This work was supported by generous funding from Shriners Children&#39;s Research Grant to S.N.T. Y.B. was supported by Shriners Hospital for Children. We also gratefully acknowledge funding to S.N.T. from the US National Institute of Health (K99/R00 HL1431149; R01HL157803; R01DK134590, R24OD034189), American Heart Association (18CDA34110049), Harvard Medical School Eleanor and Miles Shore Fellowship, Polsky Family Foundation, and the Claflin Distinguished Scholar Award on behalf of the MGH Department of Surgery and/or MGH Executive Committee on Research. Further, we acknowledge the support provided by the Massachusetts General Hospital Executive Committee of Research for awarding the Fund for Medical Discovery (FMD) award to R.J. Finally, support from &#34;Fondation des Gueules Cass&#233;es&#34; (France), Rennes University (France), CHU de Rennes (France) and the French Society of Plastic Surgery to Y.B. is greatly acknowledged. The authors thank Knight Surgery Research Laboratory for their contribution and help with the anesthesia of the animals.

All animal work was performed in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) checklist37 and was compliant with the Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) under protocol #2021N000271. Humane care was provided to the animals, following the Guide for the Care and Use of Laboratory Animals38. Five 30 kg female Yucat&#225;n minipigs were used for these experiments. The animals were obtained from a commercial source (see Table of Materials).
1. Pre-operative care and anesthesia
<ol>
	<li>Fast the animals for 12 h before&#160;general anesthesia.</li>
	<li>Initiate anesthesia by an intramuscular injection of 2-4 mg/kg of tiletamine hydrochloride and zolazepam hydrochloride and 1-2 mg/kg of xylazine, followed by inhalation of isoflurane (2-3% in oxygen) (see Table of Materials) for maintaining anesthesia during the procedure.</li>
</ol>
2. Burn wound design and randomization
<ol>
	<li>In order to study both partial and full-thickness burns with negative controls on the same animal (no inter-individual variation), create eight wounds on each pig&#39;s dorsum in the paravertebral area. Perform three full-thickness wounds, three partial-thickness wounds, and two control wounds.</li>
	<li>Number the wounds from 1 to 8, and perform randomization to allocate different burn degrees (partial thickness, full thickness, control) to different anatomical locations on the back. 
	NOTE: Randomization of each wound (depth and location) is performed to improve the significance: Pig dermis is known to vary in terms of thickness and collagen composition39. Therefore, randomization on each animal is used to decrease potential biases.</li>
</ol>
3. Tattoo wound delimitation
NOTE: The first procedure consists of creating circular tattoos on the pig&#39;s dorsum in order to localize and number the randomized wounds (Figure 1). This is performed two days before the initial burn procedure to allow for better acclimatization, but it can be performed on the day of the burn procedure.
<ol>
	<li>After anesthesia (see Step 1)&#160;and orotracheal intubation in the supine position40, place the pig in the prone position, and place soft wedges (bed sheets) under the animal for its comfort and stabilization (under the forelimbs, the umbilical area, and the hindlimbs)41. Place a forced-air warming blanket on the head and neck of the animal to maintain adequate body temperature.</li>
	<li>Place a straight line centrally on the pig&#39;s back to allow the symmetrization of the drawings.</li>
	<li>Draw two vertical lines using a dermographic pen (see Table of Materials) on both sides of the central line, placed 6.5 cm laterally from it. The two lateral lines will be used to place the center of the circles.</li>
	<li>Draw each pair of 4.5 cm diameter circles symmetrically, keeping a minimum distance of 4 cm between two distinct circles (Figure 1A).</li>
	<li>Once the drawings are done, perform a single-step skin preparation (povidone-iodine 7.5%) to decrease the skin bacterial flora.</li>
	<li>Perform the tattoo procedure42 of eight 4.5 cm circles using a needle-tattoo machine, sterile black ink, and a sterilized 5-point needle (see Table of Materials). The final result is presented in Figure 1B.</li>
	<li>In case of a delayed burn creation procedure, apply triple-antibiotic ointment on the tattoos and cover it with transparent adhesive dressings.</li>
</ol>
4. Burn wound creation and advanced wound dressing
NOTE: Burns will be created by placing the brass block in contact with the skin on the dedicated spot (randomization) for 30 s (invariable). The temperature will determine the burn depth.
<ol>
	<li>On the day of the burn procedure, once the animal is anesthetized and placed in the prone position as discussed in Step 1, place cylindric brass blocks in stainless steel containers filled with aluminum beads (see Table of Materials). Place the containers on a temperature-controlled hot plate. Set one hot plate to 65 &#176;C (for the partial thickness burns), and the second to 93 &#176;C (for the full-thickness burn, Figure 2). 
	NOTE: The container can be kept dry to avoid scalding burns (2-3 h needed to reach the temperature equilibrium) or half-filled with water (30 min to reach the temperature equilibrium). If water is used, the brass blocks need to be thoroughly dried before step 4.5 in order to avoid subsequent burns due to scalding.</li>
