Collagen in the scarless fetal skin wound: Detection with Picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a "response" to burn injury.     

A randomised controlled trial of amniotic membrane in the treatment of a standardised burn injury in the merino lamb

Burn injury is associated with disabling scar formation which impacts on many aspects of the patient's life. Previously we have shown that the fetus heals a deep dermal burn in a scarless fashion. Amniotic membrane (AM) is the outermost fetal tisue and has beeen used as a dressing in thermal injuries, though there is little data to support this use. To assess the efficacy of AM in scar minimisation after deep dermal burn wound, we conducted a randomised controlled study in the 1-month lamb. Lambs were delivered by caesarian section and the amniotic membranes stored after which lambs were returned to their mothers post-operatively. At 1 month, a standardised deep dermal burn was created under general anaesthesia on both flanks of the lamb. One flank was covered with unmatched AM, the other with paraffin gauze. Animals were sequentially euthanased from Day 3–60 after injury and tissue analysed for histopathology and immunohistochemically for α-smooth muscle actin (αSMA) content. AM resulted in reduced scar tissue as assessed histopathologically and reduced αSMA content. This study provides the first laboratory evidence that AM may reduce scar formation after burn injury.     

一种大鼠蒸气烫伤模型的建立

目的　建立一种可控深度及面积的大鼠烫伤模型。　方法　用高压蒸气消毒锅及自制烫伤支架制作高压蒸气烫伤装置 ,用压力为 0 .12MPa(1MPa=75 0 0mmHg)、直径 2 .6cm的致伤孔上分别在大鼠背部烫 3、4、5、6、7、8、9、10s,每时相点 5个创面。伤后 2 4h取标本行组织学观察 ,用Photoshop软件测量烫伤深度 ,并观察有无毛囊、汗腺附件受损、烫伤区被毛生长及创面愈合情况。　结果　烫伤深度与烫伤时间的变化呈正相关 (r =0.99)。浅Ⅱ、深Ⅱ、Ⅲ度烫伤模型的致伤时间分别为 3、5、7s。烫伤 7～ 10s创面深度虽逐渐加重 ,愈合时间却相近。　结论　该模型可以控制烫伤深度、面积 ,烧伤深度划分准确且操作简便 ,是研究创伤修复机制及评价创面用药的较好模型。     

Evidence‐based injury prediction data for the water temperature and duration of exposure for clinically relevant deep dermal scald injuries

Deep dermal burn injuries require extensive medical care; however, the water temperatures and durations of exposure that result in a severe scald injury are unknown. This study used a porcine burn model to investigate the time and temperature threshold for clinically relevant deep dermal injuries for both immersion (long duration) and spill/splash (short duration) scald events. Scald wounds were created on the flanks of anaesthetized juvenile large White pigs (27 kg). Acute tissue injury evaluations performed at 1 hour and days 1, 3, and 7 postburn (16 pigs) included: wound examination, biopsies, and laser Doppler imaging. Up to 20 burn combinations were tested including: 50–60 °C water for 1–10 minutes (immersion); and 60–90 °C water for 5 seconds (spill/splash). Burn conditions demonstrating mid‐to‐deep dermal damage histologically were followed for 21 days to assess time to reepithelialize (eight pigs). Histologically, depth of damage increased until day 3 postburn. Damage to ≥75% of the depth of dermis was associated with burns taking longer than 3 weeks to fully reepithelialize. For spill/splash (5 seconds) scalds, water at ≥75 °C showed damage to mid‐dermis or deeper by day 3; however, only burns from water ≥85 °C were not reepithelialized by day 21. For immersion scalds of equivalent duration, water at 55 °C caused significantly deeper dermal damage than 50 °C (p < 0.05) at day 3. Immersion scalds that were not fully reepithelialized by day 21 included 50 °C for >10 minutes, 55 °C for 5 minutes, 60 °C for 60 seconds, and 70 °C for > 15 seconds. This research provides valuable evidence‐based injury prediction data, which can be used to inform future burn injury prevention guidelines/legislation to reduce the risk of severe scald injuries and support medicolegal opinions for cases where an inflicted mechanism of injury is alleged.     

