Research, development and application of collagen: a review / 生物工程学报

Collagen, which widely exists in skin, bone, muscle and other tissues, is a major structural protein in mammalian extracellular matrix. It participates in cell proliferation, differentiation, migration and signal transmission, plays an important role in tissue support and repair and exerts a protective effect. Collagen is widely used in tissue engineering, clinical medicine, food industry, packaging materials, cosmetics and medical beauty due to its good biological characteristics. This paper reviews the biological characteristics of collagen and its application in bioengineering research and development in recent years. Finally, we prospect the future application of collagen as a biomimetic material.

A morphometric analysis of pathological alterations in hemorrhoidal disease versus normal controls: a controlled trial

Objective: Until today, the true pathophysiology of hemorrhoidal disease (HD) has not yet been unraveled. More and more evidence guides us towards the hypothesis that reduced connective tissue stability is associated with a higher incidence of hemorrhoids. The present study aimed to compare the quantity and quality of collagen, and vessel morphometrics, in patients with symptomatic HD compared with normal controls. Methods: Twenty-two samples of grade III and grade IV HD tissue from patients undergoing a hemorrhoidectomy between January 2004 and June 2015 were included in the study group. Samples of 15 individuals without symptomatic HD who donated their body to science and died a natural death served as controls. The quantity and quality of anal collagen, and anal vessel morphometrics were objectified. The quality of collagen was subdivided in young (immature) and old (mature) collagen. Results: Patients with HD had an increased percentage of total anal collagen (62.1 ± 13.8 versus 18.7 ± 14.5%; p = 0.0001), a decreased percentage of young collagen (0.00009 ± 0.00008 versus 0.0008 ± 0.0008%; p = 0.001), and a smaller surface area of the anal vessels (795.1 ± 1215.9 micrometre2 versus 1219.0 ± 1976.1; p = 0.003) compared with controls. The percentage of old collagen did not differ between the control and study groups (0.588 ± 0.286% versus 0.389 ± 0.242%; p = 0.06). Conclusion: The outcomes of the present study suggest that alterations in anal collagen composition may play a role in the formation of hemorrhoids. (AU)

Meat quality of the Berrichon du Cher lambs and its cross-breeds with a native breed / Qualidade da carne dos cordeiros Berrichon du Cher e seus cruzamentos com raça nativa

This study evaluated the meat of lambs from crossbreeding of Polish lowland sheep, heather and rams of the Berrichone du Cher breed (B). The lambs were kept in a medium-intensive feeding system. During fattening, they received haylage and grass hay with the addition of clover, and a mixture of cereal grains with the addition of minerals and vitamins. During the fattening period, all feeds were given at will. The slaughter and cutting of the carcasses were performed in accordance with the technology applicable in the meat industry. In the longissimus lumborum muscle, the content of dry matter, total protein, total fat and ash was determined. In addition, the pH value of 45 minutes and 24 hours after slaughter, as well as the parameters of colour and tenderness of the meat were measured. The content of collagen protein, cholesterol and selected fatty acids was also determined. As a result of the conducted analyzes, the influence of the Berrichone du Cher breed on the improvement of quantitative parameters related to the slaughter value and some parameters related to the physicochemical composition of the hybrid meat was reported.

O objetivo deste estudo foi avaliar como o cruzamento da raça Berrichon du Cher com raças nativas de ovelhas polonesas de baixa altitude e ovelhas de charneca polonesa afeta os parâmetros qualitativos da carne .O maior teor de proteína (19,86 ± 0,36%, P <0,05) em cordeiros foi encontrado no grupo Berrichon du Cher e no grupo de cordeiros ovinos (19,23 ± 0,67%, P <0,05). Por outro lado, o maior teor de colágeno total (1,10 ± 0,21%) e a maior sensibilidade do tecido muscular (10,69 ± 0,74 N cm-2), que também apresentaram o menor diâmetro das fibras musculares (24,01 ± 4,62 µm, P < 0,01) foi encontrado nos cordeiros mestiços Berrichon du Cher e Polish Heath Sheep. Tanto a raça quanto o gênero afetaram significativamente o teor de colesterol na carne de cordeiro e o maior teor de colesterol foi encontrado na carne dos cordeiros Berrichon du Cher. Seu menor teor foi demonstrado na carne de Ovinos da Baixada Polonesa com a diferença de 9,1 mg 100g-1 de tecido muscular. Um dos níveis mais baixos do isômero CLA (C 18: 2 cis-9 trans-11) também foi encontrado no grupo dos cordeiros Berrichon du Cher. Este isômero evita a formação de colesterol. Dependência semelhante em relação ao gênero também foi observada. Os resultados obtidos indicam que as raças nativas são utilizadas em cruzamentos com a raça Berrichon du Cher destinada à engorda, principalmente para melhorar parâmetros quantitativos relacionados ao valor de abate e parâmetros selecionados de propriedades físicas e químicas da carne ovina.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Collagen quantification in peri-implant soft tissues in human peri-implantitis lesions / Cuantificación de colágeno en tejidos blandos periimplantarios en lesiones de periimplantitis en humanos

