30-Evaluation of Topical Off-the-Shelf Therapies to Reduce the Need to Evacuate Battlefield-Injured Warfighters

MILITARY MEDICINE, 00, 0/0:1, 2023Evaluation of Topical Off-the-Shelf Therapies to Reduce the Need to Evacuate Battlefield-Injured WarfightersDavid Larson,<a href="#bookmark1">MS*,</a><a href="#bookmark2">†;</a>Anders H. Carlsson, <a href="#bookmark1">PhD*,</a><a href="#bookmark2">†;</a> Franklin A. Valdera, <a href="#bookmark1">MD*;</a> Matthew Burgess,<a href="#bookmark1">BS*;</a> Logan Leatherman, MS<a href="#bookmark1">*;</a> Lucy Shaffer, BS<a href="#bookmark1">*;</a> Robert J. Christy, PhD<a href="#bookmark1">*;</a> Kristo Nuutila, MSc, PhD<a href="#bookmark1">*</a>Downloaded from <a href="https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209bygueston30November2023">https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209 by guest on 30 November 2023</a>ABSTRACT Introduction:Immediate evacuation of burn casualties can be challenging in austere environments, and it is predicted to be even more diffcult in future multi-domain battlespaces against near-peer foes. Therefore, a need exists to treat burn wounds at the point of injury to protect the exposed injury for an extended period. In this study, we compare two commercially available FDA-approved therapies to the current gold standard of care (GSOC), excisional debridement followed by the application of split-thickness skin graft, and the standard for prolonged feld care, silver sulfadiazine (SSD) cream. The shelf-stable therapies evaluated were irradiated human skin (IHS) allograft and polylactic acid (PLA). Our objective was to study whether they have the potential capability to reduce the need for evacuation to a burn center for surgical intervention so that the combat power can be preserved in the feld.Materials and Methods:Sixteen burns (50 cm2 ) were created on the dorsum of four anesthetized swine. All materials were sterile, but a sterile feld was not utilized in order to simulate the prolonged feld care setting. The wounds were then treated with PLA, IHS, and SSD cream, and the remaining wounds (designated GSOC) were also treated with SSD cream. On post-operative day (POD) 3, sterile surgical debridement and skin grafting (1:4) were performed on the GSOC wounds. Burn healing was followed for either PODs 10, 14, 21, or 28, wherein one animal was humanely euthanized at each time point; each represented a time point of the healing process. A full-thickness excisional biopsy was taken from each wound immediately after euthanasia to give a cross-section view of the woundedgeto edge. Wound healing was determined by the histological analysis of wound re-epithelialization, epidermal thickness, rete ridges, and scar elevation index and macroscopically using noninvasive imaging systems.Results:The PLA and IHS treatments did not need to be reapplied to the wounds during the course of the experiment, unlike SSD, which was reapplied at each assessment time point. In terms of re-epithelialization, on POD 10, IHS and SSD were similar to the GSOC; on POD 14, all treatments were similar; on POD 21, PLA and IHS were similar to SSD; fnally, on POD 28, re-epithelialization was similar in all groups. On POD 28, scar elevation index and rete ridges/mm were similar to all groups, and epidermal and dermal thickness for PLA and IHS were similar to GSOC.Conclusions:This preclinical study demonstrated that the use of the PLA and the IHS dressings resulted in similar outcomes to the GSOC-treated burns in several key metrics of wound healing. These therapies represent a potentially useful tool in current and future battlespaces, where surgical intervention is not possible. The products are lightweight and, more importantly, stable at room temperature for their entire shelf lives. This would allow for easy storage and transport by medical practitioners in the feld.INTRODUCTIONBurns are common in all military conficts. During Operation Iraqi Freedom and Operation Enduring Freedom, a burn- injured American service member had an average time of 3 to 4 days from point of injury to receiving defnitive care at the<a id="bookmark1"></a>*United States Army Institute of Surgical Research, Fort Sam Houston, TX 78234, USA<a id="bookmark2"></a>† Metis Foundation, San Antonio, TX 78216, USAThe views expressed are those of the author(s) and do not refect the off- cial policy or position of the U.S. Army Medical Department, Department of the Army, DOD, or the U.S. Government. This work was presented at the Military Health System Research Symposium (MHSRS) in Kissimmee, FL, USA, in August 2023.doi:https://doi. org/10 . 