A human skin equivalent burn model to study the effect of a nanocrystalline silver dressing on wound healing

In this study, a deep burn wound model was established using a 3D human skin equivalent (HSE) model and this was compared to native skin. HSEs were constructed from dermis derived from abdominoplasty/breast surgery and this dermal template was seeded with primary keratinocytes and fibroblasts. The HSE model was structurally similar to native skin with a stratified and differentiated epidermis. A contact burn (60 °C, 80 °C, 90 °C) was applied with a modified soldering iron and wounds were observed at day 1 and 7 after burn. The HSEs demonstrated re-growth with keratinocyte proliferation and formation of a neo-epidermis after burn injury, whereas the ex vivo native skin did not. To assess the suitability of the 3D HSE model for penetration and toxicity studies, a nanocrystalline silver dressing was applied to the model for 7 days, with and without burn injury. The effect of silver on skin re-growth and its penetration and subcellular localization was assessed in HSEs histologically and with laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS). The silver treatment delayed or reduced skin re-growth, and silver particles were detected on the top of the epidermis, and within the papillary dermis. This novel in vitro 3D multicellular deep burn wound model is effective for studying the pathology and treatment of burn wound injury and is suitable for penetration and toxicity studies of wound healing treatments.     

Thermal injury model in the rabbit ear with quantifiable burn progression and hypertrophic scar

Hypertrophic scar is a major clinical outcome of deep‐partial thickness to full thickness thermal burn injury. Appropriate animal models are a limitation to burn research due to the lack of, or access to, animal models which address the endpoint of hypertrophic scar. Lower species, such as rodents, heal mainly by contracture, which limits the duration of study. Higher species, such as pigs, heal more similarly to humans, but are associated with high cost, long duration for scar development, challenges in quantifying scar hypertrophy, and poor manageability. Here, we present a quantifiable deep‐partial thickness burn model in the rabbit ear. Burns were created using a dry‐heated brass rod for 10 and 20 seconds at 90 °C. At the time of eschar excision on day 3, excisional wounds were made on the contralateral ear for comparison. Burn wound progression, in which the wound size expands over time is a major distinction between excisional and thermal injuries, was quantified at 1 hour and 3 days after the injuries using calibrated photographs and histology and the size of the wounds was found to be unchanged from the initial wound size at 1 hour, but 10% in the 20 seconds burn wounds at 3 days. A quantifiable hypertrophic scar, measured by histology as the scar elevation index, was present in both 20 seconds burn wounds and excisional wounds at day 35. ImageJ measurements revealed that the 20 seconds burn wound scars were 22% larger than the excisional wound scars and the 20 seconds burn scar area measurements from histology were 26% greater than in the excisional wound scar. The ability to measure both burn progression and scar hypertrophy over a 35‐day time frame suits this model to screening early intervention burn wound therapeutics or scar treatments in a burn‐specific scar model.     

Changes in the microvasculature of skin subjected to thermal injury

Guinea-pigs were anaesthetized and the caudal 50 per cent of the animal was scalded by immersion in boiling water (100°C) for 3 seconds. Skin samples were obtained from 15 minutes to 24 hours after scalding. The microvascular alterations characteristic of experimentally-induced severe inflammatory responses were studied by electron microscopy. Large intercellular gaps were present in venule endothelium by 15 minutes after injury and in capillary endothelium by 30 minutes after injury. These were detectable for at least 24 hours after the burn. Extensive destruction was observed in peripheral nerves 15 minutes after injury and in endothelial cells 4 hours after scalding. No morphological evidence of a biphasic pattern of increased vascular permeability was detected in this material. The standardized, reproducible conditions of this study could be used to evaluate the efficacy of anti-inflammmatory procedures or burn treatments of skin.     