SUMMARY: Peri-implantitis is an inflammatory lesion of bacterial etiology characterized by inflammation of the mucosa and bone loss. Chronic inflammation is characterized by neovascularization and collagen neoformation. Mast cells have been shown to participate in the inflammatory process by releasing mediators and proteases such as chymase and tryptase, important in the collagen neoformation process. Although a higher percentage of collagen has been described in periodontal disease, the literature is scarce about the percentage of collagen in peri-implantitis. The aim of this study was to quantify the percentage of collagen fibers present in the peri- implant soft tissue of patients with peri-implantitis lesions. A descriptive observational cross-sectional study was performed. Samples of peri-implant soft tissue were collected from eleven patients with peri-implantitis and then processed by Masson's Trichrome Technique. In microscopic analysis, collagen fibers were observed in all samples, with an average percentage of 39.89 %, standard deviation of 17.02 %, with a minimum value of 8.99 % and a maximum value of 75.65 % density. From these results, it can be concluded that in human peri-implantitis lesions with bone loss greater than 50 %, there is an important percentage of collagen fibers, which is interpreted as connective tissue in a permanent process of reparative response, in the presence of inflammatory infiltrate.

RESUMEN: La periimplantitis es una lesión inflamatoria de etiología bacteriana caracterizada por inflamación de la mucosa y pérdida ósea. La inflamación crónica se caracteriza por neovascularización y neoformación de colágeno. Se ha demostrado que los mastocitos participan en el proceso inflamatorio liberando mediadores y proteasas como quimasa y triptasa, importantes en el proceso de neoformación del colágeno. Aunque se ha descrito un mayor porcentaje de colágeno en la enfermedad periodontal, la literatura sobre el porcentaje de colágeno en la periimplantitis es escasa. El objetivo de este estudio fue cuantificar el porcentaje de fibras de colágeno presentes en el tejido blando periimplantario de pacientes con lesiones de periimplantitis. Se realizó un estudio observacional descriptivo transversal. Se recogieron muestras de tejido blando periimplantario de once pacientes con periimplantitis y luego se procesaron mediante la técnica tricrómica de Masson. En el análisis microscópico, se observaron fibras de colágeno en todas las muestras, con un porcentaje promedio de 39,89 %, desviación estándar de 17,02%, con un valor mínimo de 8,99 % y un valor máximo de 75,65% de densidad. De estos resultados se puede concluir que en las lesiones de periimplantitis humana con pérdida ósea superior al 50 %, existe un porcentaje importante de fibras de colágeno, que se interpreta como tejido conectivo en un proceso permanente de respuesta reparadora, en presencia de infiltrado inflamatorio.

Collagen I and III ratios and tenacity of rats muscle injured and treated with platelet-rich plasma / Relaciones de colágeno I y III y tenacidad del músculo de rata lesionado y tratado con plasma rico en plaquetas

