Burns 32 (2006) 806–820 www.elsevier.com/locate/burns
A porcine deep dermal partial thickness burn model with hypertrophic scarring Leila Cuttle a,*, Margit Kempf a, Gael E. Phillips b, Julie Mill a, Mark T. Hayes a, John F. Fraser a, Xue-Qing Wang a, Roy M. Kimble a a
Royal Children’s Hospital Burns Research Group, University of Queensland, Department of Paediatrics and Child Health, Royal Children’s Hospital, Herston, 4029, Queensland, Australia b Royal Children’s Hospital Burns Research Group & Anatomical Pathology, Royal Brisbane and Women’s Hospital, Queensland Health Pathology Services, Herston, 4029, Queensland, Australia Accepted 27 February 2006
Abstract We developed a reproducible model of deep dermal partial thickness burn injury in juvenile Large White pigs. The contact burn is created using water at 92 8C for 15 s in a bottle with the bottom replaced with plastic wrap. The depth of injury was determined by a histopathologist who examined tissue sections 2 and 6 days after injury in a blinded manner. Upon creation, the circular wound area developed white eschar and a hyperaemic zone around the wound border. Animals were kept for 6 weeks or 99 days to examine the wound healing process. The wounds took between 3 and 5 weeks for complete re-epithelialisation. Most wounds developed contracted, purple, hypertrophic scars. On measurement, the thickness of the burned skin was approximately 1.8 times that of the control skin at week 6 and approximately 2.2 times thicker than control skin at 99 days after injury. We have developed various methods to assess healing wounds, including digital photographic analysis, depth of organising granulation tissue, immunohistochemistry, electron microscopy and tensiometry. Immunohistochemistry and electron microscopy showed that our porcine hypertrophic scar appears similar to human hypertrophic scarring. The development of this model allows us to test and compare different treatments on burn wounds. # 2006 Elsevier Ltd and ISBI. All rights reserved. Keywords: Burn; Model; Porcine; Pig; Scar; Scarring; Hypertrophic; Histology; Immunohistochemistry; Scanning electron microscopy; Tensiometry; Collagen
1. Introduction Burn wounds are recognised to heal differently from incisional or excisional wounds [1–3]. In burn wounds, after the initial injury there is expansion of the initial necrosis deeper into the tissue. Progressive necrosis and initiation of the inflammatory response is not seen in incisional wounds and this extra damage leads to wound reepithelialisation being delayed. The depth of the injury, as well as the site of the wound, dictate the outcome of the * Corresponding author at: University of Queensland, Department of Paediatrics and Child Health, Royal Children’s Hospital, Herston Road, Herston, Qld 4029, Australia. Tel.: +61 7 3636 9067; fax: +61 7 3365 5455. E-mail address:
[email protected] (L. Cuttle). 0305-4179/$30.00 # 2006 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2006.02.023
wound appearance. A superficial burn will heal quickly with no scar. However, a deep dermal partial thickness burn (DDPTB) is unlikely to heal within 3 weeks and will heal with scarring. The incidence of hypertrophic scarring in humans rises from 33% if healing is delayed for 3 weeks to 78% if healing is delayed for 6 weeks [4]. To achieve a more acceptable result, a deep dermal partial thickness burn is usually debrided and split thickness skin grafted. However, this surgical intervention itself also causes scarring. Ideally, in order to heal a burn wound such as this without scarring, healing of the wound must occur within 2–3 weeks. In order to study the burn wound healing response and hypertrophic scar formation, an animal model which displays the pathophysiology of burn and forms a hypertrophic scar should be employed. Our aim was to
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create an animal model which mimics the human situation. That is, using the definition of thickness of injury as defined by Shakespeare, a deep dermal partial thickness burn [5]. These burns take longer than 2–3 weeks to heal and heal with contracted, hypertrophic scars that require long-term scar management. The burn also needed to be large enough to study and to represent a significant burn, while still being ethically sound and without initiating major systemic responses in the animal. There are many animal models used for studying burn injury and scar formation. Many studies use small animals such as rats which are relatively inexpensive [6,7]. However, rodent skin is markedly different to human skin in terms of the distribution of hair follicles and the presence of the panniculus carnosus muscle layer, which causes wounds to close predominantly via contraction rather than re-epithelialisation [7,8]. In the past our group has created fetal and lamb ovine burn models [9,10], however the lanolin and wool production in sheep are unlike human skin and make topical application of treatments difficult. Pig skin is known to be more similar to human skin, anatomically and physiologically [11,12]. A review by Sullivan et al. found that porcine models are ideal for evaluating therapeutic agents for wounds as the results are more comparative to human studies [13]. There are several established porcine models for wound healing studies, however they were not sufficient for our research. Some models are excisional rather than burn wounds [14–17] and therefore have a different pathophysiology. Many models have several wounds on the same flank of the animal, however this was undesirable for us as the systemic effects of some agents could obfuscate results. It is also well known that different anatomical positions on the body will contract to different degrees [15]. Many animal models are only used for short-term observation (up to 2 weeks). Clinically, the cosmetic and functional outcome of a burn or scar is important and so for our model we extended the length of study to specifically examine scar formation. There are some excellent models of contact burn injury [18,19], however the burns created in these ways are quite small (6.25 and 19 cm2, respectively). Our challenge was to create a good, reasonably large (approximately 50 cm2) contact injury that was uniform, with a device that was able to conform to the contours of the porcine flank. This model, once developed, was to be used for two purposes. Firstly, it was to be used to further understand the pathophysiology of burn wound healing and scar formation. Secondly, this model was to be used for the testing of various agents which could potentially improve the outcome of the burn wound. Currently, there is no standard method to evaluate the outcome of a burn wound. Various groups employ several different techniques including: macroscopic examination and photographic analysis, immunohistochemistry, electron microscopy, tensiometry, contraction analysis and histological markers. This paper will detail the analysis techniques we have employed to compare and evaluate wounds.
