Walnut ointment promotes full-thickness burning wound healing: role of linoleic acid

Purpose: To investigate the active ingredients of walnut ointment (WO) and its mechanism in repairing wounds. Methods: The ingredients of WO were detected by gas chromatography­mass spectrometry. The effect of linoleic acid (LA) was tested by in vitro Alamar Blue (AB) reagent. Image J software, histological and immunohistochemical analysis were used to confirm the healing effect of LA in the porcine skin model. The animals were euthanized after the experiment by injection of pentobarbital sodium. Results: LA, 24% in WO, promotes keratinocytes and fibroblasts proliferation, which were 50.09% and 15.07% respectively higher than control (p < 0.05). The healing rate of the LA group (96.02% ± 2%, 98.58% ± 0.78%) was higher than the saline group (82.11% ± 3.37%, 88.72% ± 1.73%) at week 3 and week 4 (p < 0.05). The epidermal thickness of the LA was 0.16 ± 0.04 mm greater and the expression of the P63 and CK10 proteins was stronger in the LA group than the control (p < 0.05). Conclusions: LA, which is the main components in WO can promote full-thickness burning wounds (FBWs) by stimulating cell proliferation and differentiation.

A modified hydrogel production protocol to decrease cellular content

Purpose: To analyze the cytotoxicity and cell in porcine-derived decellularized skin matrix. Methods: We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation. Results: After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days. Conclusions: The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.

Fibroma cutâneo atípico em uma vaca / Atypical cutaneous fibroma in a cow

Background: Cutaneous fibroma is a benign neoplasm affecting the fibroblasts and collagen matrix that develops in the dermis or subcutaneous tissue. This neoplasm is uncommon in cattle, and few reports have described the treatment and resolution of this neoplasm. Despite its benign character, a veterinarian should consider cutaneous fibroma in the differential diagnosis of skin tumors. This report aims to describe a rare case of large fibroma in the scapular region in a cow, with emphasis on the clinical-surgical and anatomopathological aspects of the condition. Case: A 3-year-old Girolando 3/4 cow was attended to at a rural property in Lagamar-MG, Brazil. According to the owner, the animal presented with a small mass in the right scapular region that grew progressively over 1 year and 6 months. Clinical examination revealed an exuberant and painless increase in volume on palpation in the proximal region of the right thoracic limb, which, in its vertical axis, extended from the proximal end of the scapula to near the olecranon tuberosity, and, in its horizontal axis, extended from the 6th intercostal space to the scapulohumeral joint, reaching the dimensions 66 cm and 62 cm, respectively. It presented with multiple nodules that were firm in consistency with extensive areas of ulceration. Neoplasia was suspected, and surgical excision was decided upon. The cow was sedated and restrained in the left lateral decubitus position. Trichotomy and antisepsis of the operative field were performed followed by an infiltrative anesthetic block around the tumor. The tumor was excised maintaining a safety margin of 1 cm. Dermorrhaphy was not possible, and healing by secondary intention was awaited. In the postoperative period, antibiotic therapy with benzathine penicillin, analgesia with meloxicamand dipyrone and daily dressing of the wound were performed. There were no postoperative complications and complete healing occurred approximately 100 days after surgery. One year after the surgical procedure, the owner reported that the cow did not present with recurrence of the neoplasm. The resected tumor weighed 11.2 kg, and, when cut, presented with solid conformation and whitish coloration. Tumor fragments were harvested, fixed in 10% formalin, and sent for histopathological examination, which revealed neoproliferation of remarkable cellular density composed of dense, well vascularized fibrocollagenous connective tissue arranged in multidirectional bundles and undulating pattern. Mild cellular pleomorphism was identified, and no mitosis figures were observed. Alcian blue staining was negative for mucopolysaccharides, differing from Masson's trichrome staining, which widely stained the fibrocollagenous tissue blue. In view of these findings, the diagnosis of cutaneous fibroma was confirmed. Discussion: Cutaneous fibromas are benign neoplasms of fibrous tissue, and they are uncommon in cattle and may be associated with bovine papillomavirus and/or trauma. Although the origin of cutaneous fibroma is not clear, the present report stands out due to the large size of the tumor mass. The complete healing of the surgical wound, the absence of recurrence one year after surgery and the return of the animal to dairy production demonstrate that the surgical treatment was adequate. The macro- and microscopic characteristics of the cutaneous fibroma in this case corroborate with other cases reported in the literature. Large cutaneous fibroma is uncommon in bovines, and may hinder surgical excision and prolong healing time, as well as the complete recovery of the animal. Moreover, the differential diagnosis with other neoplasms of fibroblastic origin is relevant, especially for those with malignant biological behavior, such as fibrosarcoma and myxosarcoma.

