Systemic and local inflammatory response after implantation of biomaterial in critical bone injuries.

PURPOSE: To evaluate inflammatory response in critical bone injuries after implantation of the biomaterial composed of hydroxyapatite (HA)/poly (lactic-coglycolic acid) (PLGA)/BLEED. METHODS: Forty-eight male Wistar rats (280 ± 20 grams) were divided into two groups: control group (CG), in which the animals do not receive any type of treatment; and biomaterial group (BG), in which the animals received the HA/PLGA/BLEED scaffold. Critical bone injury was induced in the medial region of the skull calotte with the aid of a trephine drill 8 mm in diameter. The biomaterial was implanted in the form of 1.5-mm thick scaffolds. Serum and calotte were collected at one, three and seven days. RESULTS: Biomaterial had a significant effect on the morphological structure of the bone, accelerating osteoblast activation within three days, without causing exacerbated systemic inflammation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that BG induced upregulation of osteogenic genes such as runt-related transcription factor 2, and stimulated genes of inflammatory pathways such as tumor necrosis factor-α, on the first day without overexpressing genes related to bone matrix degradation, such as tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-9. CONCLUSIONS: The HA/PLGA/BLEED® association can be used as a bone graft to aid bone repair, as it is capable of modulating expression of important genes at this stage of the repair process.

Effect of photobiomodulation on the behaviour of mesenchymal stem cells in three-dimensional cultures.

Photobiomodulation (PBM) has been proposed as a strategy to improve the regenerative capacity of human adipose-derived stem cells (hASCs). Yet, this effect has been proved in 2D culture conditions. To analyze the effect of different doses of laser irradiation (660 nm) with different levels of energy (1 J, 2 J and 6 J) on hASCs cultured at 2D and 3D conditions. We used gellan gum spongy-like hydrogels as a biomaterial to 3D culture hASCs. Different doses (1-7 daily irradiations) and energy levels (1-6 J) of PBM were applied, and the metabolic activity, viability, proliferation, and release of ROS and IL-8 was evaluated up to 7 days. In 3D, cell proliferation increased at high energy (6 J) and after a single dose of irradiation, while in 2D, metabolic activity and proliferation was enhanced only after 3 doses and independently of the energy. More than 1 dose was needed to promote ROS secretion both in 2D and 3D culture conditions. Interestingly, a decrease of IL-8 secretion was detected only in 3D after 3-7 daily irradiations. Overall, hASCs response to PBM was not only dependent on the energy level and the number of applied stimuli, but also on the in vitro culture conditions.

Carbon Black CB-EDA Nanoparticles in Macrophages: Changes in the Oxidative Stress Pathway and in Apoptosis Signaling.

The influence of black carbon nanoparticles on J774.A1 murine cells was investigated with the objective of exploring the cytotoxicity of black carbon functionalized with ethylenediamine CB-EDA. The results showed that CB-EDA has a cytotoxic profile for J774.A1 macrophages in a time- and dose-dependent manner. When phagocytosed by the macrophage, CB-EDA triggers a mechanism that leads to apoptosis. In this process, there is an increase in oxidative stress pathways due to the activation of nitric oxide and then ROS. This causes an imbalance in redox function and a disruption of membrane integrity that occurs due to high levels of LDH, in addition to favoring the release of the pro-inflammatory cytokines IL-6, IL-12, and tumor necrosis factor (TNF) in an attempt to modulate the cell. However, these stimuli are not sufficient to repair the cell and the level of mitochondrial integrity is affected, causing a decrease in cell viability. This mechanism may be correlated with the activation of the caspasse-3 pathway, which, when compromised, cleaves and induces cells death via apoptosis, either through early or late apoptosis. In view of this, the potential for cell damage was investigated by analyzing the oxidative and inflammatory profile in the macrophage lineage J774.A1 and identifying potential mechanisms and metabolic pathways connected to these processes when cells were exposed to NP CB-EDA for both 24 h and 48 h.

