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

Abstract

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 "shadow zone" 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.

Introduction

Burns are a major public health problem and affect more than 480,000 patients in the US each year, according to the National Burn Repository^1,2. 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 casualties^3,4. 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 process^13,14,15,16. 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 'shadow' or 'stasis' zone, indicating potentially reversible ischemia. This area can either deteriorate, resulting in an extended necrosis zone, or heal by reversing the apoptotic process^17,18. 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 "shadow zone" 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 nanomaterials^20,21, tissue-engineered skin^22,23, and novel epidermal culture approaches^24,25. Also, modern reconstructive surgery and fasciocutaneous flaps have improved the management of long-term after-effects, particularly burn contractures following pathologic healing of fold areas^26,27. 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 skin^30,31,32. 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 burns^33,34. 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 Yucatán 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.

Protocol

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 National Institute of Health Guide for the Care and Use of Laboratory Animals³⁸. Five 30 kg female Yucatán minipigs were used for these experiments. The animals were obtained from a commercial source (see Table of Materials).

1. Pre-operative care and sedation

1. Fast the animals for 12 h before each short sedation and/or general anesthesia.
2. Initiate sedation 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 L/min) (see Table of Materials) for maintaining sedation during the procedure.

2. Burn wound design and randomization

1. 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's dorsum in the paravertebral area. Perform three full-thickness wounds, three partial-thickness wounds, and two control wounds.
2. 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.

3. Tattoo wound delimitation

NOTE: The first procedure consists of creating circular tattoos on the pig'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.

1. After sedation 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.
2. Place a straight line centrally on the pig's back to allow the symmetrization of the drawings.
3. 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.
4. Draw each pair of 4.5 cm diameter circles symmetrically, keeping a minimum distance of 4 cm between two distinct circles (Figure 1A).
5. Once the drawings are done, perform a single-step skin preparation (povidone-iodine 7.5%) to decrease the skin bacterial flora.
6. 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.
7. In case of a delayed burn creation procedure, apply triple-antibiotic ointment on the tattoos and cover it with transparent adhesive dressings.

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.

1. On the day of the burn procedure, 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 °C (for the partial thickness burns), and the second to 93 °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.
2. 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's temperature to 65 °C or 93° 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.
3. In parallel, place the animal in the prone position for the burning procedure, similar to the tattoo procedure (step 3.1).
4. Once the animal is ready in the prone position, prepare the skin in three steps (povidone-iodine 7.5%, saline solution 0.9%, drying).
5. 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's own weight.
6. 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.
7. Perform an advanced multi-layer dressing to ensure dressing stability after the animal has recovered.
   1. 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.
   2. Spray a tincture of benzoin solution on the surrounding skin for better adherence of the transparent dressings to the skin.
   3. Wrap the animal in self-adherent adhesive wrap dressing while paying attention not to overtighten it (restriction of pulmonary volumes).
   4. Complete the dressing by adding a final layer of tubular stockinette (size adapted to the animal) (see Table of Materials).
   5. 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).
   6. Following each sedation or anesthesia, 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.

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.

1. After similar preparation of the animal, including three-step skin preparation, perform sterile draping of the back of the animal.
2. Perform 4 mm punch biopsies (see Table of Materials) on the wound to confirm the type of injury.
3. Perform the escharotomy^43,44 by a circular incision of the eschar with a sterile scalpel blade (n°15) until reaching the deep dermis.
4. 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.
5. 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.
   1. 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.
6. Put a first set of dry gauze on the wound bed to enhance local hemostasis.
7. 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.
8. Provide analgesia by changing the opioïd transdermal patch and injecting Caprofen (2-4 mg/kg, IM).

6. Follow-up wound dressings

NOTE: Subsequent dressings are performed every 2 to 7 days, depending on the experimental treatment design and the animal's tolerance. Wound dressings can be stopped after 21 days to allow re-epithelialization in a dry environment and improve the animal'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.

1. Sedate the animal for a short period and placed in the supine position with a nose cone (isoflurane 2-5 L/min).
2. Remove the previous dressings and clean the wounds using sterile 0.9% saline and sterile gauze.
3. Apply the experimental treatment if applicable.
4. 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).
5. Cover all wound locations, including control wounds, with petroleum-impregnated or equivalent gauze, dry, non-woven gauze, and adhesive transparent dressing.