SUMMARY: Severe muscle injuries are common in accidents and have a delayed recovery of muscle integrity. In these cases, muscle suture surgery is the standard treatment. However, Platelet Rich Plasma (PRP), has been widely used in orthopedic injuries due to its growth factors. Thus, the objective of the study will be to analyze the association of suture and PRP techniques in the collagen and tenacity of the injured muscle. Were used seventy rats, divided into five groups: control (C), injury control (CI), injury and suture (IS), injury and PRP (IP), injury, suture, and PRP (ISP). Were sectioned approximately 50 % of the width and 100 % of the thickness of the gastrocnemius muscle. The homologous PRP was applied 24h after the injury. On the 7th day after the injury, the animals were euthanized and their muscles subjected to mechanical testing to measure tenacity or collagen analysis to calculate the ratio between type I and III collagen. The results show a significant decrease (p <0.05) in the values of the relationship between collagens in all injured groups (CI, IS, IP, ISP) compared to group C. In injured groups, the tenacity was significantly (p <0.05) reduced compared to the control group, with no observed difference between treatments and injured groups. The amount of collagen in the injured area has increased, but it did not affect the tenacity of the muscles, which was reduced.

RESUMEN: Las lesiones musculares graves son comunes durante los accidentes y la integridad del músculo está sujeta a una larga recuperación. En esos casos la cirugía, para la sutura del músculo, es el tratamiento común, no obstante el plasma rico en plaquetas (PRP) ha sido utilizado recientemente en lesiones ortopédicas, debido a sus factores del crecimiento. El objetivo del estudio fue analizar la asociación de las técnicas de sutura y PRP en la histología y tenacidad de músculo lesionado. Fueron utilizadas 70 ratas distribuidas en cinco grupos: control (C), control lesión (CL), lesión y sutura (LS), lesión y PRP (LPRP), lesión, sutura y PRP (LSPRP). Aproximadamente en la lesión, el 50 % de la longitud y el 100 % del espesor del músculo gastrocnemio fueron seccionados. El PRP homólogo fue aplicado 24 horas después de la lesión. En el 7º día después de la lesión los animales fueron eutanasiados y las muestras fueran sometidas al ensayo mecánico para la medición de la tenacidad y análisis del colágeno, para realizar el cálculo de la relación entre los colágenos I y III. Los resultados demostraron una reducción significativa (p<0,05) en los valores de la relación entre los colágenos en todos los grupos lesionados en relación al grupo C. La tenacidad fue (p<0,05) reducida significativamente en los grupos lesionados en relación al grupo control, sin diferencia entre los tratados. En la lesión muscular hubo disminución de los valores de colágeno, aunque en los tratamientos se observó elevación de la cantidad de colágeno en la área lesionada, esta no tuvo efecto en la tenacidad de los músculos que fue disminuida en la lesión.

Types I and III parotid collagen variations and serum biochemical parameters in obese rats exposed to monosodium glutamate / Variaciones de colágeno parotídeo tipos I y III y parámetros bioquímicos séricos en ratas obesas expuestas a glutamato monosódico

SUMMARY: The objective of this study was to describe the effects of monosodium glutamate on the collagen of the parotid gland in an obesity model. 18 newborn male Sprague Dawley rats were used (first control group; second group of MSG1: 4 mg/g of monosodium glutamate weight, 5 doses, and third group of MSG2: 4 mg/g of monosodium glutamate, 5 doses, maintained for 8 and 16 weeks respectively). The content and type of collagen were analyzed, in addition to the levels of cholesterol, glucose, triglycerides and uric acid. Monosodium glutamate produced an increase in the obesity rates of the MSG2 group, in addition to an increase in blood cholesterol, glucose and uric acid levels compared to the control group. Type III collagen in the MSG2 group showed a statistically significant increase. Monosodium glutamate induced obesity, in addition to an increase in type III collagen fibers.

RESUMEN: El objetivo de este estudio fue describir los efectos del glutamato monosódico sobre el colágeno de la glándula parótida en un modelo de obesidad. Se utilizaron 18 ratas Sprague Dawley machos recién nacidas (primer grupo control; segundo grupo MSG1: 4 mg/g de peso de glutamato monosódico, 5 dosis, y tercer grupo MSG2: 4 mg/g de glutamato monosódico, 5 dosis, mantenidas durante 8 y 16 semanas respectivamente). Se analizó el contenido y el tipo de colágeno, además de los niveles de colesterol, glucosa, triglicéridos y ácido úrico. El glutamato monosódico produjo un aumento en las tasas de obesidad del grupo MSG2, además de un aumento en los niveles de colesterol en sangre, glucosa y ácido úrico en comparación con el grupo control. El colágeno tipo III en el grupo MSG2 mostró un aumento estadísticamente significativo. La obesidad inducida por glutamato monosódico, además de un aumento en las fibras de colágeno tipo III.