Efeito do extrato de Bidens pilosa L, Mel e pomadas homeopática e alopática na cicatrização de feridas cutâneas de ratos Wistar / [Effect of Bidens pilosa L, Honey extract and homeopathic and allopathic ointments on the healing of skin wounds of Wistar rats]

Cicatrização de ferida é um processo dinâmico, que tem por objetivo restaurar a continuidade do tecido lesionado. No entanto, em alguns casos, é necessário favorecer condições adequadas para viabilizar o processo fisiológico. Neste estudo foram utilizados ratos Wistar, divididos aleatoriamente entre cinco grupos, com 12 animais cada, sendo eles: grupo P (Bidens pilosa L.), grupo mel, grupo Co1 (pomada comercial alopática), grupo Co2 (pomada comercial homeopática) e grupo CT (controle). As lesões foram geradas por incisão com punch de 8mm, sendo tratadas diariamente de forma tópica. Foram eutanasiados quatro animais por grupo, no terceiro, sétimo e 14º dias do experimento, e o material coletado foi armazenado em formalina 10% e encaminhado para processamento histológico. Posteriormente, realizou-se a contagem de leucócitos mononucleares, fibroblastos e neovasos e avaliou-se a arquitetura de fibras colágenas. Os resultados da contagem foram analisados pela ANOVA, seguida pelo teste de Tukey (P<0,05). O modelo experimental proposto neste estudo demonstrou que todos os tratamentos apresentaram potencial cicatrizante, com exceção do mel. A aplicação tópica do creme do extrato de Bidens pilosa L. a 10% apresentou melhor perfil anti-inflamatório; a pomada alopática apresentou boa aderência à superfície da lesão e a pomada homeopática, grande potencial angiogênico, com menor tempo de cicatrização.(AU)

Wound healing is a dynamic process that aims to restore the continuity of injured tissue. However, in some cases it is necessary to favor adequate conditions to enable the physiological process. Wistar rats were randomly divided into 5 groups with 12 animals each, namely: group P (Bidens pilosa L.), group honey, group Co1 (commercial allopathic ointment), group Co2 (commercial homeopathic ointment) and group CT (control). The lesions were generated by an 8mm punch incision and were treated topically daily. Four animals per group were euthanized on the 3rd, 7th and 14th day of the experiment and the collected material was stored in 10% formalin and sent for histological processing, after which mononuclear, fibroblasts and neovascular leukocytes were counted and collagen fiber architecture was evaluated. Counting results were analyzed by ANOVA, followed by Tukey test (p <0.05). The experimental model proposed in this study showed that all treatments had healing potential, except honey. The topical application of 10% Bidens pilosa L. extract cream showed the best anti-inflammatory profile; Allopathic ointment showed good adhesion to the surface of the lesion and homeopathic ointment showed great angiogenic potential with shorter healing time.(AU)

Estabelecimento de linhagens celulares em felídeos silvestres Uma revisão dos efeitos das condições de cultivo e criopreservação sobre a qualidade celular / Establishment of cell lines in wild felids A review of the effects of culture and cryopreservation conditions on cell quality

Linhagens celulares, principalmente fibroblastos derivados da pele, podem ser ferramentas interessantes para a conservação e multiplicação de felídeos silvestres ameaçados de extinção. Esses animais em virtude de seu quantitativo reduzido têm nos bancos de células somáticas uma alternativa para a conservação de material biológico derivado de pequenas populações, garantindo assim o armazenamento da diversidade genética de diferentes grupos de indivíduos. Isso porque tais linhagens, quando apropriadamente estabelecidas por meio das técnicas de cultivo in vitro e criopreservação, permitem seu emprego na obtenção de embriões por clonagem por transferência nuclear de célula somática e produção de células induzidas à pluripotência. Assim, uma das etapas essenciais para o uso dessas linhagens de maneira eficiente consiste na avaliação dos efeitos das condições de cultivo in vitro e criopreservação das células, visando reconhecer os danos gerados pelas manipulações e identificar os ajustes necessários aos protocolos empregados. Portanto, o objetivo desta revisão consiste em reunir e explorar informações úteis sobre as condições de cultivo in vitro e criopreservação de células derivadas de felídeos silvestres, visando o estabelecimento de linhagens celulares na conservação e multiplicação destas espécies.(AU)

Cell lines, mainly fibroblasts derived from the skin, can be interesting tools for the conservation and multiplication of endangered wild felids. These animals, due to their reduced quantity, have somatic cell banks as an alternative for the conservation of biological material derived from small populations, thus guaranteeing the storage of the genetic diversity of different groups of individuals. This is because such lines, when properly established through in vitro culture and cryopreservation techniques, allow their use in obtaining embryos by cloning by somatic cell nuclear transfer and production of cells induced to pluripotency. Thus, one of the essential steps for the use of these lines in an efficient way consists of the evaluation of the effects of the conditions of in vitro culture and cryopreservation of the cells, aiming to recognize the damages generated by the manipulations and to identify the necessary adjustments to the employed protocols. Therefore, the aim of this review is to gather and explore useful information about in vitro culture and cryopreservation conditions of cells derived from wild felids, aiming at the establishment of cell lines in the conservation and multiplication of these species.(AU)