Representative Results

Figure 2A,B display the results of multiple burns on the dorsum of a Yucatàn minipig. Wounds (I) and (VII) are control wounds (37 °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 65 degrees. Figure 2C displays the aspect of the wounds following the escharotomy on postoperative day 3. A blisterectomy was performed on the second-degree wounds, and "en-bloc" excision of the indurated burned tissue was performed on the third-degree wounds, in order to create a viable wound bed for secondary healing. A minimal cauterization of the dermal plexus was performed if the bleeding was not spontaneously stopping. Figures 2D-F display the aspect of the wounds at subsequent time points (Week 2, Week 3, and Week 7, respectively).

Histological results are shown in Figure 3 (H&E, 5.5x and 20x). The control skin (Figure 3A,B) shows an intact epidermis (blue bracket), including a stratum corneum (blue arrow) adherent to the rest of the cutaneous tissue. The dermis shows homogeneous pale-pink eosinophilia (collagen and elastic fibers - red asterisk). Second-degree burns (Figure 3D,E) reveal loss of the stratum corneum compared to controls (cleavage plane, corresponding to the blisters). The superficial dermis shows stronger eosinophilia and fissures/fractures within the dermal structures (blue arrows). Deep blood vessels are preserved (red arrow). The third-degree burns (Figure 3G,H) show extensive injuries with increased eosinophilia of the dermis as long as severe dermal fractures (blue arrows), reaching the deep dermis to the hypodermis (adipocyte vacuity, green asterisk). Finally, Trichrome staining (Figure 3C,F,I) allows for a better assessment of the connective tissue. It uses the differences in stainability of heat-denatured collagen to show burn depth. Figure 3C clearly shows the blue features in the intact dermis of the control wound biopsy with the top epidermal layer. This is in contrast to the second-degree burn (Figure 3F), where the loss of epithelium is accompanied by collagen denaturation in the upper dermis layer (white asterisk). Finally, the third-degree wound (Figure 3I) shows the loss of the epidermal layer with extensive red staining (yellow arrow) of the dermal layer, indicating complete collagen denaturation in this layer of the skin.

Figure 1: Operative room positioning burn device setup, and study design. The animal is sedated, intubated, ventilated, and placed supine. The brass block is placed in the pre-heated container filled with aluminum beads +/- water. A thermometer is placed centrally in the brass block to allow for core temperature monitoring. The aimed temperature depends on the aimed burn depth according to the study design. To provoke partial thickness burns, a 30 s contact at 65 °C with no added pressure is recommended. For third-degree burns, a 30 s contact at 93 °C with no added pressure is recommended. Second and third-degree burns should be randomized to avoid biases due to differential skin thickness. Pre-tattooed wound location allows for easy monitoring of each randomized wound during follow-up. Please click here to view a larger version of this figure.

Figure 2: Representative results of wound aspects following randomized multi-depth burns in a Yucatan Minipig. (A) The tattoo procedure is done by placing it at the location of all 8 wound spots. Each location is numbered to facilitate monitoring. (B) Aspect of the wounds following burn creation. Wounds I and VII are controls, and wounds II, III, and VIII are partial thickness wounds. Wounds IV, V and VI are third-degree wounds. (C) Aspect of the wounds following escharotomy of the third-degree wounds on POD3. A blisterectomy was also performed on the partial thickness wounds. (D) Aspect of the wound healing on POD14 showing a clean wound bed of the third-degree wounds (II and V), and re-epithelialization islands from the skin appendages on the partial thickness wounds (III, IV, VI, VII, VIII). (E) Aspect of the wound healing process on POD21 showing contracture of the third-degree wound beds and further re-epithelialization on the second-degree wounds. (F) Aspect of the wounds on POD49 showing advanced wound contracture on the third-degree wounds and subtotal epithelialization of the partial thickness wounds with important post-inflammatory hyperpigmentation. Please note that displayed wounds could present biopsy site scars. Please click here to view a larger version of this figure.

Figure 3: Histological results. Representative results of the histological aspect of the wounds [Light microscopy 10x (A,C,D,F,G,I) and 20x (B,E,H); Hematoxylin and Eosin (A,B,D,E,G,H); Masson's Trichrome (C,F,I)]. Control skin (A,B) shows an intact epidermis (black bracket), including a stratum corneum (black arrow) adherent to the rest of the cutaneous tissue. The dermis shows homogeneous pale-pink eosinophilia (collagen and elastic fibers - red asterisk). Trichrome staining (C) reveals normal aspect of the epidermal layer and of the connective tissue and dermal collagen fibers. Second-degree burns (D,E) reveal loss of the stratum corneum compared to controls (cleavage plane, removed during blisterectomy). The superficial dermis shows stronger eosinophilia and fissures/fractures within the dermal structures (blue arrows). Deep blood vessels are preserved (red arrow). Trichrome (F) reveals loss of the epithelium, accompanied by collagen denaturation in the upper dermis layer (yellow arrow). The third-degree burns (E,F) show extensive injuries with increased eosinophilia of the dermis as long as severe dermal fractures (blue arrows), reaching the deep dermis to the hypodermis (adipocyte vacuity, green asterisk) and extensive red staining (I, yellow arrow) of the dermal layer with Trichrome, indicating complete collagen denaturation. Scale bars: A,D,G, 500 µm; B,E,H, 100 µm; C,F,I, 200 µm. Please click here to view a larger version of this figure.