Although with intact mucosa at colonoscopy, chagasic megacolons have an overexpression of Gal-3 / Embora com mucosa íntegra à colonoscopia, megacólons chagásicos apresentam superexpressão de Gal-3

ABSTRACT Objective To evaluate the density of anti-galectin-3-immunostained cells, collagen percentage, mast cell density and presence of pathological processes in intestinal muscle biopsies of patients. Methods Thirty-five patients who underwent intestinal biopsy were selected from 1997 to 2015. Patients were divided into three groups: chagasic patients with mucosal lesion (n=13), chagasic patients with intact mucosa (n=12) and non-chagasic patients with no mucosal lesion (n=10). Histological processing of the biopsied fragments and immunohistochemistry for galectin-3 were performed. Additional sections were stained with hematoxylin and eosin to evaluate the general pathological processes, picrosirius for evaluation of collagen and toluidine blue to evaluate the mast cell density. Results Patients of mucosal lesion group had a significantly higher frequency of ganglionitis and myositis when compared to the chagasic patients with intact mucosa and non-chagasic group. The density of anti-galectin-3-immunostained cells was significantly higher in the chagasic patients with intact mucosa group when compared to the non-chagasic group. The group of chagasic patients with intact mucosa presented a higher percentage of collagen in relation to the patients with mucosal lesion and to the non-chagasic group, with a significant difference. There was no significant difference in mast cell density among the three groups. Conclusion The higher density of anti-galectin-3-immunostained cells in patients in the chagasic patients with intact mucosa group suggested the need for greater attention in clinical evaluation of these patients, since this protein is associated with neoplastic transformation and progression.

RESUMO Objetivo Avaliar a densidade de células imunomarcadas por anti-galectina-3, a percentagem de colágeno, a densidade de mastócitos e a presença de processos patológicos na musculatura intestinal de pacientes biopsiados. Métodos Foram selecionados 35 pacientes submetidos à biópsia de intestino entre 1997 a 2015. Os pacientes foram divididos em três grupos: chagásicos com lesão de mucosa (n=13), chagásicos com mucosa íntegra (n=12) e não chagásicos sem lesão de mucosa (n=10). Foram realizados processamento histológico dos fragmentos biopsiados e imunohistoquímica para galectina-3. Cortes adicionais foram corados por hematoxilina e eosina, para avaliar os processos patológicos gerais, pelo picrosírius, para avaliação do colágeno, e pelo azul de toluidina, para avaliar a densidade de mastócitos. Resultados Os pacientes do grupo chagásicos com lesão de mucosa apresentaram frequência significativamente maior de ganglionite e miosite quando comparados aos dos grupos chagásico com mucosa íntegra e não chagásicos. A densidade das células imunomarcadas por anti-galectina-3 foi significativamente maior no grupo chagásicos com mucosa íntegra quando comparada ao grupo não chagásico. O grupo de chagásicos com mucosa íntegra apresentou maior percentagem de colágeno em relação aos grupos chagásicos com mucosa lesada e ao grupo de não chagásicos, com diferença significativa. Não houve diferença significativa com relação à densidade de mastócitos entre os três grupos. Conclusão A maior densidade de células imunomarcadas por anti-galectina-3 nos pacientes do grupo chagásico com mucosa íntegra sugere a necessidade de maior atenção na avaliação clínica desses pacientes, uma vez que essa proteína está associada com transformação e progressão neoplásica.

Expression of stromal elements of prostatic adenocarcinoma in different gleason scores

Abstract Purpose: To quantify and compare the expression of stromal elements in prostate adenocarcinoma of different Gleason scores with non-tumor area (control). Methods: We obtained 132 specimens from samples of prostate peripheral and transition zone. We analyzed the following elements of the extracellular matrix: collagen fibers, elastic system, smooth muscle fibers and blood vessels. The tumor area and non-tumor area (control) of the TMA (tissue microarray) were photographed and analyzed using the ImageJ software. Results: The comparison between the tumor area and the non-tumor area showed significant differences between stromal prostate elements. There was an increase of collagen fibers in the tumor area, mainly in Gleason 7. Elastic system fibers showed similar result, also from the Gleason 7. Blood vessels showed a significant increase occurred in all analyzed groups. The muscle fibers exhibited a different behavior, with a decrease in relation to the tumor area. Conclusions: There is a significant difference between the extracellular matrix in prostate cancer compared to the non-tumor area (control) especially in Gleason 7. Important modifications of the prostatic stromal elements strongly correlate with different Gleason scores and can contribute to predict the pathological staging of prostate cancer.