Comparing the reported burn conditions for different severity burns in porcine models: a systematic review

There are many porcine burn models that create burns using different materials (e.g. metal, water) and different burn conditions (e.g. temperature and duration of exposure). This review aims to determine whether a pooled analysis of these studies can provide insight into the burn materials and conditions required to create burns of a specific severity. A systematic review of 42 porcine burn studies describing the depth of burn injury with histological evaluation is presented. Inclusion criteria included thermal burns, burns created with a novel method or material, histological evaluation within 7 days post‐burn and method for depth of injury assessment specified. Conditions causing deep dermal scald burns compared to contact burns of equivalent severity were disparate, with lower temperatures and shorter durations reported for scald burns (83°C for 14 seconds) compared to contact burns (111°C for 23 seconds). A valuable archive of the different mechanisms and materials used for porcine burn models is presented to aid design and optimisation of future models. Significantly, this review demonstrates the effect of the mechanism of injury on burn severity and that caution is recommended when burn conditions established by porcine contact burn models are used by regulators to guide scald burn prevention strategies.     

Bone marrow-derived myofibroblasts recruited to the upper dermis appear beneath regenerating epidermis after deep dermal burn injury

Fibroblasts and myofibroblasts migrating to sites of tissue repair after injury may not only be locally recruited but could also be recruited from the bone marrow. However, the characteristics and functional roles, if any, of these cells in wound healing are poorly understood. Here, we show unequivocally that bone marrow-derived fibroblasts do contribute to deep dermal burn wound healing. Bone-marrow stromal cells were collected from femurs of male Lewis rats, cultured for a week, and then the adherent cells were labeled with the fluorescent marker PKH-26. These cells stained positive for alpha-smooth muscle actin and prolyl 4-hydroxylase, but did not express RM-4 (a macrophage marker), CD34, or cytokeratin, characteristic of myofibroblastic differentiation. When injected intravenously into Lewis rats, they homed to the bone marrow. Five days after transplantation, a deep dermal burn was made on the back of the rat, and biopsies were taken 7, 10, and 14 days later. PKH-positive cells were not found at day 7, but by day 10, they were easily detected mainly in the upper dermis close beneath the regenerating epidermis. These PKH-positive cells still stained for alpha-SMA and prolyl 4-hydroxylase, but not RM4. Thus, it is suggested that myofibroblasts originating in the bone marrow contribute not only to promotion of granulation but also enhancement of dermal-epidermal interaction after thermal injury.     

Skin flap survival after superficial and deep partial-thickness burn injury

Whether a flap can be raised successfully in a body region that has been subjected to burn injury remains an issue. The aim of this study was to investigate the survival of skin flaps that were elevated after superficial and deep partial-thickness burn injury in a rat model. Sixty-five rats were divided into five groups: Group 1 (N = 15) was the control group, group 2 (N = 10) included rats with superficial partial-thickness burns that had flaps elevated on day 0, group 3 (N = 15) was comprised up of rats with superficial partial-thickness burns that had flaps elevated on day 4, group 4 (N = 10) included rats with deep partial-thickness burns that had flaps elevated on day 0, and group 5 (N = 15) was comprised of rats with deep partial-thickness burns that had flaps elevated on day 4. Caudally based dorsal flaps consisting of skin and panniculus carnosus were elevated in all groups, and the amount of surviving tissue on each flap was quantified. The surviving areas of flaps elevated on postburn days 0 and 4 in superficial partial-thickness burn zones (groups 2 and 3) were larger than those of flaps that were elevated on postburn days 0 and 4 in deep partial-thickness burn zones (groups 4 and 5). The surviving portions of flaps that were elevated on day 4 in superficial partial-thickness burn zones (group 3) were similar to the surviving areas of flaps in the control group (group 1), and were larger than those of all other groups (groups 2, 4, and 5). In this rat model, flaps were elevated in superficial dermal burn zones with successful outcomes. However, raising flaps in deep dermal burn zones was not a reliable method.     