Efeito do extrato de Bidens pilosa L, mel e pomadas homeopática e alopática na cicatrização de feridas cutâneas de ratos Wistar / [Effect of Bidens pilosa L, honey extract and homeopathic and allopathic ointments on the healing of skin wounds of Wistar rats]

Cicatrização de ferida é um processo dinâmico, que tem por objetivo restaurar a continuidade do tecido lesionado. No entanto, em alguns casos, é necessário favorecer condições adequadas para viabilizar o processo fisiológico. Neste estudo foram utilizados ratos Wistar, divididos aleatoriamente entre cinco grupos, com 12 animais cada, sendo eles: grupo P (Bidens pilosa L.), grupo mel, grupo Co1 (pomada comercial alopática), grupo Co2 (pomada comercial homeopática) e grupo CT (controle). As lesões foram geradas por incisão com punch de 8mm, sendo tratadas diariamente de forma tópica. Foram eutanasiados quatro animais por grupo, no terceiro, sétimo e 14º dias do experimento, e o material coletado foi armazenado em formalina 10% e encaminhado para processamento histológico. Posteriormente, realizou-se a contagem de leucócitos mononucleares, fibroblastos e neovasos e avaliou-se a arquitetura de fibras colágenas. Os resultados da contagem foram analisados pela ANOVA, seguida pelo teste de Tukey (P<0,05). O modelo experimental proposto neste estudo demonstrou que todos os tratamentos apresentaram potencial cicatrizante, com exceção do mel. A aplicação tópica do creme do extrato de Bidens pilosa L. a 10% apresentou melhor perfil anti-inflamatório; a pomada alopática apresentou boa aderência à superfície da lesão e a pomada homeopática, grande potencial angiogênico, com menor tempo de cicatrização.(AU)

Wound healing is a dynamic process that aims to restore the continuity of injured tissue. However, in some cases it is necessary to favor adequate conditions to enable the physiological process. Wistar rats were randomly divided into 5 groups with 12 animals each, namely: group P (Bidens pilosa L.), group honey, group Co1 (commercial allopathic ointment), group Co2 (commercial homeopathic ointment) and group CT (control). The lesions were generated by an 8mm punch incision and were treated topically daily. Four animals per group were euthanized on the 3rd, 7th and 14th day of the experiment and the collected material was stored in 10% formalin and sent for histological processing, after which mononuclear, fibroblasts and neovascular leukocytes were counted and collagen fiber architecture was evaluated. Counting results were analyzed by ANOVA, followed by Tukey test (p <0.05). The experimental model proposed in this study showed that all treatments had healing potential, except honey. The topical application of 10% Bidens pilosa L. extract cream showed the best anti-inflammatory profile; Allopathic ointment showed good adhesion to the surface of the lesion and homeopathic ointment showed great angiogenic potential with shorter healing time.(AU)

Estabelecimento de linhagens celulares em felídeos silvestres – Uma revisão dos efeitos das condições de cultivo e criopreservação sobre a qualidade celular / Establishment of cell lines in wild felids – A review of the effects of culture and cryopreservation conditions on cell quality

Linhagens celulares, principalmente fibroblastos derivados da pele, podem ser ferramentas interessantes para a conservação e multiplicação de felídeos silvestres ameaçados de extinção. Esses animais em virtude de seu quantitativo reduzido têm nos bancos de células somáticas uma alternativa para a conservação de material biológico derivado de pequenas populações, garantindo assim o armazenamento da diversidade genética de diferentes grupos de indivíduos. Isso porque tais linhagens, quando apropriadamente estabelecidas por meio das técnicas de cultivo in vitro e criopreservação, permitem seu emprego na obtenção de embriões por clonagem por transferência nuclear de célula somática e produção de células induzidas à pluripotência. Assim, uma das etapas essenciais para o uso dessas linhagens de maneira eficiente consiste na avaliação dos efeitos das condições de cultivo in vitro e criopreservação das células, visando reconhecer os danos gerados pelas manipulações e identificar os ajustes necessários aos protocolos empregados. Portanto, o objetivo desta revisão consiste em reunir e explorar informações úteis sobre as condições de cultivo in vitro e criopreservação de células derivadas de felídeos silvestres, visando o estabelecimento de linhagens celulares na conservação e multiplicação destas espécies.