Miopatia White Striping em diferentes linhagens de frangos de corte e suas consequências sobre a composição e a qualidade da carne / The White Striping myopathy in different broiler strains and its influence on composition and meat quality

Avaliou-se a incidência da miopatia White Striping (WS) em três linhagens de frangos de corte machos e suas consequências sobre a composição química e a qualidade física da carne. Para determinação das porcentagens de incidência dos diferentes graus da miopatia WS (normal, moderada e severa) nas linhagens, foi utilizado um delineamento inteiramente ao acaso (DIC), com três tratamentos (linhagens A, B e C) e cinco repetições de 300 peitos cada. Para as variáveis químicas e físicas, utilizou-se um DIC em esquema fatorial 3 x 3 (três linhagens x três níveis de WS), com 15 repetições cada. Os dados foram submetidos à análise de variância e, em casos de diferenças significativas, utilizou-se o teste de Tukey (P≤0,05). As aves da linhagem A apresentaram 84,81% de peitos classificados como normais e 9,91% de peitos classificados como moderados, valores respectivamente maiores e menores que nas duas demais linhagens. Os peitos classificados com miopatia WS severa foram mais pesados e apresentaram maiores porcentagens de lipídios e umidade, e menor concentração de proteína bruta. As concentrações de colágeno total, termo solúvel e insolúvel variaram com o nível de acometimento por WS nos filés, com comportamento específico para cada linhagem.(AU)

The incidence of the White Striping (WS) myopathy in three male broiler strains and its consequences on the chemical composition and physical quality of the meat was evaluated. A completely randomized design (DIC) with three treatments (strains A, B and C) and five replicates of 300 breasts each were used to determine the incidence rates of the different degrees of WS myopathy (normal, moderate and severe) in each strain. For the chemical and physical variables, a 3 X 3 factorial scheme was used (three strains X three WS levels), with 15 replicates. The data were submitted to ANOVA and in cases of significant differences the Tukey test (P< 0.05) was used.The A strain chickens presented 84.81% of breasts classified as normal and 9.91% of breasts classified as moderate, respectively higher and lower values than in the other two strains. Breasts classified with severe WS myopathy were heavier and had higher percentages of lipids and moisture, and lower concentration of crude protein. However, the concentrations of total, soluble and insoluble collagen had interaction effects between lineage and WS level in the fillets, and therefore, the same behavior was not observed in the samples of the different strains.(AU)

Efeito do congelamento por três meses sobre a coloração, gordura e concentração de colágeno da carne de peito de frangos de corte acometidos pela miopatia White Striping / Effect of freezing for three months on the color, fat and collagen concentration of breast meat from broilers affected by White Striping myopathy

Foram utilizadas amostras de peito de frangos de corte acometidos pela miopatia “White Striping” (estrias brancas em seus diferentes graus de severidade) para análise de coloração (L*, a* e b*), lipídeos totais e concentração de colágeno. Amostras de filés acometidos pela miopatia foram avaliadas logo após o abate e outras foram armazenadas congeladas (-20°C) durante três meses. Com a severidade da miopatia ocorre o aumento da concentração de gordura e, consequentemente, alteração da coloração da superfície do peito, além da redução do percentual de colágeno. O congelamento da carne de peito por três meses provoca alterações de cor da superfície dos filés. A produção de exsudato, oriunda do descongelamento, resulta no aumento da concentração de componentes como colágeno solúvel e lipídeos.