	<li>Insert a thermometer into the brass cylinder via its central opening to monitor its core temperature, and confirm the surface temperature with a temperature gun. If needed, increase the hot plate&#39;s temperature to 65 &#176;C or 93&#176; C in the cylinder despite minor heat dissipation. 
	NOTE: Aluminum foil can be used to cover the container and further minimize heat dissipation, reaching the desired temperature faster.</li>
	<li>In parallel, place the animal in the prone position for the burning procedure, similar to the tattoo procedure (step 3.1).</li>
	<li>Once the animal is ready in the prone position, prepare the skin in three steps (povidone-iodine 7.5%, saline solution 0.9%, drying).</li>
	<li>Grasp the brass block with heat-resistant gloves and place it on the dedicated tattoo spot, depending on the randomization. Start the timer as soon as the brass block touches the skin. Be careful not to apply more pressure to the skin than the brass block&#39;s own weight.</li>
	<li>After 30 s, remove the brass block from the skin and put it back in its dedicated heating container. Monitor the temperature until it reaches the target, and repeat the procedure for all the wounds.</li>
	<li>Perform an advanced multi-layer dressing to ensure dressing stability after the animal has recovered.
	<ol>
		<li>Place a petroleum-impregnated gauze on each burn location, including controls, and cover it with a dry non-woven gauze and a large transparent adhesive dressing.</li>
		<li>Spray a tincture of benzoin solution on the surrounding skin for better adherence of the transparent dressings to the skin.</li>
		<li>Wrap the animal in self-adherent adhesive wrap dressing while paying attention not to overtighten it (restriction of pulmonary volumes).</li>
		<li>Complete the dressing by adding a final layer of tubular stockinette (size adapted to the animal) (see Table of Materials).&#160;Alternatively, custom fitted swine jackets could&#160;be used&#160;if the animal is unable to keep the dressing clean for the duration of study</li>
		<li>Ensure correct analgesia by placing a transdermal opioid (fentanyl) patch on the neck of the animal after injecting a single dose of buprenorphine (0.05-0.1 mg/kg, IM) and a single dose of carprofen (2-4 mg/kg, IM).</li>
		<li>Following each anesthetization, monitor animals closely up to 2 h after recovery. Use a warm pad if needed, and provide food freely after a full standing position is reached.</li>
	</ol>
	</li>
</ol>
5. Full-thickness burn escharotomy
NOTE: Between 1 and 3 days postoperatively, the animals will receive full-thickness surgical excision of the eschar following the third-degree burns.
<ol>
	<li>After similar preparation of the animal including anesthesia, intubation, and placing the animal in supine position, prepare the skin via the three step skin preparation, perform sterile draping of the back of the animal.</li>
	<li>Perform 4 mm punch biopsies (see Table of Materials) on the wound to confirm the type of injury.</li>
	<li>Perform the escharotomy43,44 by a circular incision of the eschar with a sterile scalpel blade (n&#176;15) until reaching the deep dermis.</li>
	<li>Grasp one side of the eschar firmly with a pair of sterile Adson tissue forceps (with 2/1 teeth) (see Table of Materials) and hitch it upwards to reveal the deep limit of the eschar.</li>
	<li>Continue the escharotomy using the scalpel blade while staying in the same plane within the deep dermis/underlying fascia. A bleeding dermis is considered a sign of tissue viability.
	<ol>
		<li>If an arteriole is cut during the process, perform a minimal cauterization using bipolar coagulation forceps (see Table of Materials). CAUTION: Extensive cauterization of the created wound bed can result in delayed healing and should be strictly avoided.</li>
	</ol>
	</li>
	<li>Put a first set of dry guaze on the wound bed for 5 minutes to enhance local hemostasis before placing the definitive dressing.</li>
	<li>Dress the wounds similarly as previously (step 4.7), with the addition of several dry gauze to fill the cavity. Apply the same layers as previously mentioned: Tincture benzoin spray, adhesive transparent dressing, self-adhesive wrap, tubular stockinette.</li>
	<li>Provide analgesia by changing the opio&#239;d transdermal patch and injecting Caprofen (2-4 mg/kg, IM).</li>
</ol>
6. Follow-up wound dressings
NOTE: Subsequent dressings are performed every 2 to 7 days, depending on the experimental treatment design and the animal&#39;s tolerance. Wound dressings can be stopped after 21 days to allow re-epithelialization in a dry environment and improve the animal&#39;s tolerance. Alternatively, if the treatment group necessitates a moist or wet environment, the dressings can be prolonged until the end of the study. The follow-up period was extended for up to 10 weeks in order to study both the acute and prolonged healing processes.
<ol>
	<li>Anesthetize&#160;the animal for a short period and placed in the supine position with a nose cone (isoflurane 2-5 L/min).</li>
	<li>Remove the previous dressings and clean the wounds using sterile 0.9% saline and sterile gauze.</li>
	<li>Apply the experimental treatment if applicable.</li>
	<li>Similarly, perform subsequent biopsies during this procedure if needed by the study. The animal should then receive pain medication such as carprofen (2-4 mg/kg, IM, 24 h effect).</li>
	<li>Cover all wound locations, including control wounds, with petroleum-impregnated or equivalent gauze, dry, non-woven gauze, and adhesive transparent dressing.</li>
</ol>