Cell lines, mainly fibroblasts derived from the skin, can be interesting tools for the conservation and multiplication of endangered wild felids. These animals, due to their reduced quantity, have somatic cell banks as an alternative for the conservation of biological material derived from small populations, thus guaranteeing the storage of the genetic diversity of different groups of individuals. This is because such lines, when properly established through in vitro culture and cryopreservation techniques, allow their use in obtaining embryos by cloning by somatic cell nuclear transfer and production of cells induced to pluripotency. Thus, one of the essential steps for the use of these lines in an efficient way consists of the evaluation of the effects of the conditions of in vitro culture and cryopreservation of the cells, aiming to recognize the damages generated by the manipulations and to identify the necessary adjustments to the employed protocols. Therefore, the aim of this review is to gather and explore useful information about in vitro culture and cryopreservation conditions of cells derived from wild felids, aiming at the establishment of cell lines in the conservation and multiplication of these species.

The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients

Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.(AU)

Expression dynamics of caveolin-1 in fibroblasts of newborn rats with chronic lung disease and its impact on lung fibroblast proliferation

Purpose: To evaluate the changes of caveolin-1 in lung fibroblasts in newborn Wistar rats when exposed to hyperoxic conditions, as well as lung fibroblasts cell cycle. Methods: One hundred newborn Wistar rats were randomly divided (50 rats/group) into experimental and control groups, exposed to hyperoxic conditions or normal air, respectively. The fraction of inspired oxygen (FiO2) in the experimental group was 90%, whereas this value was 21% in the control group. Lung fibroblasts were collected on days 3, 7, and 14 of the experiment. Caveolin-1 expression dynamics in lung fibroblasts was assayed in each group by immunofluorescence and Western blot analyses. Flow cytometry (FCM) was used to assess the proportions of lung fibroblasts at different stages of the cell cycle. Results: On day 3, no significant difference in caveolin-1 expression was observed between the hyperoxic and control groups; however, on days 7 and 14, caveolin-1 expression was significantly lower in the hyperoxic group than in the control (P 0.05). No apparent differences were observed in caveolin-1 expression in the control group at the different time points. Using FCM analysis, we showed that the proportion of lung fibroblasts in G0/G1 phase in the hyperoxic group decreased compared to that of the control group on day 7, while the proportion of S-phase cells increased (P 0.05). These differences were more significant when the groups were compared on day 14 (P 0.01). Conclusion: After seven days the exposure to hyperoxic conditions, lung fibroblasts proliferated and caveolin-1 expression decreased.(AU)

EXPRESSÃO GÊNICA EM EMBRIÕES CLONES DE VEADO-CATINGUEIRO (Mazama gouazoubira) PRODUZIDOS POR TRANSFERÊNCIA NUCLEAR DE CÉLULAS SOMÁTICAS INTERESPECÍFICA (TNCSi) UTILIZANDO CITOPLASTOS BOVINOS

A biodiversidade animal vem diminuindo como reflexo da excessiva exploração dos recursos naturais. Dentre esses animais, os cervídeos apresentam um número considerável de espécies inclusas, sendo o veado-catingueiro (Mazama gouazoubira) uma espécie vulnerável. Como instrumento para conservar espécies ameaçadas, é possível utilizar as biotécnicas reprodutivas, como a transferência nuclear de células somáticas interespecífica (TNCSi). Portanto, esse trabalho teve como objetivo comparar a expressão gênica de embriões clones intraespecíficos (bovino-bovino), interespecíficos (cervídeo-bovino) e embriões bovinos oriundos de fecundação in vitro (FIV). O cultivo celular (fibroblasto) foi realizado de 7-14 dias a partir de amostras criopreservadas de pele de veado-catingueiro e de bovino já existentes no laboratório. Posteriormente, oócitos bovinos foram maturados, desnudados, e utilizados na reconstrução embrionária nos grupos (citoplasto/carioplasto): cervídeo/bovino e bovino/bovino e posteriormente ativados. Os embriões reconstruídos juntamente com os oriundos de FIV, utilizados como grupo controle, foram mantidos a -80º C até avaliação da expressão gênica. Os genes OCT4, T-FAM, GJA1, BAX e BCL2, relacionados à qualidade embrionária, foram análisados por PCR em tempo real tendo como referência os genes, GAPDH, B-ACTINA e 16S. Para análise estatística, a quantificação relativa da expressão dos genes foi realizada utilizando o método 2-Ct. Os perfis de expressão foram apresentados como média (± desvio padrão) da quantificação relativa de mRNA e avaliados pelo teste de Kruskal-Wallis ou Mann-Whitney, quando conveniente, considerando um nível de significância de 5%. Não houve diferença (p > 0,05) no desenvolvimento embrionário entre intraTNCS e interTNCS (72,8% e 65,5% de taxa de clivagem, 38,7% e 43,2% de taxa de mórula e 11,3% e 5,9% de taxa de blastocistos, respectivamente). Ao analisar a expressão do gene OCT4, observou-se diferença (p < 0,05) nos grupos intraTNCS e inter TNCS quando comparados com o grupo FIV. A expressão do T-FAM no grupo intraTNCS obteve uma expressão significativamente maior quando comparado com o grupo FIV (p <0,05). No entanto, essa diferença não foi observada entre os grupos FIV e interTNCS, assim como os grupos intraTNCS e interTNCS (p >0,05). A expressão do gene GJA1 foi estatisticamente semelhante (p > 0,05) em todos os grupos experimentais. O gene BAX obteve uma expressão estatisticamente diferente entre todos os grupos, sendo mais expresso no grupo intraTNCS (p <0,05). Por sua vez, o gene BCL2 obteve uma expressão próxima de valores nulos em todos os grupos. A partir dos resultados obtidos conclui-se que a diferença observada na expressão de alguns genes nos embriões cervídeo-bovino não interferiu no desenvolvimento, sendo possível afirmar que os citoplastos bovinos são capazes de reprogramar os carioplastos do veado-catingueiro. Cabe frisar que, apesar dessa reprogramação é interessante a realização de estudos que avaliem a reprogramação epigenética nesses embriões. Essa pesquisa ajudará a elucidar os mecanismos envolvidos na interação núcleo-citoplasma e que resultam na ativação ou inativação de genes importantes para o desenvolvimento embrionário e formação de indivíduos.