Systemic and local inflammatory response after implantation of biomaterial in critical bone injuries

Purpose: To evaluate inflammatory response in critical bone injuries after implantation of the biomaterial composed of hydroxyapatite (HA)/poly (lactic-coglycolic acid) (PLGA)/BLEED. Methods: Forty-eight male Wistar rats (280 ± 20 grams) were divided into two groups: control group (CG), in which the animals do not receive any type of treatment; and biomaterial group (BG), in which the animals received the HA/PLGA/BLEED scaffold. Critical bone injury was induced in the medial region of the skull calotte with the aid of a trephine drill 8 mm in diameter. The biomaterial was implanted in the form of 1.5-mm thick scaffolds. Serum and calotte were collected at one, three and seven days. Results: Biomaterial had a significant effect on the morphological structure of the bone, accelerating osteoblast activation within three days, without causing exacerbated systemic inflammation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that BG induced upregulation of osteogenic genes such as runt-related transcription factor 2, and stimulated genes of inflammatory pathways such as tumor necrosis factor-α, on the first day without overexpressing genes related to bone matrix degradation, such as tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-9. Conclusions: The HA/PLGA/BLEED® association can be used as a bone graft to aid bone repair, as it is capable of modulating expression of important genes at this stage of the repair process.

Functionalized Titanium Nanoparticles Induce Oxidative Stress and Cell Death in Human Skin Cells.

Purpose: Nanoparticles are resources of advanced nanotechnology being present in several products. Titanium dioxide nanoparticles are among the five most widely used NP currently expanding their benefits from the oil industry to the areas of diagnostic medicine due to their properties and small size. However, its impact on human health is still controversial in the literature. We aimed to evaluate the cytotoxicity of a new titanium NP functionalized with sodium carboxylic ligand (COOH-Na+) in human keratinocytes (HaCaT) and human fibroblasts (HDFn). Methods: The physical-chemical characterization was performed by the transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential techniques, respectively. MTT and LDH assays were used to assess cytotoxicity and cell membrane damage respectively, ELISA to identify the inflammatory profile and, reactive oxygen species assay and cytometry to detect reactive oxygen species and their relationship with apoptosis/necrosis mechanisms. Results: The results demonstrated a decrease in cell viability at the highest concentrations tested for both cell lines, but no change in LDH release was detected for the HaCaT. The cell membrane damage was found only at 100.0 µg/mL for the HDFn. It was demonstrated that cytotoxicity in the highest concentrations evaluated for both cell lines for the 72 h period. The HDFn showed damage to the cell membrane at a concentration of 100 µg/mL followed by a significant increase in reactive oxygen species production. No inflammatory profile was detected. The HaCaT showed apoptosis when exposed to the highest concentration evaluated and HDFn showed both apoptosis and necrosis for the same concentration. Conclusion: Thus, it is possible to conclude that the cytotoxicity mechanism differs according to the cell type evaluated, with HDFn being the most sensitive line in this case, and this mechanism can be defined in a dose and time dependent manner, since the highest concentrations also triggered death cell.

Toxicological effects of the mixed iron oxide nanoparticle (Fe3O4 NP) on murine fibroblasts LA-9.

The increase in large-scale production of magnetic nanoparticles (NP) associated with the incomplete comprehensive knowledge regarding the potential risks of their use on environmental and human health makes it necessary to study the biological effects of these particles on organisms at the cellular level. The aim of this study to examine the cellular effects on fibroblast lineage LA-9 after exposure to mixed iron oxide NP (Fe3O4 NP). The following analyses were performed: field emission gun-scanning electron microscopy (SEM-FEG), dynamic light scattering (DLS), zeta potential, ultraviolet/visible region spectroscopy (UV/VIS), and attenuated total reactance-Fourier transform infrared (ATR-FTIR) spectroscopy analyses for characterization of the NP. The assays included cell viability, morphology, clonogenic potential, oxidative stress as measurement of reactive oxygen species (ROS) and nitric oxide (NO) levels, cytokines quantification interleukin 6 (IL-6) and tumor necrosis factor (TNF), NP uptake, and cell death. The size of Fe3O4 NP was 26.3 nm when evaluated in water through DLS. Fe3O4 NP did not reduce fibroblast cell viability until the highest concentration tested (250 µg/ml), which showed a decrease in clonogenic potential as well as small morphological changes after exposure for 48 and 72 hr. The NP concentration of 250 µg/ml induced enhanced ROS and NO production after 24 hr treatment. The uptake assay exhibited time-dependent Fe3O4 NP internalization at all concentrations tested with no significant cell death. Hence, exposure of fibroblasts to Fe3O4 NP-induced oxidative stress but not reduced cell viability or death. However, the decrease in the clonogenic potential at the highest concentration demonstrates cytotoxic effects attributed to Fe3O4 NP which occurred on the 7th day after exposure.