Yucatan Minipig Burn Wound Model for Analysis of Multi-Depth Burns

The authors have no conflict of interest to declare.

Wound healing following burn injuries is a long process that can take up to several months, with various treatment options and considerations for patient care2,13. In order to study it, a reliable and reproducible model is needed. Several animal models have been described, mainly including rodents29,45,46 and swine29,4...

Burns are a major public health problem and affect more than 480,000 patients in the US each year, according to the National Burn Repository1,2. This leads to more than 50,000 yearly hospitalizations for non-fatal complex cases requiring in-depth care2. Moreover, burns are a fundamental cause of military mortality and morbidity and are responsible for 10% to 30% of military casualties3,4. The management of burns has remained nearly unchanged for a long time, despite its immense and diverse impacts on patients, ranging from physical to psychological and emotional5.
Initial diagnosis and evaluation of burn injuries lead to a baseline classification according to the type of burns (first, second, and third) or the depth of the affected tissue (superficial, partial thickness, and deep burns)6,7,8. Partial-thickness burns (first and second-degree) involve the epidermis and different depths of the dermis (superficial or deep dermis, i.e., superficial and deep second-degree burns)9. In particular, damage to the appendages in the deep dermis excludes the possibility of re-epithelialization from the adnexal epithelium10. By definition, full-thickness burns reach the subcutaneous fat, fascia and/or the underlying muscle (third-degree burns), and sometimes the bone (also referred to as fourth-degree burns)11,12.
Following hospitalization, burn patients receive special care involving a strategy consisting of a delicate balance between tissue debridement and preservation. The damaged and/or secondarily infected soft tissue needs to be progressively removed until healthy tissue is exposed, allowing for the use of specific dressings and skin grafts to improve the healing process13,14,15,16. Yet, caution is required during surgery to avoid unintentional removal of healing tissue and reduce complications for optimal recovery. Biologically, burns exhibit a central necrotic area encircled by a &#39;shadow&#39; or &#39;stasis&#39; zone, indicating potentially reversible ischemia. This area can either deteriorate, resulting in an extended necrosis zone, or heal by reversing the apoptotic process17,18. This varying severity of burns presents challenges for surgeons to assess accurately, complicating the balance between conservative treatments and surgical excision19. To date, no efficient tool is available to help characterize this &#34;shadow zone&#34; preceding burn conversion. Developing such tools is crucial for optimizing this delicate balance.
Several treatments have been tested to help decrease secondary burn conversion. Yet, no specific therapy is currently available in the clinic18. Other examples of advances in burn treatments include the development of modern wound dressings and nanomaterials20,21, tissue-engineered skin22,23, and novel epidermal culture approaches24,25. Also, modern reconstructive surgery and fasciocutaneous flaps have improved the management of long-term after-effects, particularly burn contractures following pathologic healing of fold areas26,27. These advancements give promising prospects for burn patients, improving their treatment strategies and quality of life, but recent results show that the functional impact still remains substantial, both in the physical and psychological spheres28. Taken together, the demand for innovative advancements in both burn diagnosis and burn treatment is substantial.
Overall, many approaches are aiming at improving the diagnosis, management, and treatment of complex burn cases, and researchers need a reproducible and relevant model to test these new approaches. Due to its biological complexity, involving several organs and systemic reactions, no in vitro model proved itself relevant to study the burn wound process29. Rodent models have shown major discrepancies with humans due to major differences in biology, skin architecture, elasticity, and lack of adherence to the underlying structures29. In contrast, the swine model has proven to be relevant due to the structural similarity of swine skin to human skin30,31,32. It presents with a similar vascularization, elastic fiber composition, and renewal timing. Moreover, the hair follicle and apocrine annexes allow for islanded re-epithelialization, as can be observed in clinical superficial burns33,34. More specifically, Yucatan minipig models provide interesting features, making them relevant to studying pigmented skin35 and long-term outcomes with minimal physical changes36.