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2. Methods 2.1. Animal surgery All animal work was approved by the institutional animal ethics committee. Large White juvenile pigs of 15–20 kg (approximately 8 weeks of age) were used for the study. Pigs TM were administered 1 mg/kg of Stresnil prior to transport to reduce the stress of new environment and mixing with potentially unknown pigs and were delivered to the animal house at least 5 days before the beginning of the experiment. They were fed on a moistened standard pellet diet and allowed to drink water ad libitum. Anaesthesia was induced with an intramuscular dose of 13 mg/kg ketamine and 1 mg/kg xylazil, and was maintained with halothane via a size 4 laryngeal mask airway [20]. The hair on the back and flanks was clipped and the skin rinsed with clean water prior to wounding. Buprenorphine (0.01 mg/kg) was administered as an analgesic on induction. The animals were positioned on a flat table, lying on one side with the flank for burn creation upward. 2.2. Burning device Wounds were created using a Pyrex laboratory Schott Duran bottle with the bottom removed and replaced with plastic wrap, which was secured with tape around the base (Fig. 1). The burning device was filled with 300 mL of sterile
Fig. 1. The thermal injury device, which consists of a Schott Duran bottle with the bottom removed and replaced with plastic wrap. The plastic wrap is secured to the bottle with tape. Here, the bottle is filled with approximately 300 mL of water and a temperature probe has been inserted into the water through the lid of the bottle. The flexible base allows the device to fit the contours of the pig flank, allowing a uniform burn to be created.
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water and then placed in a microwave oven and heated until it was above the desired temperature. The temperature of the water inside the bottle was monitored with a digital thermometer (N19-Q1436 Dick Smith, Australia, range 50 to 100 8C (0.5%)), with the probe inserted down through the lid of the device. When the water was at the desired temperature, the device was placed on the pig flank in a specific anatomical position (a flat area halfway between the front leg and the caudal edge of the ribcage, on the dorsal flank). The device was held in place by the same person for the desired time. Two burns were created on each animal, one on each flank. The base of the bottle had a diameter of 8.5 cm. 2.3. Temperature and duration of burn In order to create a deep dermal partial thickness burn injury, water at different temperatures and durations were tested. Initially, a starting temperature of 76 8C was used in the anticipation that the burn in the pig model would be created using a similar temperature and duration as that used for the lamb model created previously (82 8C for 10 s) [9]. Various temperatures were tested for 10 s duration: 76, 78, 80, 82, 84, 86, 90, 92 and 94 8C. The highest temperatures of 92, 94 and 96 8C were also applied for 20 s duration. These TM wounds were created and then dressed with Jelonet (an TM inert paraffin gauze, Smith and Nephew) and Melolin (Smith and Nephew). Two days after the burn, the animals were euthanized and the wounds collected for histological analysis. Sections of tissue were fixed in 10% formalin and embedded in paraffin. Paraffin sections were then stained with haematoxylin and eosin and examined by an experienced histopathologist. The histopathologist determined the depth of injury, looking for acute damage markers such as inflammatory migration into appendages and damage to the epidermis. On the basis of this analysis, further testing was done using 92 8C for 10, 15 and 20 s. Six days after the burn the animals were euthanized and the wounds collected and analysed. This time, the histopathologist examined the depth of apoptotic and necrotic damage in the dermis, specifically looking for damage to the appendages. The type of injury we were trying to create was damage down to the base of the hair follicles, whilst still allowing for some living cells for re-growth. 2.4. Porcine model creation Once the correct temperature and duration of contact to create a deep dermal partial thickness burn was determined, two animals were kept until 99 days after the burn to examine scar formation. Another four animals were burned and kept until 6 weeks after the burn. All TM TM wounds were dressed with Jelonet and Melolin and examined and changed on a weekly basis. To protect the wound area, specially made vinyl garments were fastened to the pigs with Velcro and straps (Fig. 2). The garments
Fig. 2. The specially designed waterproof dressing garment used to protect the wounds. The garment is fastened under the belly with Velcro and around the neck with a strap and clip. The elasticised sections and Velcro allow for growth and movement.