神经肽P物质与烫伤创面愈合关系的实验研究

目的　探讨神经肽P物质(SP)与烫伤创面愈合之间的关系。　方法　(1)制作大鼠不同深度烫伤模型,分别于伤后1、3、7、14d致死,用放射免疫法测定创面SP含量。(2)将大鼠肉芽组织成纤维细胞(GTF)用不同培养液培养,分为空白对照组、SP组及SP SP受体拮抗剂(Spantide)组。体外检测SP及Spantide对GTF增殖活性[以吸光度(A)值表示]及凋亡率的影响。　结果　(1)伤后1d,浅Ⅱ、深Ⅱ、Ⅲ度烫伤创面SP含量分别为(145±78)、(94±48)、(53±27)ng/g,深Ⅱ度创面与其余两者比较,差异有统计学意义(P<0 01).浅Ⅱ度创面伤后3、7dSP含量显著增高;深Ⅱ度创面伤后7、14dSP含量显著增高;Ⅲ度创面伤后SP含量无显著变化。(2)SP增强GTF增殖活性(空白对照组A为0. 21±0. 05,SP组A为0. 36±0 07,P<0. 01)并抑制其凋亡,Spantide可抑制SP对GTF的作用。　结论　SP可促进GTF增殖,创面SP含量与创面损伤程度及愈合能力关系密切。     

Effect of the thromboxane synthetase inhibitor dazmegrel (UK 38,485) on skin blood flow in deep partial thickness burns

We have previously demonstrated more rapid wound healing in deep partial thickness burn guinea-pigs treated with intramuscular injection of the lower dosage of the thromboxane synthetase inhibitor Dazmegrel. In striking contrast, systemic and topical application of larger dosage of Dazmegrel inhibited or did not improve wound healing in this study, a deep partial thickness burn model of guinea-pig was used to evaluate the effect of Dazmegrel given systemically using dosage of 3.4 mg, 10 mg and 30 mg/kg/day on dermal perfusion measured by India ink injection or by Xe-133 clearance. There was no improvement of dermal perfusion in any of the groups receiving Dazmegrel. The beneficial effect of Dazmegrel on wound healing at a dosage of 3.4 mg/kg/day was not due to improved local dermal perfusion but, rather, resulted from a systemic immune effect on the animal. The Xe-133-determined blood perfusion showed a significantly diminished blood perfusion in the burn wound at 7h post-burn, and a higher burn skin blood flow at 24h post-burn. Their finding is consistent with the report from other laboratory that microvascular perfusion in zone-of-stasis burns, immediately post-burn, gradually diminishes to nil over the next 16 hours, to be followed by reperfusion between 16 and 96 hours postburn.     

Blood vessel occlusion in peri‐burn tissue is secondary to erythrocyte aggregation and mitigated by a fibronectin‐derived peptide that limits burn injury progression

Although vascular occlusion has long been noted in peri‐burn tissue, the literature is inconsistent regarding the nature of the occlusion, with articles in the 1940s claiming that erythrocytes were the culprit and in the 1980s–1990s that microthrombi were responsible. To better define the nature of vessel occlusion, we studied two porcine burn models, a hot comb horizontal injury model and a vertical injury progression model. In both cases, tissue from the first two days after burn were stained with hemotoxylin and eosin, or probed for platelets or for fibrinogen/fibrin. Erythrocytes, identified as nonstained, clumped, anuclear, 5 µm cells, occluded most blood vessels (BVs) in both burn models. In contrast, platelet or fibrinogen/fibrin antibodies stained BV occlusions minimally at early time points, and only up to 16% of deep dermal BVs at 48 hours in the hot comb model and up to 7% at 24 hours in the vertical injury progression model. Treatment of animals with a fibronectin‐derived peptide (P12), which limits burn injury progression and can dilate peripheral microvasculature, reduced erythrocyte occlusion by at least 50%, speeded healing and reduced scarring. Early erythrocyte aggregation, rather than thrombosis, explains the ineffectiveness of anticoagulants to prevent burn injury progression.     