Evaluation of Collagen Fibers, MMP2, MMP9, 8-OHdG and Apoptosis in the Aorta of Ovariectomized LDL Knockout Mice Submitted to Aerobic Exercise / Avaliação de Fibras de Colágeno, MMP2, MMP9, 8-OH-Dg e Apoptose na Aorta de Ratas LDL Nocaute Ovariectomizadas Submetidas a Exercício Aeróbico

Abstract Background: In menopause, there is greater cellular exposure to oxidative stress, related to the decreased antioxidative effects of estrogen. These metabolic changes favor the progression of cardiovascular diseases, such as atherosclerosis. Abnormal function of the aorta - the most important artery - is associated with many cardiovascular diseases. Collagen, especially types I and III, is one of the most important aortic wall components and it can be affected by many factors, including menopause. The 8-OHdG is one of the main markers of DNA oxidative damage induced by reactive oxygen species (ROS). Objective: We aimed to investigate effects of moderate aerobic training on the ascending aorta of LDL-knockout (LDL-KO) and ovariectomized female mice. Methods: A total of 15 C57BL/6 mice and 15 LDL-KO mice were divided into experimental groups. The thickness and volume density of types I and III collagen fibers were performed by morphoquantitative analysis, whereas the MMP-2 and MMP-9 and 8-OHdG were detected by immunohistochemistry and apoptosis was detected by the TUNEL assay. The significance level for all tests was p < 0.05. Results: Exercise causes an increase in the thickness of the aorta in LDL-KO groups, particularly accentuated in the ovariectomized groups. The type I collagen fibers showed an increase in volume density influenced by training in both Control groups and in the LDL-KO group. Type III collagen density decreased in both groups. The MMP-2 showed moderade immunostaining in the tunica media in LDL-KO groups, which did not occur in the control groups and the MMP-9 stained irregularly in all tissues. The marker 8-OhdG was stronger in the exercise training groups. Additionally, the ovariectomy, the exercise training and the LDL-KO treatments increased apoptosis. Conclusion: These results suggest that moderate-intensity aerobic exercise in ovariectomized mice associated to an increase in LDL rate possibly increases oxidative stress and apoptosis induction.

Resumo Fundamento: Na menopausa, há maior exposição celular ao estresse oxidativo, relacionada à diminuição dos efeitos antioxidantes do estrogênio. Essas alterações metabólicas favorecem a progressão das doenças cardiovasculares, como a aterosclerose. A função anormal da aorta - a artéria mais importante - está associada a muitas doenças cardiovasculares. O colágeno, especialmente os tipos I e III, é um dos mais importantes componentes da parede da aorta e pode ser afetado por muitos fatores, incluindo a menopausa. Por sua vez, 8-OHdG é um dos principais marcadores de danos oxidativos do DNA induzidos por espécies reativas de oxigênio (EROS). Objetivo: Investigar os efeitos do treinamento aeróbico moderado na aorta ascendente de camundongos fêmeas, nocaute para LDL (LDL-KO) e ovariectomizadas. Métodos: Um total de 15 animais C57BL/6 e 15 animais LDL-KO foram divididos em grupos experimentais. A espessura e a densidade de volume das fibras de colágeno tipos I e III foram realizadas por análise morfoquantitativa; MMP-2 e MMP-9 e 8-OHdG foram detectadas por imunohistoquímica; e a apoptose foi detectada pelo ensaio TUNEL. O nível de significância adotado para todos os testes realizados foi p < 0,05. Resultados: o exercício causa aumento da espessura da aorta em grupos LDL-KO, particularmente acentuada em grupos ovariectomizados. As fibras de colágeno de tipo I mostraram aumento da densidade de volume influenciado pelo treinamento em animais controle e LDL-KO. A densidade do colágeno tipo III diminuiu em ambos os grupos. A MMP-2 mostrou imunomarcação moderada na túnica média em animais LDL-KO; em grupos controle, a MMP-9 marcou irregularmente em todos os tecidos. O marcador 8-OHdG foi mais forte nos grupos de treinamento de exercícios. Além disso, a ovariectomia, o treinamento físico e os tratamentos de LDL-KO aumentaram a apoptose. Conclusão: Esses resultados sugerem que exercícios aeróbicos de intensidade moderada em camundongos ovariectomizados associados ao aumento da taxa de LDL, possivelmente, aumentam o estresse oxidativo e a indução da apoptose.