The purpose of this article is to describe a reliable multi-degree burn model in Yucat&#225;n pigs, enabling the study of several second and third-degree burns on the same subject. This provides a relevant and reproducible model for studying diagnostic and therapeutic innovations for the management of burns. Further, this model features different burn types and severity, a long-term follow-up allowing the study of burn contracture and pathologic healing, and pigmented skin differential behavior, which is known to have specific characteristics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adson tissue forceps</td>
			<td>Jarit</td>
			<td>130-234</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum beads</td>
			<td>Lab Armor</td>
			<td>42370-002</td>
			<td>Lab Armor Beads&#160;</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride</td>
			<td>Ranbaxy Pharmaceuticals</td>
			<td>NDC:12469-0757-01</td>
			<td>Buprenex Injectable</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer</td>
			<td>NADA 141-199</td>
			<td>Rymadyl 50mg/ml injectable&#160;</td>
		</tr>
		<tr>
			<td>Cylindric brass block</td>
			<td>Hand-made</td>
			<td>N/A</td>
			<td>Engineering drawing included in the manuscript</td>
		</tr>
		<tr>
			<td>Dermographic pen</td>
			<td>McKesson</td>
			<td>Surgical Skin Marker Sterile</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable #15 surgical scalpels</td>
			<td>Medline</td>
			<td>MDS15315</td>
			<td>Scalpel blades</td>
		</tr>
		<tr>
			<td>Fentanyl patch</td>
			<td>Mylan</td>
			<td>NDC:60505-7082</td>
			<td>Fentanyl Transdermal System</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal</td>
			<td>NDC:66794-013-25</td>
			<td>Isoflurane, USP</td>
		</tr>
		<tr>
			<td>McPherson Bipolar coagulation forceps</td>
			<td>Bovie</td>
			<td>A842</td>
			<td>Reusable, autoclavable</td>
		</tr>
		<tr>
			<td>Miltex assorted biopsy punches (3,4 and 5 mm)</td>
			<td>Integra</td>
			<td>33-38</td>
			<td>Biopsy punches- size to adapt to the study</td>
		</tr>
		<tr>
			<td>Non woven gauze</td>
			<td>Starryshine</td>
			<td>GZNW22</td>
			<td>2 x 2&#34; non woven 4 ply medical gauze pads</td>
		</tr>
		<tr>
			<td>Povidone-Iodine</td>
			<td>Betadine</td>
			<td>NDC:0034-9200-88</td>
			<td>Surgical scrub 7.5%&#160;</td>
		</tr>
		<tr>
			<td>Sterile isotonic sodium chloride solution 0.9%</td>
			<td>Aqualite System</td>
			<td>RL-2095</td>
			<td>Sterile saline solution</td>
		</tr>
		<tr>
			<td>Tattoo ink</td>
			<td>Spaulding &#38; Rogers</td>
			<td>Black - 2 oz - #9053</td>
			<td></td>
		</tr>
		<tr>
			<td>Tattoo marker</td>
			<td>Spaulding &#38; Rogers</td>
			<td>Special Electric Tattoo Marker</td>
			<td></td>
		</tr>
		<tr>
			<td>Tattoo needle</td>
			<td>Spaulding &#38; Rogers</td>
			<td>1310251</td>
			<td>Tattoo 5 point needle</td>
		</tr>
		<tr>
			<td>Tegaderm Transparent Film Dressing</td>
			<td>3M</td>
			<td>1.628</td>
			<td>Large transparent adhesive dressing</td>
		</tr>
		<tr>
			<td>Temperature-controlled hot plate</td>
			<td>Cole-Parmer</td>
			<td>03407-11</td>
			<td>StableTemp hot plate stirrer</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>American Scientific</td>
			<td>U14295</td>
			<td>Tube mercury thermometerr</td>
		</tr>
		<tr>
			<td>Tiletamine and zolazepam hydrochloride</td>
			<td>Zoetis</td>
			<td>NDC:54771-9050</td>
			<td>Telazol</td>
		</tr>
		<tr>
			<td>Tincture of Benzoin Spray</td>
			<td>Smith&#38;Nephew</td>
			<td>407000</td>
			<td>Adhesive layer spray</td>
		</tr>
		<tr>
			<td>Triple Antibiotic ointment</td>
			<td>Fougera</td>
			<td>NDC 0168-0012-31</td>
			<td>Triple antibiotic ointment</td>
		</tr>
		<tr>
			<td>Tubular stockinette</td>
			<td>Medline</td>
			<td>NONNET02</td>
			<td>Curad Medline Latex Free Elastic Nets</td>
		</tr>
		<tr>
			<td>Warming blanket</td>
			<td>3M</td>
			<td>Bair Hugger 750 warming unit</td>
			<td></td>
		</tr>
		<tr>
			<td>Xeroform Occlusive Gauze Strip</td>
			<td>Covidien</td>
			<td>8884433301</td>
			<td>Xeroform petrolatum wound dressings</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Vetone</td>
			<td>NDC:13985-704-10</td>
			<td>AnaSed LA</td>
		</tr>
		<tr>
			<td>Yucat&#224;n minipigs (female, 30 kg)</td>
			<td>Sinclair Bio Resources</td>
			<td>N/A</td>
			<td>Full pigmentation&#160;</td>
		</tr>
	</tbody>
</table>