Figure 4: Engineering drawing of the brass block used for burn wound creation. (A) Illustration displaying the dimensions of the brass block that was used for building the block. A diameter of 40 mm allows for optimal-sized wounds while allowing for designing 8 wounds on a 20-30 kg Yucatan minipig. (B) Diagram showing the final aspect of the brass block with a central tunnel allowing for the thermometer to measure the core temperature of the block. (C) Image highlighting the brass block heated to 65 °C, as displayed by the mercury thermometer. Please click here to view a larger version of this figure.

Discussion

Wound healing following burn injuries is a long process that can take up to several months, with various treatment options and considerations for patient care^2,13. In order to study it, a reliable and reproducible model is needed. Several animal models have been described, mainly including rodents^29,45,46 and swine^29,47. The porcine model is more relevant because of its similarities to human skin structure^34,48. The size of an adult pig means that several wounds can be performed on the same animal, increasing statistical significance and eliminating inter-individual variability between experimental treatments and their controls⁷. We recommend choosing the subjects by weight and not necessarily by age. The current model model allows performing up to 8 wounds while staying under 10% of the total body surface. This enables for staying away from hemodynamic consequences, as well as manageable pain.

A critical point is related to the breed. While most of the existing swine protocols use Yorkshire pigs^29,31,47,48, we developed the Yucatan Minipig model for several reasons. Firstly, this minipig breed shows little growth over the follow-up months^36,49. This allows for minimizing the growth-related wound size variation and, therefore, for better intra-individual comparison. The lower weight variation compared to Yorkshire pigs also ensures better optimization and dosing of the anesthesia drugs. Secondly, the high amount of melanin is relevant for studying pigmented skin reactions to burn injuries and healing, suggesting better applicability to human phototypes 3 to 6^35,50,51. Rice et al. found substantial healing differences between Yucatan and domestic white pigs following dermabrasions as a treatment for chemical burns⁵². Moreover, higher phototypes clinically showed higher rates of pathologic scarring, such as hypertrophic and keloids^53,54,55, although the Red Duroc breed seems to remain the most accurate for hypertrophic scar assessment^34,56. In addition, the pigmented healing epidermis also makes it easier to measure the re-epithelialization process from the external margins as well as from the annexes. Finally, this model shows clear signs of post-inflammatory hyperpigmentation (Figure 2D-F), which is also of interest for further research to address. Other authors used this minipig model to study pharmacological dermal toxicity and phototoxicity and found impressive predictive values for safety and efficacy to human outcomes of 89% and 100%, respectively⁵⁷. If these features emphasize the relevance of the Yucatan minipig model to studying wound healing, the literature in the specific field of burns remains poor. Therefore, it seemed necessary to describe a reproducible model to enable the study of burn wound healing and the various diagnostic and therapeutic interventions.

Previous protocols have been described to study burns in the swine model. Branski et al.⁵⁸ published a model in 2008 for full-thickness thermal contact burns using an aluminum bar heated to 200 °C. Their technique, inspired by several previous publications, implemented a controlled pressure by using a 50 mL syringe attached to the aluminum bar. Although this feature seems relevant since pressure influences the extent of the burn injuries, the precision of their device could lack accuracy (pressure by the operator on the syringe itself not considered, and pressure level based on changes in the piston position). More recently, Kim et al.⁵⁹ and Seswandhana et al.⁶⁰ demonstrated reproducible burn wounds using a custom device with an electrically heated device and temperature control. Both their devices included pressure monitoring measuring downward forces. The feedback loop, as described by Kim, makes it possible to maintain both core and surface temperatures during contact. Both authors showed a good correlation between burn depth and pressure, emphasizing the importance of pressure control. We chose not to apply any supplementary pressure to the brass block other than its weight, and the operator's action is simply stabilizing. The objective was to simplify the model and minimize the number of variables. Fan et al.⁶¹ described a similar model using a modified soldering iron filled with glycerin, which has the disadvantage of creating supplementary interfacial thermal resistance. The blocks were made from brass with a specific heat capacity (c) of 395 W/m*K and a thermal conductivity (k) of 134 J/kg*K¹. Compared to other common metals such as iron (c = 490 W/m*K, k = 66 J/kg*K) and aluminum (c = 949 W/m*K, k = 240 J/kg*K), these properties allow reaching the desired temperature faster and also allow maximum heat to be transferred to the skin quickly to produce the burns. To enhance reproducibility, Figure 4 shows the features of the brass block that was used for burn making. Figure 4A shows the engineering drawing of the brass block with all the dimensions in mm. As shown, the outer diameter of the block is 40 mm x height 151 mm. The dotted line shows a coaxial hole through the block for inserting a thermometer, with a hole diameter of 9 mm and 128 mm depth. Figure 4B,C show the thermometer inserted into the brass block for core temperature measurement.