Swimming training potentiates the recovery of femoral neck strength in young diabetic rats under insulin therapy

OBJECTIVE: To test whether swimming training benefits femoral neck strength in young diabetic rats under insulin therapy. METHODS: A total of 60 male Wistar rats (age: 40 days) were divided equally into the following six groups: control sedentary, control exercise, diabetic sedentary, diabetic exercise, diabetic sedentary plus insulin and diabetic exercise plus insulin. Diabetes was induced with a unique intraperitoneal injection (60 mg/kg body weight) of streptozotocin. Seven days after the injection and after 12 hours of fasting, the animals with blood glucose levels ≥300 mg/dL were considered diabetic. Seven days after the induction of diabetes, the animals in the exercise groups were subjected to progressive swimming training (final week: 90 min/day; 5 days/week; 5% load) for eight weeks. The animals in the insulin groups received a daily dose of insulin (2-4 U/day) for the same period. RESULTS: Severe streptozotocin-induced diabetes reduced the structural properties of the femoral neck (trabecular bone volume, trabecular thickness and collagen fiber content). The femoral neck mechanical properties (maximum load and tenacity) were also impaired in the diabetic rats. Insulin therapy partially reversed the damage induced by diabetes on the structural properties of the bone and mitigated the reductions in the mechanical properties of the bone. The combination of therapies further increased the femoral neck trabecular bone volume (∼30%), trabecular thickness (∼24%), collagen type I (∼19%) and type III (∼13%) fiber contents, maximum load (∼25%) and tenacity (∼14%). CONCLUSIONS: Eight weeks of swimming training potentiates the recovery of femoral neck strength in young rats with severe streptozotocin-induced diabetes under insulin therapy.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

Effects of tadalafil to prevent injury on corpus cavernosum after vascular or nervous peri-prostatic bundle injury. Experimental model in rats

Abstract Purpose: To evaluate the effects of tadalafil (TD) in preventing histological alterations of the corpus cavernosum caused by isolated lesions of cavernous nerve (ILCN) and artery (ILCA) in rats. Methods: Fifty male Wistar rats were randomly assigned in five groups: G1: control; G2: bilateral ILCN; G3: bilateral ILCA; G4: ILCN+TD; G5: ILCA+TD. The cavernous bodies were submitted to histomorphometry, immunohistochemistry and biochemical analysis. Results: Nerve density was significantly higher in G2 and G4 compared to control (22.62±2.84 and 19.53±3.47 vs. 15.72±1.82; respectively, p<0.05). Smooth muscle density was significantly lower in G2 and G3 in comparison to G1 (12.87±1.90 and 18.93±1.51 vs. 21.78±1.81, respectively; p<0.05). A significant decrease in the sinusoidal lumen area was observed in G2 compared to controls (5.01±1.62 vs. 9.88±3.66, respectively; p<0.05) and the blood vessel density was increased in G2 and G3 (29.32±4.13 e 20.80±2.47 vs. 10.13±2.71, p<0.05). Collagen density was higher in G3 compared to G1 (93.76±15.81 vs. 64.59±19.25; p<0.05). Conclusions: Histomorphometric alterations caused by ILCN were more intense than those produced by vascular injury, but the collagen analyses showed more fibrosis in animals with ILCA. TD was effective in preventing the majority of the alterations induced by the periprostatic bundle injury.

Muscle strength training is better than the use of growth hormone (GH) in bone health of Wistar rats / Entrenamiento de la fuerza muscular es mejor que el uso de la hormona del crecimiento (GH) en salud ósea del ratas Wistar

SUMMARY: The objective of this study was to evaluate the effects of growth hormone (GH) and muscle strength training (ST) on the composition of bone tissue of Wistar rats through Raman spectroscopy. In total, 40 male rats were randomly distributed into four groups: (N = 10) control (C), control with the application of GH (GHC), strength training (T), and strength training with the application of GH (GHT). The training consisted of four series of 10 water jumps, performed three times a week, with an overload corresponding to 50 % of body weight and duration of four weeks. GH was applied at a dose of 0.2 IU / kg in each animal three times a week and every other day. After four weeks, the animals were euthanized and the right femurs collected for analysis of the bone structure. Raman spectroscopy (ER) was used to observe the following compounds from their respective bands: Calcium Carbonate-Triglycerides (fatty acids) 1073 cm-1, Collagen type I 509 cm-1, Bone-DNA Phosphate (Protein) 589 cm-1, Phosphate Phospholipids 1078 cm-1. For the statistical analysis, the Shapiro-Wilk and ANOVA One-Way variance analysis normality tests were performed, followed by the Tukey post-test. The results showed an increase in the concentrations of calcium carbonate-triglycerides (fatty acids), type I collagen, bone phosphate-DNA (protein), and phosphate phospholipids in all experimental groups, with or without ST and/or GH , But only the isolated training group differed significantly from the control group (P <0.05). It was concluded that all treatments could promote bone tissue gain, however, only the T group demonstrated a significant difference in the mineral compounds analyzed.