烫伤大鼠不同深度创面组织中表皮干细胞分布的初步研究

目的初步观察烫伤大鼠不同深度创面组织中表皮干细胞的分布情况。方法将32只SD大鼠分别造成Ⅰ、浅Ⅱ、深Ⅱ和Ⅲ度烫伤，伤后24h取创面组织标本，制作切片。采用细菌蛋白一生物素标记法(LSAB)进行免疫组织化学染色，以α2、β1整合素及角蛋白10(K10)作为一抗，观察不同创面组织中表皮干细胞的表达和分布情况。结果Ⅰ度烧伤创面组织中，K10阳性细胞分布于表皮的棘细胞层、颗粒层、透明层，α2、β1整合素阳性细胞分布于表皮基底层，数量多。浅Ⅱ度创面中，α2、β1整合素阳性细胞位于残留的基底层和皮肤附属器(主要是毛囊)中，数量较少。深Ⅱ度创面中，α2、β1整合素阳性细胞仅存在于真皮深层健存的皮肤附属器中，数量很少。Ⅲ度创面中，罕见α2、β1整合素阳性细胞。结论表皮干细胞在烧伤创面中的分布与烧伤深度有关，残存的表皮干细胞可能是创伤修复过程中再上皮化的细胞来源。     

烫伤大鼠创面皮肤中神经肽P物质的变化

目的　观察皮肤创面神经肽P物质 (SP)的变化规律并探讨其来源。　 方法　运用免疫组化检测烫伤大鼠皮肤创面、创周和远处未损伤皮肤内含SP的神经分布密度的改变及其神经肽的分布 ,运用原位杂交技术观察其mRNA表达情况。　结果　烫伤后 15min在皮肤创面、创周和远处未损伤皮肤含SP的神经分布密度明显下降 (P <0.0 5 ),烫伤后 6～ 12h达到最低值 ,以后逐渐恢复。相比之下 ,在创周恢复过程中SP出现较早 ;在创面和创周的真皮层 ,SP免疫反应阳性的的巨噬细胞样大细胞从局部血管中游出 ,烫伤后 12h该细胞与局部含SP的神经关系密切 ,伤后 2 4h该细胞染色增强、破碎并释放大量SP免疫反应阳性的颗粒状物质 ,而后这些细胞在烫伤后 4 8h消失。原位杂交结果显示 ,伤后 6h相同大小细胞内表达编码合成P物质前体的前速激肽A(PPTA)mRNA。　 结论　皮肤烧伤后 ,SP不仅从皮肤内神经末梢释放 ,也可能从局部炎性细胞合成释放     

复合皮混合移植治疗深Ⅱ度烧伤患者创面疗效观察

目的观察深Ⅱ度烧伤患者创面削痂术后应用复合皮混合移植治疗的效果。方法对23例烧伤患者的30个深Ⅱ度烧伤肢体在伤后3d内分次行削痂术,削至浅筋膜后移植大张异体脱细胞真皮基质,然后切取大张自体刃厚皮(0.10～0.25mm)覆盖于其上。术后10—12d计算移植皮片的存活率,记录创面愈合时间。观察随访3—6个月时患者的肢体外观及功能恢复情况。取1例患者随访3个月时的愈合创面皮肤标本,行病理学观察。结果本组患者复合皮片成活率为93%,7%的皮片因术中固定较差,移植后自体刃厚皮与异体脱细胞真皮基质分离致皮片坏死,或因感染致皮片溶解。随访3—6个月,移植部位皮肤外观、弹性及功能恢复良好。病理学观察显示,成活皮片表皮、真皮结构正常。结论烧伤后早期削痂立即移植复合皮是治疗深Ⅱ度创面的有效方法。     

A porcine deep dermal partial thickness burn model with hypertrophic scarring

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 degrees C for 15s 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.     