1093/milmed/usad390Published by Oxford University Press on behalf of the Association of Mil- itary Surgeons of the United States 2023. This work is written by (a) US Government employee(s) and is in the public domain in the US.<a id="bookmark3"></a>U.S. Military’s burn center.<a href="#bookmark4">1</a> However, future multi-domain operations against near-peer foes will not have this advantage; evacuation of casualties is expected to be delayed for days <a id="bookmark5"></a>and even up to a week<a href="#bookmark6">.2,3</a> Given that the future battlespace will be quite different from Operation Iraqi Freedom/Opera- tion Enduring Freedom, there is a critical need for materials to address the inability to debride and graft burns at the point <a id="bookmark7"></a>of injury.<a href="#bookmark8">4</a>The current standard of care (SOC) for partial- and full- thickness burns in a prolonged feld care (PFC) setting is silver sulfadiazine (SSD) cream that is applied to wounds twice <a id="bookmark9"></a>daily (BID) for 3–5 days<a href="#bookmark10">.5</a> Once a patient has been evacu- ated, the gold standard of care (GSOC) is defned as surgical debridement of the dead tissue followed by the application of autologous split-thickness skin graft (STSG). Although deep partial-thickness (DPT) burns may heal without surgicalMILITARY MEDICINE, Vol. 00, Month/Month 2023 1Off-the-Shelf Therapies for Battlefeld Burnsintervention, it can take as much as 3 to 4 weeks for wound closure to occur. Early intervention and the GSOC greatly reduce the risk of infection, scarring, and physical disabil- <a id="bookmark11"></a>ity.<a href="#bookmark6">2</a><a href="#bookmark12">,6–</a><a href="#bookmark13">9</a>To mimic the GSOC in the feld, the ideal topical therapy for treating burns in the battlespace should be an easy-to-use, lightweight, cost-effective, single-component, and single- application product. Additionally, it should provide rapid wound coverage to protect the injured area from contamina- tion and create a moist environment that reduces pain and <a id="bookmark14"></a><a href="#bookmark15">prevents burn progression.10–</a><a href="#bookmark16">14</a>Importantly, any therapy used in PFC must be easy to apply by inexperienced personnel and left in place for several days. There currently exist commer- cially available advanced wound care products, such as skin substitutes and matrices, for the treatment of burns. These therapies provide barrier function, protect against infection, promote re-epithelialization, and thus may be more effective in the PFC of wounded servicemembers than the current SOC, <a id="bookmark17"></a>SSD cream<a href="#bookmark18">.11–</a><a href="#bookmark19">20</a>In our previous study, we compared fve commercially available off-the-shelf (OTS) therapies in the treatment of <a id="bookmark20"></a>DPT burns in a PFC scenario to the current SOC.<a href="#bookmark21">21</a>DPT burns, unlike full-thickness burns, do not require evacuation from theater; as such, we investigated products that might allow service members to remain in situ. However, DPT is often debrided and grafted to avoid development of hypertrophic <a id="bookmark22"></a>burn scars<a href="#bookmark23">.22</a>Two shelf-stable OTS therapies were selected in our initial study: polylactic acid (PLA) and irradiated human skin (IHS)<a href="#bookmark21">.21</a> PLA (trade name: Suprathel® , PolyMedics Innovations GmbH, Denkendorf, Germany) is an epidermal skin substitute with properties that mimic natural epithe- lium. It is produced from a synthetic copolymer of dl-lactide <a id="bookmark24"></a>(&gt;70%), trimethylene carbonate, and ε-caprolactone<a href="#bookmark25">.23,24</a>IHS (trade name: GammaGraft, Promethean Life Sciences, Inc., Pittsburgh, PA) is an irradiated, shelf-stable, and sterile