Animal biodiversity has been decreasing as a result of the excessive exploitation of natural resources. Among these animals, deer have a considerable number of included species, and the brocket deer (Mazama gouazoubira) is a vulnerable species. As a tool to conserve endangered species, it is possible to use reproductive biotechniques, such as interspecific somatic cell nuclear transfer (iSCNT). Therefore, this work aimed to compare the gene expression of clone embryos obtained by intraspecific (bovine- bovine), interspecific (cervid- bovine) and bovine embryos from in vitro fertilization (IVF). Cell culture (fibroblast) was carried out for 7-14 days from cryopreserved samples of brocket deer and bovine skin already existing in the laboratory. Later, bovine oocytes were matured, denuded, and used in embryonic reconstruction in the groups (cytoplast/karyoplast): cervid/bovine and bovine/bovine and later activated. Embryos reconstructed together with those from IVF, used as a control group, were kept at -80º C until evaluation of gene expression. The OCT4, T-FAM, GJA1, BAX and BCL2 genes, related to embryo quality, were analyzed by real-time PCR having as reference the genes, GAPDH, B-ACTIN and 16S. For statistical analysis, the relative quantification of gene expression was performed using the 2-Ct method. Expression profiles were presented as mean (± standard deviation) of the relative quantification of mRNA and evaluated by the Kruskal-Wallis or Mann-Whitney test, when convenient, considering a significance level of 5%. There was no difference (p > 0.05) in embryonic development between intraSCNT and interSCNT (72.8% and 65.5% cleavage rate, 38.7% and 43.2% morula rate and 11.3% and 5.9% blastocyst rate, respectively). When analyzing the expression of the OCT4 gene, a difference (p < 0.05) was observed in the intraSCNT and interSCNT groups when compared to the IVF group. The expression of T-FAM in the intraSCNT group had a significantly higher expression when compared to the IVF group (p < 0.05). However, this difference was not observed between the IVF and interSCNT groups, as well as the intraSCNT and interSCNT groups (p >0.05). GJA1 gene expression was statistically similar (p > 0.05) in all experimental groups. The BAX gene had a statistically different expression between all groups, being more expressed in the intraSCNT group (p < 0.05). In turn, the BCL2 gene had an expression close to null values in all groups. From the results obtained, it can be concluded that the difference observed in the expression of some genes in the cervid-bovine embryos did not interfere in the development, being possible to affirm that the bovine cytoplasts are capable of reprogramming the brocket deer karyoplasts. It should be noted that, despite this reprogramming, it is interesting to carry out studies that assess the epigenetic reprogramming in these embryos. This research will help to elucidate the mechanisms involved in the nuclear-cytoplasm interaction and that result in the activation or inactivation of genes important for embryonic development and formation of individuals.

Fibrogenesis and epithelial coating of skin wounds in rats treated with angico extract (Anadenanthera colubrina var. cebil)

PURPOSE: To evaluate the effects of angico bark extract (Anadenanthera colubrina var. cebil) in the healing process of the skin of rats.METHODS: Twenty adult male rats were divided into four groups of five animals each, according to the respective postoperative days, as follow: G4, G7, G14 and G21. Each group received two incisions on skin and subcutaneous tissue in the right and left antimere of the thoracic region, separated by a distance of 2 cm. The right lesion was treated daily with saline and the left with the angico alcoholic extract (5%). At the end of each experimental period, the animals were euthanized and fragments of the wound area with the edges were removed, fixed in 10% formaldehyde solution and processed for paraffin embedding. Histological sections (5 m of thickness) were stained with hematoxylin and eosin (HE), Gomori trichromic and picrosisirus red for morphological and morphometric analyses. Statistical analysis was done by ANOVA and Tukey-Kramer test (p 0.05).RESULTS: Morphological analysis showed larger fibroblasts and a higher concentration of collagen fibers in skyn wounds treated with the angico extract. Morphometric analysis demonstrated a significant increase in the number of fibroblasts at 7th and collagen in 7th and 14th days (p 0.01) in wounds treated with the angico extract.CONCLUSION: The angico alcoholic extract (Anadenanthera colubrina var. cebil) induces the acceleration of wound healing in skin wounds of rats.(AU)