Analysis of cytotoxicity and genotoxicity in a short-term dependent manner induced by a new titanium dioxide nanoparticle in murine fibroblast cells.

The extensive use of titanium dioxide nanoparticles (TiO2 NPs) in cosmetics, food, personal care products, and industries brought concerns about their possible harmful effects. Nowadays it has become important to assess TiO2 NPs toxic effects as a way to understand their primary risks. In the cellular environment, after cell uptake, TiO2 NPs were described to induce reactive oxygen species (ROS) production, unbalance oxidative state, and activate apoptosis in several cell lines. Therefore, we aimed to evaluate the cytotoxicity and genotoxicity of a new TiO2 NP surface-functionalized with sodium carboxylic ligands in a murine fibroblast cell line (LA-9). TEM and DLS analyses were performed to define nanoparticle physicochemical characteristics. We evaluated the metabolic activity and LDH released after 24 h exposition to determine cytotoxic effects. Also, we evaluated DNA damage, intracellular reactive oxygen species (ROS) production, and apoptosis induction after 24 h exposure. The TiO2 NP impaired the cell membrane integrity at 1000 µg/mL, induced intracellular ROS production and late apoptosis at 24 h. The genotoxic effects were observed at all conditions tested at 24 h. Indeed, in fibroblasts exposed at 100 µg/mL was observed early apoptosis cells. The intracellular ROS content was increased in a dose-dependent manner. Thus, short-term exposure to TiO2 NP promoted cytotoxicity, genotoxicity and activated apoptosis pathways based on the potential role of oxygen species in the fibroblasts cell line.

Comparison of two different biomaterials in the bone regeneration (15, 30 and 60 days) of critical defects in rats.

PURPOSE: To evaluate and compare two types of different scaffolds in critical bone defects in rats. METHODS: Seventy male Wistar rats (280 ± 20 grams) divided into three groups: control group (CG), untreated animals; biomaterial group 1 (BG1), animals that received the scaffold implanted hydroxyapatite (HA)/poly(lactic-co-glycolic) acid (PLGA); and biomaterial group 2 (BG2), animals that received the scaffolds HA/PLGA/Bleed. The critical bone defect was induced in the medial region of the skull calotte with the aid of an 8-mm-diameter trephine drill. The biomaterial was implanted in the form of 1.5 mm thick scaffolds, and samples were collected after 15, 30 and 60 days. Non-parametric Mann-Whitney test was used, with the significance level of 5% (p ≤ 0.05). RESULTS: Histology revealed morphological and structural differences of the neoformed tissue between the experimental groups. Collagen-1 (Col-1) findings are consistent with the histological ones, in which BG2 presented the highest amount of fibers in its tissue matrix in all evaluated periods. In contrast, the results of receptor activator of nuclear factor kappa-Β ligand (Rank-L) immunoexpression were higher in BG2 in the periods of 30 and 60 days, indicating an increase of the degradation of the biomaterial and the remodeling activity of the bone. CONCLUSIONS: The properties of the HA/PLGA/Bleed scaffold were superior when compared to the scaffold composed only by HA/PLGA.

New Multi-Walled carbon nanotube of industrial interest induce cell death in murine fibroblast cells.