Burn depth assessments by photoacoustic imaging and laser Doppler imaging

Diagnosis of burn depths is crucial to determine the treatment plan for severe burn patients. However, an objective method for burn depth assessment has yet to be established, although a commercial laser Doppler imaging (LDI) system is used limitedly. We previously proposed burn depth assessment based on photoacoustic imaging (PAI), in which thermoelastic waves originating from blood under the burned tissue are detected, and we showed the validity of the method by experiments using rat models with three different burn depths: superficial dermal burn, deep dermal burn and deep burn. On the basis of those results, we recently developed a real‐time PAI system for clinical burn diagnosis. Before starting a clinical trial, however, there is a need to reveal more detailed diagnostic characteristics, such as linearity and error, of the PAI system as well as to compare its characteristics with those of an LDI system. In this study, we prepared rat models with burns induced at six different temperatures from 70 to 98 °C, which showed a linear dependence of injury depth on the temperature. Using these models, we examined correlations of signals obtained by PAI and LDI with histologically determined injury depths and burn induction temperatures at 48 hours postburn. We found that the burn depths indicated by PAI were highly correlative with histologically determined injury depths (depths of viable vessels) as well as with burn induction temperatures. Perfusion values measured by LDI were less correlative with these parameters, especially for burns induced at higher temperatures, being attributable to the limited detectable depth for light involving a Doppler shift in tissue. In addition, the measurement errors in PAI were smaller than those in LDI. On the basis of these results, we will be able to start clinical studies using the present PAI system.     

Muscle contractile properties in severely burned rats

Burn induces a sustained catabolic response which causes massive loss of muscle mass after injury. A better understanding of the dynamics of muscle wasting and its impact on muscle function is necessary for the development of effective treatments. Male Sprague–Dawley rats underwent either a 40% total body surface area (TBSA) scald burn or sham burn, and were further assigned to subgroups at four time points after injury (days 3, 7, 14 and 21). In situ isometric contractile properties were measured including twitch tension (Pt), tetanic tension (Po) and fatigue properties. Body weight decreased in burn and sham groups through day 3, however, body weight in the sham groups recovered and increased over time compared to burned groups, which progressively decreased until day 21 after injury. Significant differences in muscle wet weight and protein weight were found between sham and burn. Significant differences in muscle contractile properties were found at day 14 with lower absolute Po as well as specific Po in burned rats compared to sham. After burn, the muscle twitch tension was significantly higher than the sham at day 21. No significant difference in fatigue properties was found between the groups. This study demonstrates dynamics of muscle atrophy and muscle contractile properties after severe burn; this understanding will aid in the development of approaches designed to reduce the rate and extent of burn induced muscle loss and function.     

Burn wound assessment in porcine skin using indocyanine green fluorescence.

BACKGROUND: An accurate assessment of deep dermal burns within the first week after burn is still an unresolved clinical problem. Infrared-excited fluorescence of indocyanine green was examined as a method of early determination of burn depth. METHODS: Burns of varying depths were placed on the paraspinal region, flank, and abdomen of swine using a heated brass block. Fluorescence images of the burns were recorded 1, 24, 48, and 72 hours later. RESULTS: The ratio of fluorescence in 64 burn wounds relative to adjacent normal tissue identified wounds that healed and did not heal within 21 days with an accuracy of 100%, after accounting for the age of the burn. Higher fluorescence ratios were observed in newly placed burns relative to older burns having comparable depths. CONCLUSION: Deep partial-thickness burns were differentiated from deep dermal full-thickness burns in a porcine skin burn model independent of body location. Diagnosis was possible between 1 and 72 hours after injury.