a swine burn model for investigating the healing process in multiple depth burn wounds

Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face significant challenges. Among these challenges, the extent of the burned soft tissue can evolve in the early phase, creating a delicate balance between conservative treatments and necrosing tissue removal. Thermal burns are the most common type, and burn depth varies depending on multiple parameters, such as temperature and exposure time. Burn depth also varies in time, and the secondary aggravation of the &#34;shadow zone&#34; remains a poorly understood phenomenon. In response to these challenges, several innovative treatments have been studied, and more are in the early development phase. Nanoparticles in modern wound dressings and artificial skin are examples of these modern therapies still under evaluation. Taken together, both burn diagnosis and burn treatments need substantial advancements, and research teams need a reliable and relevant model to test new tools and therapies. Among animal models, swine are the most relevant because of their strong similarities in skin structure with humans. More specifically, Yucatan minipigs show interesting features such as melanin pigmentation and slow growth, allowing for studying high phototypes and long-term healing. This article aims to describe a reliable and reproducible protocol to study multi-depth burn wounds in Yucatan minipigs, enabling long-term follow-up and providing a relevant model for diagnosis and therapeutic studies.

Author Spotlight: A Multi-Depth Porcine Model for Comprehensive Study of Burn Injuries and Healing Processes

A Swine Burn Model for Investigating the Healing Process in Multiple Depth Burn Wounds

The objective of our research is to provide a reliable and robust model to study multiple burn injuries and its healing process. This is relevant for both diagnosis and treatment research. We successfully established a multi depth model of burns that accurately mimics the burns in humans.We standardized this protocol by providing specific details of one, making device, temperature and time of contact, which makes it easy to reproduce the model by other researchers in the field. By using this model, we allow studying the healing process with a long-term follow-up while minimizing intra-individual variability. In addition, the high melanin content of the skin allows advanced research relevant to high prototypes.Swine models of burn injury are important due to their similarities with human burns. Our model allows us to observe the changes in the early phase of healing when treatment decisions need to be made, as well as in the late phase when scar formations need to be minimized for aesthetic and functional recovery. Our lab at Massachusetts General Hospital is developing non-invasive biomedical devices to assess burn severity and healing potential at various stages, aiming to improve clinical burn management through insights from initial experiments.

Figure 2A,B display the results of multiple burns on the dorsum of a Yucat&#224;n minipig. Wounds (I) and (VII) are control wounds (37 &#176;C). Second-degree wounds (II; III and VIII) present with intense redness and blisters. In contrast, third-degree wounds (IV; V; and VI) are pale and indurated to palpation. It is to be noted that wound VIII looks intermediate between second and third degree: for the purpose of an ongoing study, we increased the contact time to 45 s at 6...

Read this Scientific Article Protocol about Author Spotlight: A Multi-Depth Porcine Model for Comprehensive Study of Burn Injuries and Healing Processes at JoVE.com

This protocol describes a reproducible multi-depth burn wound model in a Yucatan minipigs.

Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face ...

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697 Views.  Massachusetts General Hospital, Harvard Medical School. The objective of our research is to provide a reliable and robust model to study multiple burn injuries and its healing process. This is relevant for both diagnosis and treatment research. We successfully established a multi depth model of burns that accurately mimics the burns in humans.We standardized this protocol by providing specific details of one, making device, temperature and time of contact, which makes it easy to reproduce the model by other researchers in the field. By usin...