One limit of the current model is the persistence of non-burned zones due to the rib reliefs, which is mainly problematic in partial thickness burns (Figure 2). This could influence the re-epithelialization process by providing a central source of keratinocyte migration. However, it seems difficult to avoid this issue while using hard materials to create the burns. In addition, the total surface of unburnt areas is minor, and the 4 cm diameter of the brass blocks provides enough wound surface to perform studies with long follow-ups despite repeated punch biopsies.

Another major variable is the timing of the escharotomy in full-thickness burns, planned on POD3 in this protocol. This delayed time point was chosen in order to target the shadow zone featuring delayed tissue injuries, also referred to as burn wound conversion in clinical practice^62,63. The objective was to remove all injured tissue, which was facilitated by the cohesiveness of the eschar at this time point. This timing also allows for studying early interventions aiming to decrease delayed injuries of the "shadow zone" such as anti-inflammatory agents^64,65,66, stem cell therapy^18,67,68, cold therapy⁶⁹, or anticoagulation^65,70,71 for up to 3 days. In addition, while most of the authors described short follow-up periods of 2 to 4 weeks^29,47, an extended follow-up period of 8 to 10 weeks was adopted here, allowing one to study not only the acute phase but also the secondary healing steps.

During this extended follow-up, postoperative care and wound dressing are critical: The team needs to be trained to provide adequate care to the animal. The Yucatán breed often presents dry skin and needs to receive moistening lotion several times a week in order to decrease pruritus, which is a sign of discomfort and can lead to subsequent self-injuries of the wounds. The multi-layer dressing presented in this manuscript is the result of several years of experience in similar models within our center^7,72. The animals don't show signs of discomfort or pain. No infection was found in the study, but daily monitoring for the first two weeks is still needed in order to detect these potential complications early. This emphasizes safety and animal welfare compliance, which are of the utmost importance in modern research. We chose to discontinue dressings after 3 weeks to allow air exposure and dry re-epithelialization^73,74. However, in the modern era of nanoparticle-based²⁰ and crosslinked gels³⁵ to enhance burn wound healing, continuing occlusive dressings can be implemented into this protocol.

Finally, one major characteristic of this model is that it allows for studying different wound depths in the same subject. The histology results confirm the protocol's relevance in this respect. This feature is of interest for diagnosis purposes, such as new technologies to help measure burn depth, deterioration during the initial phase, or improvement during the healing process, but also for assessing new therapies and their differential action in first-, second-, and third-degree burns. Since the healing process is substantially different depending on the initial burn depth, this model allows for measuring the differential effects of potential therapies while avoiding interindividual variations. However, one limitation is that mixing several burn types on the same animal makes it impossible to assess systemic biomarkers such as metalloproteinases, pro-inflammatory and anti-inflammatory cytokines, which are known to vary with wound depth and total burned surface area⁷⁵.

In conclusion, the current Yucatan Minipig burn model stands out for its ability to provide a reliable platform to study wound healing following burn injuries. Leveraging the physiological proximity between porcine and human skin, this pig model offers distinct advantages, including controlled intra-individual comparisons and the ability to accommodate multiple types of wounds on a single animal. Reproducible burn induction techniques, timing of interventions, and extended follow-up duration are critical points that enhance the clinical relevance and reliability of this model. The Yucatan skin allows for studying phenomena specific to pigmented skin, such as hypertrophic scarring and post-inflammatory hyperpigmentation. Novel diagnostic tools aiming to orientate the clinician with "shadow zone" burn conversion and modern treatments such as nanoparticles or 3D-printed skin grafts are examples illustrating the need for such a precious preclinical model to study acute, delayed, and long-term burn phases.

Materials

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