human skin allograft. The allograft is stored in a peni- cillin/gentamycin solution, which acts as a preservative to pre- <a id="bookmark26"></a>vent microbial contamination of the product<a href="#bookmark27">.24,25</a> Our results showed that both PLA and HIS-treated burns re-epithelialized effciently 28 days after initial burns (83% and 100%, respec- tively). Treatment with these therapies also resulted in good quality of healing in terms of maturation of the newly devel- oped epidermis. Furthermore, the results showed that IHS and PLA prevented burn wound progression and protected burns from pathogens by creating a physical barrier against envi- ronmentalbacteria<a href="#bookmark21">.21</a>Therefore, the purpose of this study is to compare these two OTS therapies to the current GSOC. Our objec- tive is to identify whether these OTS products would have the ability to temporize burn wounds to allow spontaneous healing and eliminate the need for debride- ment and autografting of DPT, potentially reducing the need to evacuate burned warfghters from the theater of operations.MATERIALS AND METHODSStudy DesignAnimalsAnimal procedures for this study were approved by the US Army Institute of Surgical Research (USAISR) Institutional Animal Care and Use Committee (IACUC, Protocol #A-22- 002). Four pathogen-free Yorkshire Hybrid Pigs (Midwest Research, Glencoe, MN, USA) weighing 55–70kg were used for this study. All animals were individually housed at the USAISR animal facility, acclimated for at least 72 h, and fasted 8 h before planned procedures. Anesthesia and analge- sia were used for primary burn creation and all planned wound biopsies as described in our previous communication<a href="#bookmark21">.21</a> This study consisted of four pigs that were followed until either post-operative days (PODs) 10, 14, 21, or 28; each pig rep- resented a time point of the healing process. Animals were humanely euthanized under surgical anesthesia according to IACUC policies (<a href="#bookmark28">Fig. 1A)</a>.Downloaded from <a href="https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209bygueston30November2023">https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209 by guest on 30 November 2023</a>Burn creationThe dorsum and fanks of the anesthetized swine were trimmed with an electric razor and then chemically depilated. The skin was then cleaned with saline and sterilized with 70% isopropyl alcohol. Sixteen 50 cm2 rectangular (10 × 5 cm) wounds were drawn using a surgical marker on the porcine dorsum. The number, size, and placement of burns were deter- mined based on the prior studies, and the dimension of a representative 55 kg pig with Total body surface area burned less than 10%. At least 3 cm separated all burns to mini- mize site-to-site infuence. A thermocouple burn device with a square brass plate (USAISR, San Antonio, TX) heated to 100 。C was applied to the skin at a constant pressure for 17 s to create DPT burns. Temperature probes and infrared cameras were used to confrm consistent temperatures. Burn wound edges were tattooed (Superior Tattoo, Phoenix, AZ, USA) to delineate wounds<a href="#bookmark21">.21</a>Therapies and applicationNonsurgical debridement was done 1 h after burn cre- ation using gauze wetted with sterile saline to remove excess eschar. Subsequently, the burns were randomized to receive (1) PLA (Suprathel® , PolyMedics Innovations GmbH, Denkendorf, Germany) therapy, (2) IHS (Gamma- Graft, Promethean Life Sciences, Inc., Pittsburgh, PA) ther- apy, (3) PFC standard of care (SSD cream, Ascend Laborato- ries, LLC, Parsippany, NJ), and GSOC (SSD cream + surgical debridement + grafting) (n = 4/treatment/animal) (<a href="#bookmark28">Fig. 1)</a>. To increase the power and reproducibility, as well as to min- imize variations and experimental errors, the randomized block design was used. It accounts for differences in healing parameters by alternating the anatomical location of treat- ments on the dorsum allowing comparison of the wounds <a id="bookmark29"></a>receiving various treatments<a href="#bookmark30">.26</a> All materials were kept ster- ile, but a sterile feld was not utilized to simulate the PFC2 MILITARY MEDICINE, Vol. 00, Month/Month 