Avaliação in vitro de propriedades biológicas de Senna occidentalis (L) Link / Evaluation of in vitro biological properties of Senna occidentalis (L) Link

Espécies de Senna são amplamente utilizadas por tribos americanas, africanas e indianas, principalmente para tratar a fraqueza, a constipação, as desordens do fígado e também em preparações tópicas para infecções de pele. A Senna occidentalis (L.) Link é um arbusto perene nativo da América do Sul encontrado em regiões tropicais. Este trabalho avaliou a atividade antimicrobiana de extratos aquosos e hidroalcoólicos de diferentes partes da planta. A atividade antimicrobiana foi estabelecida frente aos microrganismos padrões farmacêuticos por espectrofotometria e técnica de microdiluição. A Escherichia coli apresentou sensibilidade apenas a componentes extraídos das sementes, os quais podem ser de natureza proteica. O espectro mais amplo de atividade antimicrobiana foi obtido com o extrato hidroalcoólico das sementes, principalmente contra Pseudomonas aeruginosa. A toxicidade in vitro utilizando fibroblastos de camundongo indicou que este extrato pode ser um ingrediente biocompatível para formulações de uso tópico. Já o extrato hidroalcoólico de partes aéreas demonstrou ser potencialmente citotóxico.(AU)

Senna species have been widely used by American, African and Indian ethic groups mainly in the treatment of feebleness, constipation, liver disorders and skin infections. Senna occidentalis (L.) Link is a perennial shrub native to South America and indigenous to tropical regions throughout the world. Current study evaluated the antimicrobial activity of aqueous and hydroalcoholic extracts from S. occidentalis prepared from different parts of the plant. Antimicrobial activity was assessed against standard pharmaceutical microorganisms by spectrophotometry and microdilution technique. Escherichia coli was sensitive only to compounds extracted from seeds which may be proteinaceous. A broader antimicrobial spectrum was demonstrated by the hydroalcoholic extract of seeds, mostly against Pseudomonas aeruginosa. The in vitro toxicity using mouse fibroblasts indicated that the extract might be a biocompatible ingredient for topical formulations, while the hydroalcoholic extract of aerial parts demonstrated to be potentially cytotoxic.(AU)

TGF- B1 on induced osteogenic differentiation of human dermal fibroblast

PURPOSE: To evaluate the role of transforming growth factor beta 1 (TGF-β1) on the induced osteogenic differentiation of human dermal fibroblasts. METHODS: We performed four groups with cultured dermal fibroblasts according to the culture medium: CONTROL (DMEM culture medium); TGF-β1 (DMEM culture medium with 10 ng/ml of TGF-β1); OSTEOG (DMEM culture medium with 0.5 µg/ml of ascorbic acid, 10 mmol/l of β-glycerophosphate and 10 nmol/L of dexamethasone); and OSTEOG/TGF-β1 (osteogenic medium with 10 ng/ml of TGF-β1). Alkaline phosphatase (ALP) activity and the amount of osteocalcin (OC) in the supernatant, as well as the capability to form calcium phosphate deposits, were analysed for 28 days RESULTS: There were significant differences (p<0.05) between CONTROL and TGF-β1 groups in comparison with OSTEOG and OSTEOG/TGF-β1 groups in the ALP activity and OC amount. Although, both osteogenic groups had the same behavior with regard the expression curve during the experimental time, the OSTEOG/TGF-β1 group achieved significantly higher ALP and OC levels and showed no significant difference in the levels of mineralized deposits and in comparison with the levels found in the OSTEOG group. CONCLUSION: The addition of transforming growth factor beta 1 to the osteogenic culture medium increased the activity of alkaline phosphatase and the amount of osteocalcin, but TGF-β1 did not alter the presence of mineralized calcium phosphate deposits. .(AU)

Isolamento e caracterização de células doadoras derivadas da pele para a transferência nuclear / Isolation and characterization of skin-derived donor cells for nuclear transfer

