. Singer et al has studied the effect of apoptosis and necrosis (oncosis) of cells in the stasis zone.³⁶ In a rat thermal injury model apoptotic cells were depicted as cleaved caspase-3 (CC3a) positive, and cytoplasmic translocation of high-mobility group box-1 (HMGB-1) nuclear complex indicated necrotic cells. CC3a positive staining was utilized to reveal apoptosis of basal keratinocytes and cells neighboring hair follicles 30 minutes’ postinjury. At later times, 24 and 48 hours postinjury, only a minimal level of CC3a staining was associated with hair follicle cells. Diffuse staining for cytoplasmic HMGB-1 at 24 hours postinjury, with even lower levels 48 hours postinjury was found within hair follicles, keratinocytes, dermal fibroblasts, and interspace sebaceous glands. Thus, the authors demonstrated a time dependent role for apoptosis at earlier stages and oncosis at later stages in the conversion of a partial-thickness burn to full-thickness injury. While we did not directly study apoptosis in the porcine skin experiments described in this study we have previously shown that NB-201 can reduce the degree of hair follicle cell apoptosis in rat skin when compared to saline-treated controls.¹⁴

p38MAPK activates many proapoptotic elements and triggers production of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α by keratinocytes.^23,25 Analysis of rat skin burn tissue biopsies for soluble mediators by ELISA and messenger RNA expression by real-time reverse-transcriptase polymerase chain reaction showed that in animals treated with SB202190, a p38MAPK inhibitor, there was lower production of IL-1β, IL-6, and TNF-α. Inhibition of p38 mitogen-activated protein kinase suppressed production of macrophage inflammatory protein 2 along with skin myeloperoxidase activity consistent with lowered neutrophil migration into the burn wound. In addition, SB202190 tempered apoptosis of hair follicle cells after burn injury and inhibited microvascular damage as measured by vascular albumin leakage. Topical treatment of burn wounds with NB-201 in our rat and now pig models of partial-thickness burn injury have confirmed these anti-inflammatory response findings of Ipaktchi et al.^{14,15,23–26} Reduced microvascular injury could result from decreased neutrophil adhesion to the vascular endothelium, since neutrophil sequestration into the burn wound was suppressed.

There are some potential limitations present in our study that must be acknowledged. We decided to not randomize the position of wound treatments among the 10 burn wounds. This was done to minimize spillover effects of adjacent treatments. The drawback to not randomizing is the potential for contamination underneath the distal edges of our dressing in relation to the front and rear of the pig. In addition, there could be variance in the skin characteristics along the back going from cephalad to caudad. We did attempt to place the burn wounds centrally within the “back” region of the pig. The porcine model may not exactly mimic burn wound progression as seen in humans. The pig lacks eccrine glands and its immune system is not in complete homology with humans. Other factors such as poor blood flow, nutritional deficiencies, and older age can also influence burn wound progression and healing. Lastly, cooling did occur as the copper bar was applied to the skin; however, the effect of this is likely minimal as we measured the surface temperature at 78°C after 30 seconds of skin contact.

In summary, topical therapy of partial-thickness thermal burn wounds with NB-201 significantly attenuated progression to full-thickness burn injury when compared to silver sulfadiazine- and saline- (control) treated burn wounds. Twenty and 40% NB-201-treated burned skin healed to gross visual inspection and histopathologic measurements by day 21 that were consistent with normal unburned skin.

Funding: This work was supported by National Institutes of Health grant K08-GM078610 to M.R.H. with joint support from the American College of Surgeons and the American Association for the Surgery of Trauma. The work was also supported by a Michigan Corporate Relations Network grant to M.R.H. with matching funding from BlueWillow Biologics.

Conflict of interest statement. S.C.: Employee of BlueWillow Biologics. J.R.B., Jr.: Stockholder in BlueWillow Biologics. V.A.D., E.L., B.L., S.C.W., and M.R.H. have no conflicts of interest.