2023<a id="bookmark28"></a>Off-the-Shelf Therapies for Battlefeld Burns<img src="/media/202408//1724838601.586122.png" />Downloaded from <a href="https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209bygueston30November2023">https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209 by guest on 30 November 2023</a>FIGURE 1. (A) The study fowchart and wound layout. (B) Burns were randomized to receive the GSOC, PFC, PLA, or IHS treatments.setting. Therapies were placed on all experimental wounds on day 0, 1 h postburn according to the manufacturer’s instruc- tions. SSD was applied in a thick layer and affxed in place by Tegaderm (3 M, Saint Paul, MN), according to tactical combat casualty care guidelines. The GSOC group was also covered with SSD on POD 0. PLA and IHS were covered with a silicone non-adhesive (Rylon-1, Bio Med Sciences, Allen- town, PA). Changing of secondary dressings and the surgical jacket occurred on follow-up days. All treatments were then covered with non-adherent gauze, secured with self-adherent bandages and a surgical swine jacket. Wounds were assessed on PODs 3, 7, 10, 14, 21, and 28. SSD wounds were cleaned with gauze and saline, and SSD was reapplied at each time point on the wounds until POD 14. The OTS therapies were replaced only if they shifted off the wound. Assessments were carried out using digital photographs, noninvasive imaging, and histology (<a href="#bookmark28">Fig. 1)</a>.Debridement and skin graftingSurgical debridement was performed on POD 3 for GSOC- treated wounds. A sterile surgical feld was created around the wound and graft donor sites, and sharp surgical debridement was conducted to excise the eschar until punctate bleeding was observed. Up to four STSGs were harvested from dorsolateral thigh using a dermatome (Zimmer, Dover, OH). The thickness of the skin grafts was 0.30 mm, and they were subsequently meshed at a ratio of 1:4 using a sterile skin graft mesher (Zim- mer). The meshed STSGs were placed over the GSOC wounds and secured in place by sterile skin staplers. The grafted burn wounds were held in place and covered by strips of Tegaderm (3 M). STSG secondary dressings were replaced as needed as a result of shifting or saturation. Donor site wounds were covered with sterile gauze or Xeroform (Cardinal Health, Dublin, OH) and stockinetteaffxed with sterile skin staples.MILITARY MEDICINE, Vol. 00, Month/Month 2023 3Off-the-Shelf Therapies for Battlefeld BurnsOUTCOME MEASUREMENTSBurn DepthUltrasound images were taken from all wounds 1 hand 3 days postburn to determine burn depth using a linear transducer (GE Logiq E9, GE Healthcare, Chicago, IL). Wounds were measured starting from the dorsal end towards the ventral end, holding the transducer perpendicular to the wound surface. Wound depth was determined by the depth of the damage observed.Wound ContractionThe SilhouetteStar® imaging device (Aranz Medical Ltd,New Zealand) was used to measure wound contraction bycomparing the wound area on days 21 and 28 to the orig- inal area on day 0. Wounds were recorded and measured with a three-path laser, and changes in the wound area were measured using the software’s boundary tracking tools.Wound Elevation, Pigmentation, and Skin PoresThe Antera 3D imaging device (Miravex, Dublin, Ireland) was used to capture three-dimensional wound images on day 28. Collected images were analyzed and measured for wound elevation (mm3 ), pigmentation (melanin), and pores (pore count/cm2 ) using the Antera 3D imaging software (Miravex).Histological AnalysesWound biopsies were harvested after humane euthanasia. A full-thickness excisional biopsy was taken from each wound immediately after euthanasia to give a cross-sectional view of the woundedgeto edge. Tissues were fxed for at least 48 h in 10% neutral-buffered formalin, dehydrated, and embed- ded in paraffnfor histological analysis. A microtome (Leica Biosystems, Buffalo Grove, IL, USA) was utilized to cre- ate 5-7 rm thick sections stained with hematoxylin and eosin (H&amp;E) and Mason’s trichrome. Histological sections were scanned using an Axioscan 7 (Carl Zeiss AG, Oberkochen, BW, Germany). Excisional biopsies were used to assess the microscopic wound healing using Image J