O sucesso das biotecnologias reprodutivas assistidas, especialmente a clonagem por transferência nuclear de células somáticas, envolve múltiplos fatores e cada um destes pode influenciar a eficiência final. Em geral, os protocolos de isolamento, caracterização e criopreservação de células das espécies a serem clonadas constituem a etapa inicial para a obtenção de descendência clone viável, além de contribuir para outros estudos relacionados à biotecnologia celular. Neste contexto, o sucesso desta etapa pode ser alcançado pela associação da escolha do tipo celular, estabelecimento de altas taxas de viabilidade, atividade proliferativa e metabólica após o cultivo in vitro e criopreservação, além da sincronia do ciclo celular antes da reconstrução do embrião clone. Os fibroblastos e outras células derivadas da pele oriundas da região auricular de fetos ou animais adultos representam a fonte de fácil manipulação e mais adequada para a obtenção de carioplastos. Adicionalmente, avanços nesta área têm mostrado a possibilidade da produção de células pluripotentes a partir de células somáticas modificadas. Assim, o objetivo deste manuscrito é realizar uma revisão sobre os aspectos práticos da obtenção e estabelecimento de células doadoras de núcleo derivadas da pele para a reconstrução embrionária, descrevendo os fatores relacionados em cada procedimento que interferem no resultado final.(AU)

The success of assisted reproductive biotechnology, especially cloning by somatic cell nuclear transfer, involves multiple factors and each of these may influence the final efficiency. In general, the protocols for the isolation, characterization and cryopreservation of cells of the species to be cloned consist in initial step for obtaining viable clone offspring, and contribute to other studies on cell biotechnology. In this context, the success of this step may be achieved by association of the choice of cell type, establishment of high rates of viability, metabolic and proliferative activities after in vitro culture and cryopreservation, and cell cycle synchronization prior to embryo reconstruction. Fibroblasts and other skin derived-cells from ear region of fetal or adult animals represent a source of easy handling and better appropriate for obtaining caryoplasts. Additionally, advances in this area are showing the possibility of induced pluripotent cells from modified somatic cells. Thus, the aim of this manuscript is to describe the practical aspects of obtaining and establishing skin-derived donor cells for embryo reconstruction, with emphasis to the factors listed in each procedure that affect the final outcome.(AU)

Isolamento e caracterização de células doadoras derivadas da pele para a transferência nuclear / Isolation and characterization of skin-derived donor cells for nuclear transfe

O sucesso das biotecnologias reprodutivas assistidas, especialmente a clonagem por transferência nuclear de células somáticas, envolve múltiplos fatores e cada um destes pode influenciar a eficiência final. Em geral, os protocolos de isolamento, caracterização e criopreservação de células das espécies a serem clonadas constituem a etapa inicial para a obtenção de descendência clone viável, além de contribuir para outros estudos relacionados à biotecnologia celular. Neste contexto, o sucesso desta etapa pode ser alcançado pela associação da escolha do tipo celular, estabelecimento de altas taxas de viabilidade, atividade proliferativa e metabólica após o cultivo in vitro e criopreservação, além da sincronia do ciclo celular antes da reconstrução do embrião clone. Os fibroblastos e outras células derivadas da pele oriundas da região auricular de fetos ou animais adultos representam a fonte de fácil manipulação e mais adequada para a obtenção de carioplastos. Adicionalmente, avanços nesta área têm mostrado a possibilidade da produção de células pluripotentes a partir de células somáticas modificadas. Assim, o objetivo deste manuscrito é realizar uma revisão sobre os aspectos práticos da obtenção e estabelecimento de células doadoras de núcleo derivadas da pele para a reconstrução embrionária, descrevendo os fatores relacionados em cada procedimento que interferem no resultado final.

The success of assisted reproductive biotechnology, especially cloning by somatic cell nuclear transfer, involves multiple factors and each of these may influence the final efficiency. Ingeneral, the protocols for the isolation, characterization and cryopreservation of cells of the species to be cloned consist in initial step for obtaining viable clone offspring, and contribute to other studies on cell biotechnology. In this context, the success of this step may be achieved by association of the choice of cell type, establishment of high rates of viability, metabolic and proliferative activities after in vitroculture and cryopreservation, and cell cycle synchronization prior to embryo reconstruction. Fibroblasts and other skin derived-cells from ear region of fetal or adult animals represent a source of easy handling and better appropriate for obtaining caryoplasts. Additionally, advances in this area are showing the possibility of induced pluripotent cells from modified somatic cells. Thus, the aim of this manuscript is to describe the practical aspects of obtaining and establishing skin-derived donor cells for embryo reconstruction, with emphasis to the factors listed in each procedure that affect the final outcome.

Human beta defensin-4 and keratinocyte growth factor gene expression in cultured keratinocyte and fibroblasts of burned patients

To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients.(AU)

Keratinocyte growth factor, interleukins (1 beta, 6, 8, 10, 12), and tumor necrosis factor alpha in culture medium of dermal fibroblast of burned patients