The search for new nanomaterials has brought to the multifactorial industry several opportunities for use and applications for existing materials. Carbon nanotubes (CNT), for example, present excellent properties which allow us to assume a series of applications, however there is concern in the industrial scope about possible adverse health effects related to constant exposure for inhalation or direct skin contact. Thus, using cell models is the fastest and safest way to assess the effects of a new material. The aim of this study was to investigate the cytotoxic profile in LA9 murine fibroblast lineage, of a new multi-walled carbon nanotube (MWCNT) that was functionalized with tetraethylenepentamine (TEPA) to obtain better physical-chemical characteristics for industrial use. The modifications presented in the CNT cause concern, as they can change its initial characteristics, making this nanomaterial harmful. HR-TEM, FE-SEM and zeta potential were used for the characterization. Cytotoxicity and cell proliferation tests, oxidative and nitrosative stress analyzes and inflammatory cytokine assay (TNF-α) were performed. The main findings demonstrated a reduction in cell viability, increased release of intracellular ROS, accompanied by an increase in TNF-α, indicating an important inflammatory profile. Confirmation of the data was performed by flow cytometry and ImageXpress with apoptosis/necrosis markers. These data provide initial evidence that OCNT-TEPA has a cytotoxic profile dependent on the concentration of LA9 fibroblasts, since there was an increase in free radicals, inflammation induction and cell death, suggesting that continuous exposure to this nanoparticle can cause damage to different tissues in the organism.

Biphasic Dose/Response of Photobiomodulation Therapy on Culture of Human Fibroblasts.

Objective: The objective of this study was to evaluate the effects of application of different fluences and energies of laser in the 24-, 48-, and 72-h periods in fibroblasts originating from human skin (HFF-1). Methods: The cell used as a template for cell proliferation was HFF-1. For the photobiomodulation (PBM) application, a 660 nm laser with a power of 40 mW and energies of 0.84, 1.40, 5.88, and 6.72 J was used. Five experimental groups were studied: one control group (CG) with simulated PBM and four groups that received PBM in different doses. The changes observed after laser irradiation were evaluated by cell viability (trypan blue) and proliferation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] tests. Intergroup comparisons were performed using two-way analysis of variance and the Tukey post hoc test (software GraphPad Prism 7.0). Results: In the trypan blue test, the total number of cells was significantly different between the irradiated groups and the CG at all times studied. The total number of cells increased in laser group (LG)1 (0.84 J) and LG2 (1.40 J) and decreased in LG4 (6.72 J). The mitochondrial activity increased significantly in LG1 and LG2 at 48 and 72 h and decreased in LG3 (5.88 J) and LG4 (6.72 J) compared with CG. Conclusions: The results indicate that the lower doses (0.45 and 0.75 J/cm2) of PBM induce the highest mitochondrial activity and cellular viability.

Effect of photobiomodulation associated with cell therapy in the process of cutaneous regeneration in third degree burns in rats.

Due to the complexity involved in the healing process of full thickness burns, the literature looks for alternatives to optimize tissue reconstruction. The objective of this study was to explore the action of photobiomodulation therapy associated with MSCs in the healing process of third degree burns. A total of 96 male Wistar rats were used, distributed in four groups with 24 animals each: Control Group, Laser Group, Cell Therapy Group, and Laser Group and Cell Therapy. The burn was performed with aluminum plate (150 °C). We performed analysis of wound contraction, histology, immunohistochemistry, birefringence analysis, and immunoenzymatic assay to evaluate tissue quality. Our results demonstrate that the association of the techniques is able to accelerate the repair process, modulating the inflammatory process, presenting a cutaneous tissue with better quality. Thus, we conclude that the use of photobiomodulation therapy associated with cell therapy is a promising treatment in the repair of total thickness burns.

Effects of mesenchymal stromal cells on motor function and collagen in the skeletal muscles of rats with type I diabetes.

The present study aimed to evaluate the effects of mesenchymal stromal cell (MSC) transplantation on motor function and collagen organization in the muscles of rats with type 1 diabetes mellitus. Male Wistar rats were randomly assigned to three groups: control (C), diabetic (DM) and diabetic treated with MSCs (DM-MSCs). Diabetes was induced by streptozotocin (50 µg/kg). Bone marrow cells were isolated from the tibia and femur. After 10 weeks of DM induction, the DM-MSC rats received four i.p. injections of MSCs (1 × 106). Ten weeks after MSC transplantation, motor performance was evaluated by the rotarod test and the anterior tibial (TA) muscles were collected for morphometric and quantification of collagen birefringence by polarizing microscopy analysis. Motor performance of the DM group was significantly reduced when compared to the C group and increased significantly in the DM + MSC group. The TA muscle mass was significantly reduced in the DM and DM + MSC groups compared to the C group. The connective tissue increased in the DM group compared to the C group and decreased in the DM + MSC group. The percentage collagen birefringence decreased significantly in the DM group when compared to the C group and increased in the DM + MSC group. Motor performance was positively correlated with collagen birefringence and negatively correlated with percentage of connective tissue. The results indicate that MSC transplantation improves both motor function and the collagen macromolecular organization in type 1 DM.