software (U.S. NIH, Bethesda, MD, USA).Microscopic wound healing was determined by histologi- cal analysis of wound re-epithelialization, burn wound depth, dermal thickness, epidermal thickness, rete ridges, and scar elevation index (SEI). Epidermal thickness was measured as the thickness of the stratum corneum and the cellular epi- dermis (millimeters) from fve representative areas. Dermal thickness was measured as the thickness of dermis from the surface down to hypodermis (millimeters) from fve repre- sentative areas. A rete ridge per millimeter ratio was deter- mined by counting the number of rete ridges on the length of the re-epithelialized area. SEI was measured as the ratio of total scar tissue thickness to the thickness of healthy tis- sue around the scar. Re-epithelialization-% was calculated asthe sum of the new epithelium divided by the original wound area.STATISTICAL ANALYSISStatistical analysis was performed with GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Data are pre- sented as mean ± SEM. The Shapiro–Wilk normality test was used to test whether the data are normal. All samples passed the normality test allowing us to state that no signifcant depar- ture from normality was found. Comparison of treatments was performed using unpaired one-way ANOVA with Tukey’s multiple comparison test, and P-values of &lt; .05 were con- sidered statistically signifcant. N number for each treatment group was 4–8.Downloaded from <a href="https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209bygueston30November2023">https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209 by guest on 30 November 2023</a>RESULTSThe PLA and IHS treatments did not need to be reapplied to the wounds during the course of the experiment, unlike PFC SOC SSD cream, which was reapplied at each POD.Burn DepthUltrasound imaging was used to confrm that the created burn wounds were similar in depth. The results showed that all burn wounds were DPT on day 0 and progressed in depth by 3 days regardless of the treatment (<a href="#bookmark31">Fig. 2)</a>.Re-epithelializationBurn wound re-epithelialization was measured from the H&amp;E-stained wound strips on days 10, 14, 21, and 28. Re- epithelialization on POD 10 for PLA, IHS, SSD, and STSG was 21.94 ± 5.70%, 10.42 ± 3.82%, 48.41 ± 10.36%, and 12.62 ± 3.03%, respectively. SSD had signifcantly higher percent re-epithelization (P &lt; .01) than IHS and STSG. Re- epithelialization on POD 14 for PLA, IHS, SSD, and STSG was 77.25 ± 17.82%, 48.89 ± 10.37%, 47.09 ± 16.48%, and 66.9 ± 14.91%, respectively; no statistically signifcant differ- ences were found. Re-epithelialization on POD 21 for PLA, IHS, SSD, and STSG was 70.03 ± 15.94%, 44.99 ± 9.02%, 38.88 ± 11.27%, and 100.00 ± 0.00%, respectively. STSG had signifcantly higher percent re-epithelization than IHS and SSD (P &lt; .05 and P &lt; .01, respectively). On POD 28, PLA, IHS, SSD, and STSG percent re-epithelization was 69.57 ± 8.96%, 68.66 ± 13.95%, 76.84 ± 15.21%, and 100.00 ± 0.00%, respectively. No statistically signifcantdif- ferences were seen in percent re-epithelialization on POD 28 <a href="#bookmark32">(Fig. 3)</a> <a href="#bookmark33">(Table S1)</a>.Epidermal ThicknessEpidermal thickness was measured on PODs 21 and 28 utiliz- ing histology. The thickness of stratum corneum and cellular epidermis of wounds treated with PLA, IHS, SSD, and GSOC were 0.28 ± 0.06, 0.31 ± 0.03, 0.29 ± 0.05, and 0.22 ± 0.03 on day 21 and 0.45 ± 0.03, 0.38 ± 0.05, 0.5 ± 0.03, and 0.31 ± 0.04 mm on day 26 respectively. Statistically signif- icant differences were observed between SSD and STSG4 MILITARY MEDICINE, Vol. 00, Month/Month 2023<a id="bookmark31"></a>Off-the-Shelf Therapies for Battlefeld Burns<img src="/media/202408//1724838601.686767.png" />Downloaded from <a href="https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209bygueston30November2023">https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209 by guest on 30 November 2023</a>FIGURE 2. Representative ultrasound images of burns. Burn depth was determined using an ultrasound probe by evaluating the depth of the damage observed. <a id="bookmark32"></a>Red dashed lines indicate the depth of the damage immediately afterburn creation (day 0) and on day 3 postburn.