PURPOSE: To evaluate the level of cytokines and keratinocyte growth factor (KGF) or Fibroblast Growth Factor 7 (FGF-7) in the culture medium of cultured human dermal fibroblasts from patients with large burn in comparison to small burn. METHODS: Fibroblasts of 10 patients (four large burns, four small burns and two controls) were initiated by the enzymatic method using collagenase. Cytokines and KGF in the supernatant of the culture medium was measured by, respectively, flow cytometry using Cytometric Bead Array Human Inflammation kit (CBA, BD Biosciences, USA) and the enzyme immunoassay method using the Quantikine (r) Human KGF. The experiments were performed in triplicate. RESULTS: The expression of IL-12 protein in patients with large burns showed a tendency to increase. IL- 6, IL- 10, and IL- 1beta were observed no difference. For IL - 8, TNF - alpha and KGF was observed a significant difference between the expression in large and small burned patient. CONCLUSION: That IL-8, TNF-alpha and KGF showed higher expression in cultured fibroblasts of large burned patients.(AU)

Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.(AU)

Keratinocyte growth factor, tumor necrosis factor-alpha and interleukin-1 beta gene expression in cultured fibroblasts and keratinocytes from burned patients

PURPOSE: To evaluate the gene expression of KGF, TNF-alpha and IL-1 beta in skin fibroblasts and keratinocytes cultured from burned patients. METHODS: Three patients with large burns and three patients with small burns, as well as two controls, were included. The cell culture was initiated by the enzymatic method. After extraction and purification of mRNA, qPCR was used to assess the gene expression of KGF, TNF-alpha and IL-1 beta. RESULTS: The expression of KGF was increased on average 220-fold in large burns and 33.33-fold in small burns in fibroblasts, and 11.2-fold in large burns and 3.45-fold in small burns in keratinocytes compared to healthy patients (p<0.05). Expression of TNF-alpha was not observed. IL-1 beta is down-regulated in fibroblasts of burned patients, and much more repressed in small burns (687-fold, p<0.05). In keratinocytes, the repression of IL-1 beta expression occurs in patients with small burns (28-fold), while patients with large burns express this gene intensively (15-fold). CONCLUSIONS: The study showed a quantitative pattern in the expression of KGF gene, which is more expressed according to the size of the burn. TNF-alpha was not expressed. A qualitative pattern in the expression of IL-1 beta gene was demonstrated.(AU)

Isolamento e análise do potencial de células tronco MUSE (multilineage-differentiating stress-enduring) derivadas de bovinos e bubalinos adultos

As pesquisas com células tronco são importantes por causa das variadas aplicações. As células tronco adultas são de fácil obtenção, porém possuem um menor potencial. A pele é o maior órgão do corpo e são variadas as células que compõe os tecidos, dentre elas, fibroblastos e SKPs estão em maior quantidade na região da derme. SKPs são células multipotentes encontradas na região da derme que possuem o potencial de diferenciação em diversas células, como da linhagem neuronal e células germinativas. Estudos recentes relataram que é possível obter células na pele com capacidade diferenciação nas três linhagens germinativas e marcadores específicos como SSEA-3 e CD105 e que essas células podem ser separadas por meio de estresse com tripsina, são chamadas de MUSE. Assim, o objetivo deste trabalho é estabelecer células MUSE e gerar SKPs a partir de fibroblastos fetais de bovinos, visando futura aplicação em biotecnologias reprodutivas. Para isso, fibroblastos fetais bovinos foram submetidos a estresse de 3 horas a 37ºC, seguido de 18 horas a 4ºC e cultivados em meio DMEM/F12 mais fatores de crescimento e B27 durante 3 e 6 dias. As avaliações foram quantificação do numero de esferas formadas, ensaio de fosfatase Alcalina e imonocitoquímica. Os resultados mostraram que as células MUSE foram estabelecidas e geraram esferas a partir de fibroblastos fetais bovinos com marcação positivas células multipotentes. Porém, foram negativas para fosfatase alcalina que é marcador de pluripotência, além de serem de menor qualidade quando comparadas ao grupo controle, indicando que não são indicadas para formação de SKPs

Stem cell research is important because of the varied applications. At adult stem cells are easy to obtain, but have a lower potential. The skin is the largest organ of the body and the cells that make up the tissues are varied, fibroblasts and SKPs are in greater quantity in the region of the dermis. SKPs are cells multipotents found in the dermis region that have the potential of differentiation in cells, such as neuronal lineage and germ cells. Recent studies reported that it is possible to obtain cells in the skin with differentiation capacity in the three lineages specific markers such as SSEA-3 and CD105 and that such cells can be separated by trypsin stress, are called MUSE. Thus, the of this work is to establish MUSE cells and to generate SKPs from fetal fibroblasts of for future application in reproductive biotechnologies. For this, fibroblasts were subjected to stress of 3 hours at 37ºC, followed by 18 hours at 4ºC and cultured in DMEM / F12 medium plus growth factors and B27 for 3 and 6 days. At quantification of the number of beads formed, Alkaline phosphatase assay and immunocytochemistry. The results showed that MUSE cells were established and generated beads from bovine fetal fibroblasts with positive cell marking multipotent However, they were negative for alkaline phosphatase which is a marker of pluripotency, besides being of lower quality when compared to the control group, indicating that they are not indicated for formation of SKPs.