Photobiomodulation effect on the proliferation of adipose tissue mesenchymal stem cells.

The use of mesenchymal stem cells (MSCs) in tissue engineering has been extensively investigated. The greater the proliferation of this cellular group, the greater the regenerative and healing capacity of the tissue to which they belong. In this context, photobiomodulation (PBM) is an efficient technique in proliferation of distinct cell types. However, its parameters and mode of action are still unclear and require further investigation. This study aimed to evaluate the PBM action with different energies in MSCs of adipose tissue (hASCs). We used hASCs, seeded in 24-well plates, with 3 × 104 cells per well, in culture media. We used a total of four experimental groups, one with hASCs and simulated PBM and three other groups, which received PBM irradiation at 24, 48, and 72 h, with a 660-nm laser and power of 40 mW and energy of 0.56, 1.96, and 5.04 J. We performed analyses of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor) and trypan blue to evaluate cell proliferation and viability, 1 h after PBM irradiation. Software Graph PadPrism 7.0 was used. Intergroup comparisons were performed with ANOVA two-way and we used the Tukey post hoc test. Mitochondrial activity evaluated by MTT revealed the statistical difference in the first 24 h for group with more high energy when compared to control group; and in the 72 h for two irradiated groups when compared to the control group. The trypan blue test showed significant differences at the end of the experiment for two irradiated groups LG1 (4.52 × 104 ± 0.2) and LG2 (4.85 × 104 ± 0.8), when compared to the control group (1.87 × 104 ± 0.7). Both tests failed to be statistically different at the end of the experiment for groups LG1 and LG2 and observed a reduction in cellular mitochondrial growth and activity for group LG3. We conclude that PBM with energy close to 0.56 and 1.96 J promote proliferation of hASCs, and higher energy, such as 5.04 J, can be harmful.

Photobiomodulation on critical bone defects of rat calvaria: a systematic review.

Bone defects following trauma represent a high impact on the quality of life of millions of people around the world. The aim of this study was to review photobiomodulation (PBM) action in the treatment of bone critical defects in rat calvaria, related to evaluation of the current protocols applied. One hundred and forty-seven articles related to the subject were found by searching the main databases (Pubmed, Lilacs, Web of Science, and Scopus) considering the period of publication until the year 2017, and only 14 corresponded the inclusion criteria established for this systematic review. The main parameters of the PBM were expressed in Table 1. In addition, it was possible to observe the use of two different wavelengths (red and infrared), which are considered therapeutic. Most of the evaluated articles presented positive results that describe a greater amount of neoformed bone, an increase in collagen synthesis, and a contribution to microvascular reestablishment. However, two studies report no effect on the repair process when the PBM was used. In addition, we observed considerable variations between the values of power, fluence, and total energy, which make it difficult to compare the results presented between the selected studies. It was possible to conclude that the infrared laser was more effective in positively stimulating the bone repair process of critical defects. Furthermore, a discrepancy was found in the parameter values used, which made it difficult to choose the best protocol for the treatment of this type of lesion.

Evaluation of the low-level laser therapy application parameters for skin burn treatment in experimental model: a systematic review.

Burn is defined as a traumatic injury of thermal origin, which affects the organic tissue. Low-level laser therapy (LLLT) has gained great prominence as a treatment in this type of injury; however, the application parameters are still controversial in the literature. The aims of this study were to review the literature studies that use LLLT as a treatment in burns conducted in an experimental model, discuss the main parameters used, and highlight the benefits found in order to choose an appropriate therapeutic window to be applied in this type of injury. The selection of the studies related to the theme was carried out in the main databases (PubMed, Cochrane Library, LILACS, Web of Science, and Scopus in the period from 2001 to 2017). Subsequently, the articles were then chosen that fell within the inclusion criteria previously established. In the end, 22 were evaluated, and the main parameters were presented. The analyzed studies presented both LLLT use in continuous and pulsed mode. Differences between the parameters used (power, fluence, and total energy) were observed. In addition, the protocols are distinct as to the type of injury and the number of treatment sessions. Among the results obtained by the authors are the improvements in the local microcirculation and cellular proliferation; however, a study reported no effects with LLLT as a treatment. LLLT is effective in accelerating the healing process. However, there is immense difficulty in establishing the most adequate protocol, due to the great discrepancy found in the applied dosimetry values.