<img src="/media/202408//1724838601.815548.png" />FIGURE 3. Re-epithelialization: (A) Postburn day 10, (B) postburn day 14, (C) postburn day 21, and (D) postburn day 28.(P &lt; .05) on POD 28. No other statistically signifcant differ- ences were found (<a href="#bookmark34">Fig. 4A &amp; B)</a> <a href="#bookmark33">(Table S1)</a>.Dermal ThicknessDermal thickness of wounds treated with PLA, IHS, SSD,and GSOC were 5.55 ± 0.89, 3.47 ± 0.49, 4.78 ± 0.62, and3.88 ± 0.50 mm on POD 21 and 7.22 ± 0.07, 5.94 ± 1.14, 8.10 ± 0.41, and 4.87 ± 0.64 on POD 28 mm, respectively. Statistically signifcant differences were observed between SSD and STSG (P &lt; .05) on POD 28. No other statis- tically signifcant differences were found <a href="#bookmark34">(Fig. 4A &amp; B)</a> <a href="#bookmark33">(Table S1)</a>.MILITARY MEDICINE, Vol. 00, Month/Month 2023 5<a id="bookmark34"></a>Off-the-Shelf Therapies for Battlefeld Burns<img src="/media/202408//1724838601.9077172.png" />Downloaded from <a href="https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209bygueston30November2023">https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209 by guest on 30 November 2023</a>FIGURE 4. Quality of healing: (A) Microscopic quality of healing in terms of epidermal thickness, dermal thickness, number of rete ridges, and SEI on days 21 and 28. (B) Representative Masson’s trichrome images of the burns on day 28 post injury. (C) Macroscopic quality of healing in terms of epidermal thickness burn wound contraction (SilhouetteStar® imaging device, Aranz Medical Ltd), wound elevation (Antera 3D imaging device, Miravex), wound pores (Antera 3D imaging device, Miravex),and pigmentation (Antera 3D imaging device, Miravex) on day 28 postburn.Rete RidgesEpidermal rete ridges were measured on PODs 21 and 28 utilizing histology. Measurements of rete ridges per mil- limeter for wounds treated with PLA, IHS, SSD, and STSG were 1.91 ± 0.77, 3.48 ± 0.70, 3.41 ± 1.11, and 2.23 ± 0.07 on POD 21 and 1.61 ± 0.2, 1.89 ± 0.53, 1.82 ± 0.47, and 2.42 ± 0.35 on POD 28, respectively. No statistically signif- cant differences were found (<a href="#bookmark34">Fig. 4A &amp; B)</a> <a href="#bookmark33">(Table S1)</a>.Scar Elevation IndexSEI was measured on PODs 21 and 28 of using the H&amp;E-stained wound strips. Measurements of SEI on POD 21 for wounds treated with PLA, IHS, SSD, and STSG were 2.02 ± 0.39, 1.35 ± 0.19, 1.58 ± 0.16, and 1.33 ± 0.16,respectively. No statistically signifcant differences were found. On POD 28, measurements of SEI in the PLA, IHS, SSD, and STSG-treated burns on POD 28 were 3.15 ± 0.07, 2.69 ± 0.38, 3.248 ± 0.26, and 2.58 ± 0.18, respectively. No statistically signifcant differences were found (<a href="#bookmark34">Fig. 4A &amp; B)</a> <a href="#bookmark33">(Table S1)</a>.Wound ContractionWound contraction was measured on day POD 28 usingmacroscopic images of the burn wounds. Wound contrac- tion in the PLA, IHS, SSD, and STSG-treated burns on POD 28 was 37.46 ± 3.31, 13.98 ± 4.74, 18.08 ± 5.99, and 14.40 ± 9.50%, respectively. No statistically signifcant dif- ferences were observed (<a href="#bookmark34">Fig. 4C)</a> <a href="#bookmark33">(Table S1)</a>.6 MILITARY MEDICINE, Vol. 00, Month/Month 2023Off-the-Shelf Therapies for Battlefeld BurnsWound ElevationWound elevation was measured on day 28 using a 3D cameraand reported in units of volume (mm3 ). Intact skin was used as a reference, and it had an elevation of 5.73 ± 0.25 mm3 . The burn wounds treated with PLA, IHS, SSD, and STSG had elevated 21.93 ± 2.57, 40.82 ± 12.46, 38.94 ± 4.17, and 47.29 ± 6.42 mm3 , respectively. All treatment groups, exclud- ing the PLA, exhibited statistically signifcant elevation in comparison to intact skin (<a href="#bookmark34">Fig. 4C)</a> <a href="#bookmark33">(Table S1)</a>.PigmentationPigmentation was measured as the relative variation of melanin in the burn wounds on day 28 postburn using a 3D camera. Intact skin was used as a reference, exhibit- ing 0.05 ± 0.00 relative variation of melanin. The burn wounds treated with PLA, IHS, SSD, and STSG demonstrated 0.38 ± 0.04, 0.25 ± 0.03, 0.41 ± 0.09, and 0.22 ± 0.05 rel- ative variation of melanin, respectively. In