were semi-waterproof and the elastic and Velcro allowed for movement and growth. 2.5. Dressings and sedation For weekly dressing changes, the animals were sedated with an intramuscular dose of ketamine/xylasine at 40% of the dose required to induce anaesthesia (5.2 mg/kg ketamine/ 0.4 mg/kg xylasine). Dressings were removed and the wounds were washed with 0.4% chlorhexidine solution and cotton gauze. The wounds were examined and a clinical description of the wound was noted. Photographs were taken of the wound using a Canon EOS 300D digital SLR camera with a macroring flash-light attached. To ensure the camera was at a standard distance from the wound, a template was used to mark four dots on the skin surface, and these were lined up with the focusing spots inside the camera viewfinder. A cyan/ magenta/yellow/black (CMYK) colour scale was placed beside the wound so that the colours in the photos could be standardised against each other. The perimeters of the wounds were traced using a TM Visitrak device (Smith and Nephew). Briefly, a clear plastic grid is placed over the wound and a felt pen used to trace the outline of the wound edge. The grid is then placed TM on the Visitrak device and the perimeter traced again using the device stylus. The device then calculates the total area of the wound in square centimetre. 2.6. Euthanasia The animals were euthanized at either 6 weeks or day 99 after the burn with 15 mL of sodium pentobarbitone (Lethabarb). Tissue sections were collected from the burn and the unburned control areas and fixed in 10% formalin for paraffin embedding. Sections were also collected in phosphate buffered saline (PBS) for electron microscopy and tensiometry analysis.
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2.7. Histology: light microscopy and Sirius red staining Paraffin sections of 4 mm thickness were stained with haematoxylin and eosin (H&E) and examined under a Nikon EP600 microscope fitted with a Spot RT slider cooled CCD camera. Digital images were captured from each section. The thickness of the skin sections was measured digitally from the images using Image Pro Plus v4.1.29 software (Media Cybernetics). The amount of organising granulation tissue was also measured electronically in each section using Image Pro Plus software (Fig. 3). Organising granulation tissue appears more basophilic in haematoxylin and eosin stained tissue. It can be easily identified and distinguished from the residual dermal tissue by its more cellular appearance. To examine collagen morphology, samples were prepared as described previously [10]. Briefly, paraffin sections cut to a 7 mm thickness were dewaxed in xylene, and then washed though a gradient of ethanol to water. The sections were then incubated in 0.1% Sirius Red F3BA (BDH Laboratory Supplies, England) in saturated picric acid for 1 h at room temperature. After washing in water, they were placed in 0.1N HCl for 2 min and then after another rinse in water the sections were dehydrated through ethanol and xylene before being permanently mounted in Depex. Sections were examined under the microscope described above, which had been fitted with a polarising filter (Nikon). Digital images were captured from each section. 2.8. Tensile strength The tensile strength of burned and control skin 6 weeks after the burn was measured using an industrial tensiometer (Universal Instrument for Tests on Materials,
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SUN/500N, Biolab Aust) with a crosshead speed of 1000 mm/min. Software used was Graphwork 3. Strips of skin approximately 12 mm wide were tested, with results adjusted for the dimensions of each piece of skin. The area stretched between the machine clamps was 4 cm long and seven strips each of control and burned skin were tested. The force and energy required to break the tissue as well as the maximum extension of the tissue before breakage were recorded. Results were expressed as mean standard error of the mean from the seven replicates. 2.9. Scanning electron microscopy (SEM) The surface structure of the collagen in the dermis was detected using scanning electron microscopy, as reported by others [21,22]. Skin samples from the day 99 burned tissue (which had been fixed in formalin) were incubated in 0.5% trypsin/0.53 mM EDTA (Gibco) for 5 days at room temperature, and the epidermis was removed. This procedure was not sufficient to remove the epithelium from control tissue from the same animal which had also been fixed. Control tissue collected from a different animal 6 weeks after injury was incubated with trypsin–EDTA overnight at 4 8C for removal of epithelium. Skin blocks were dried through an ethanol gradient, and submerged under liquid carbon dioxide in a critical point dryer (Balzers) at