Bacterial cellulose membrane used as biological dressings on third-degree burns in rats.

INTRODUCTION: Burn injuries represent a high risk of morbidity and mortality. The wound healing process is complex and requires the participation of different types of cells. Therefore, new biomaterials, which innovate the wound healing process, are being investigated. OBJECTIVE: The aim of this study was to investigate the use of bacterial cellulose both in its pure state and enriched with lidocaine in full-thickness burns in rats. METHODS: Thirty rats (Wistar) (260 ± 20 gramas) divided into control group (CG), bacterial cellulose membrane group (MG) and bacterial cellulose membrane enriched with lidocaine group (MLG) were used. The burns were induced using a 150°C heated soldering iron, held on the animal neck for 10 seconds. The biomaterial was applied immediately after injury and skin samples were collected on the tenth day of the treatment. The level of significance of p⩽0.05 was used for the conclusion of the statistical analysis. RESULTS: The groups treated with the biomaterials, a histological pattern compatible with a more advanced repair stage showing skin appendages, mild inflammatory infiltrate, better collagen fiber organization and mild immunostaining COX-2 and MMP-9 was observed, when compared to the control group that did not receive any type of treatment. CONCLUSION: Thus, was concluded that the bacterial cellulose-based biomaterial both in its pure state and enriched with lidocaine optimizing the full-thickness burn wound healing in rats.

Alternative animal model for studies of total skin thickness burns.

PURPOSE: To present an alternative experimental model of third degree burn of easy reproducibility. METHODS: Eighteen male Wister rats were randomly divided into three groups, 6 of which were allocated to each group. A soldering iron coupled to an aluminum plate was used to produce burn, at a temperature of 150ºC, with different exposure times per group. Group 5 (G5) animals were burned at 150°C with exposure time of 5 seconds; Group 10 (G10) the animals were burned at 150°C with exposure time of 10 seconds and group 15 (G15) the animals were burned at 150°C with exposure time of 15 seconds. RESULTS: Histopathological analyzes showed that all three groups had similar morphological characteristics, with total thickness involvement. CONCLUSION: The technique is effective to reproduce a third degree burn and suggests the temperature of 150ºC with 5 seconds of exposure in order to minimize the risks to the animals.

Alternative animal model for studies of total skin thickness burns

Purpose: To present an alternative experimental model of third degree burn of easy reproducibility. Methods: Eighteen male Wister rats were randomly divided into three groups, 6 of which were allocated to each group. A soldering iron coupled to an aluminum plate was used to produce burn, at a temperature of 150ºC, with different exposure times per group. Group 5 (G5) animals were burned at 150°C with exposure time of 5 seconds; Group 10 (G10) the animals were burned at 150°C with exposure time of 10 seconds and group 15 (G15) the animals were burned at 150°C with exposure time of 15 seconds. Results: Histopathological analyzes showed that all three groups had similar morphological characteristics, with total thickness involvement. Conclusion: The technique is effective to reproduce a third degree burn and suggests the temperature of 150ºC with 5 seconds of exposure in order to minimize the risks to the animals.(AU)

Alternative animal model for studies of total skin thickness burns

Abstract Purpose: To present an alternative experimental model of third degree burn of easy reproducibility. Methods: Eighteen male Wister rats were randomly divided into three groups, 6 of which were allocated to each group. A soldering iron coupled to an aluminum plate was used to produce burn, at a temperature of 150ºC, with different exposure times per group. Group 5 (G5) animals were burned at 150°C with exposure time of 5 seconds; Group 10 (G10) the animals were burned at 150°C with exposure time of 10 seconds and group 15 (G15) the animals were burned at 150°C with exposure time of 15 seconds. Results: Histopathological analyzes showed that all three groups had similar morphological characteristics, with total thickness involvement. Conclusion: The technique is effective to reproduce a third degree burn and suggests the temperature of 150ºC with 5 seconds of exposure in order to minimize the risks to the animals.