comparison to the intact skin, the differences in the PLA and the SSD- treated burn wounds were statistically signifcant <a href="#bookmark34">(Fig. 4C)</a> <a href="#bookmark33">(Table S1)</a>.Pores on SkinThe number of pores on the healing burn wound was mea-sured on day 28 using a 3D camera. Pores on skin are smallopenings around hair follicles and glands that help gases and liquids move through the surface of your skin. The number of pores on intact skin (26.50 ± 0.97) was used as a reference. The burn wounds treated with PLA, IHS, SSD, and STSG demonstrated 31.58 ± 1.61, 34.85 ± 4.41, 30.33 ± 1.10, and 30.10 ± 0.05 relative variation of melanin, respectively. No statistically signifcant differences were observed <a href="#bookmark34">(Fig. 4C)</a> <a href="#bookmark33">(Table S1)</a>.DISCUSSIONThe current GSOC of deep and large burns consists of surgical debridement of the burned area followed by the application of STSG. The inability to surgically debride burn injuries at the point of injury requires the investigation of currently existing therapies tofll gaps in pre-hospital care for use in a PFC set- <a id="bookmark35"></a>ting.<a href="#bookmark36">27</a>Therefore, the purpose of this study was to examine the potential of two OTS dressings (PLA and IHS) to reduce the need to evacuate burned servicemembers to a defnite care for surgical intervention.We continued our work on testing the effcacy of PLA and IHS that performed well in our initial experiment<a href="#bookmark21">.21</a> We uti- lized a standardized preclinical burn model that simulated the battlespace setting by treating burns in a nonsterile fashion; saline-soaked gauze was used to non-surgically debride burns according to the Joint Trauma System’s PFC guidelines before application of treatments on day 0. We expanded on our initial study by comparing these therapies to the GSOC. The STSGs in the GSOC-treated burns were meshed 1:4 to mimic a sce- nario when the availability of healthy donor skin is scarce.Importantly, we extended the burn time from 15 s in Varon et al. (2022)<a href="#bookmark21">21</a> to 17 s and increased to burned area to create deeper and larger burns. This is all the more important when accounting for the similarities in healing outcomes observed between the OTS dressings and the GSOC by this experiment. Our primary endpoints were in re-epithelialization and quality of healing.A key metric in burn wound healing is the timely restora- <a id="bookmark37"></a>tion of the skin barrier function<a href="#bookmark38">.28</a> Wound closure showed some promising outcomes when comparing PLA and IHS to the current PFC SOC and to the GSOC. On PODs 10 and 14, PLA and IHS had similar re-epithelialization to GSOC- treated burns, and on day 28, no signifcant differences were found. These results showing that re-epithelialization was similar in groups that were not debrided and grafted were important. Currently, when burn injury occurs in austere envi- ronment, the warfghterisfrst transported to a higher role of care for surgical intervention, which may alone take several days. It has also been predicted that service members may be forced to remain in theater for up to 7 days after the pro- <a id="bookmark39"></a>cedure, further delaying return to duty.<a href="#bookmark6">2</a><a href="#bookmark40">,3,29</a> In addition to wound closure, quality of healing was studied microscopi- cally using different histological parameters and macroscopi- cally using noninvasive imaging systems. The results demon- strated that no signifcant differences in terms of maturation of the newly formed epidermis (epidermal thickness and rete ridges) or scarring (SEI, wound elevation, and contraction) were observed between the treatment groups.Downloaded from <a href="https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209bygueston30November2023">https://academic.oup.com/milmed/advance-article/doi/10.1093/milmed/usad390/7287209 by guest on 30 November 2023</a>PLA and IHS are shown here to be the promising therapies in the search for existing FDA-approved therapies for burns in a PFC setting. In several key metrics, we have shown that they behave similarly to the current SOC for PFC, as well as the GSOC. PLA has also been shown to have improved scar quality and reduced pain scores when compared to silver <a id="bookmark41"></a>dressings<a href="#bookmark42">.30</a> In accordance with our results, this suggests that PLA maybe an effective burn care product in the battlespace, potentially allowing the warfghter to needlesspain medica- tions. IHS has been previously used in the Operation Iraqi <a id="bookmark43"></a>Freedom as a temporary wound coverage with good results<a href="#bookmark44">.31</a> 	This study has some limitations. Although this study did not observe the effects of bacterial colonization of wounds, infections are an important component of many battlefeld injuries, including burn wounds.<a href="#bookmark19">20</a>The use of chemical depila- tory to remove hair may have cleaned the dorsum of the pig preventing microbial colonization of wounds, failing to mimic conditions in combat injury. The current SOC for PFC involves BID cleaning (twice daily) of burn wounds and reap- plication of SSD.<a href="#bookmark10">5</a>Limiting the hardship to the study animals prevented us from following this SOC. Though it is possible that scrubbing these wounds may have interfered with healing, we replicated PFC conditions as much as was practicable. Fur- thermore, this study, utilizing four animals, each with sixteen burns, assumes that wound healing is consistent across indi- viduals. It is known that person to person healing may vary, and it is possible that this could be a confounding factor inMILITARY MEDICINE, Vol. 00, Month/Month 2023 7Off-the-Shelf Therapies for Battlefeld Burnsour study. However, the pattern of healing displayed by our <a id="bookmark45"></a>animals mimics previous swine burn models<a href="#bookmark13">.9,13,21,27</a>CONCLUSIONSThis preclinical study demonstrated that the use of the PLA and the IHS dressings resulted in similar outcomes to the GSOC (surgical debridement + autografting)-treated burns in several key metrics of wound healing, suggesting that their utilization may reduce the need to debride and autograft and thus expedite return to duty. Importantly, these therapies have <a id="bookmark4"></a>a feld-deployable potential; they are single application, sin- gle component, lightweight, stable at room temperature, and <a id="bookmark6"></a>readily available on the market. Despite their higher costs, they only need to be applied once, whereas SSD requires mul- <a id="bookmark40"></a>tiple reapplications, potentially making them useful for battle- feld medicine. However, these advantages necessitate further<a id="bookmark8"></a>study to determine their potential in human burn victims in aPFC setting.ACKNOWLEDGMENTS<a id="bookmark33"></a>None declared.SUPPLEMENTARY MATERIAL<a href="https://academic.oup.com/milmed/article-lookup/doi/10.1093/milmed/usad390#supplementary-data">Supplementary material</a>is available at Military Medicine online.FUNDING<a id="bookmark13"></a>This work was supported by the USAMRDC (Award# K_031_2020).CONFLICT OF INTEREST STATEMENTAll authors declare no conficts of interest.DATA AVAILABILITY STATEMENTThe data that support the fndings of this study are available on request fromthe corresponding author (K.N.) upon a reasonable request.CLINICAL TRIAL REGISTRATIONNot applicable.<a id="bookmark46"></a>INSTITUTIONAL REVIEW BOARD (HUMAN <a id="bookmark16"></a>SUBJECTS)Not applicable.INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)The research was conducted in compliance with Animal Welfare Act, the implementing Animal Welfare regulations, and the principles of the Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC) approved all research conducted in this study (Protocol #A-22-002). The facility where research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.INSTITUTIONAL CLEARANCEInstitutional clearance was approved.DECLARATIONSAll authors declare no confict of interest.INDIVIDUAL AUTHOR CONTRIBUTION STATEMENTA.H.C., R.J.C., and K.N conceptualized and designed the study. Experi- mental work and animal studies were performed by D.L., A.H.C., F.A.V., M.B., L.L., L.S., and K.N. D.L., A.H.C., L.L., R.J.C., and K.N. analyzed and interpreted the data. 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