RESUMEN: El objetivo del estudio fue avaluar el efecto de la aplicación de la hormona del crecimiento (GH) y entrenamiento de la fuerza muscular (EF) en la composición del tejido óseo de ratas Wistar a partir de la espectroscopía Raman. Fueron utilizadas 40 ratas machos distribuidas de forma aleatoria en cuatro grupos (n=10): control (C), control y aplicación de GH (GHC), entrenamiento de la fuerza muscular (EF) y entrenamiento de la fuerza muscular y aplicación del GH (GHE). El entrenamiento fue consistió en cuatro series de 10 saltos acuáticos, realizados tres veces en la semana, con sobrecarga correspondiente a 50 % de la masa corporal y durante cuatro semanas. El GH fue aplicado en la dosificación de 0,2 UI/kg en cada animal, tres veces en la semana y en días alternados. Después de cuatro semanas, los animales fueran eutanasiados y retirados los fémures derechos para un análisis de la estructura ósea. La espectroscopía Raman fue utilizada para observar los siguientes compuestos a partir de las respectivas bandas: Carbonato de Calcio-Triglicéridos (ácidos grasos) 1073 cm-1, Colágeno Tipo I 509 cm-1, Fosfato Óseo-DNA (Proteína) 589 cm1, Fosfato Fosfolípidos 1078 cm-1. Para el análisis estadístico, fueron realizadas las pruebas Shapiro-Wilk y el análisis de variancia ANOVA One-Way, seguida de test post hoc de Tukey. Los resultados revelaran aumento de la concentración de Carbonato de Calcio-Triglicéridos (ácidos grasos), Colágeno Tipo I, Fosfato Óseo- DNA (Proteína), Fosfato Fosfolípidos en todos los grupos experimentales, asociados o no a la realización del EF y/o aplicación del GH. Además, solamente el grupo EF mostró diferencia significativa del grupo C (p<0,05). Es posible concluir que todos los tratamientos mostraran aumentos en el tejido óseo, sin embargo, solamente el grupo T demostró una diferencia significativa en los compuestos minerales analizados.

Tongue development in stillborns autopsied at different gestational ages / Desenvolvimento da língua em natimortos autopsiados em diferentes idades gestacionais

Abstract Objectives: This study aimed to analyze, through the morphometric method, the perimeter and length of the tongue, the collagen fibers, and the perimeter of blood vessels at different gestational ages and fetal weights. Material and methods: Tongues (n = 55) of stillborns autopsied at 23-40 weeks of gestational age were macroscopically analyzed, and their length and perimeter were measured. Fifty-five tongue fragments were collected through a longitudinal section in the region that accompanies the median lingual sulcus and histologically processed. Slides were stained with picrosirius and immunolabeled with CD31 antibody. Quantification was performed on collagen fibers under polarized light, and on the perimeter of vessels with the CD31. Results: A positive and significant correlation of gestational age with tongue perimeter and length was found. There was a positive and significant correlation between collagen fibers and gestational age, as well as between gestational age and the perimeter of blood vessels. Between collagen fibers and fetal weight, a positive and significant increase was observed. Regarding the correlation between the perimeter of blood vessels and the fetal weight, an increase was observed. Conclusion: As gestational age advances, there is an increase in tongue perimeter and length, in the percentage of collagen fibers, and in vascular perimeter, demonstrating that tongue formation is directly related to tongue growth and development.

Resumo Objetivo: Analisar, por meio do método morfométrico, o perímetro e o comprimento da língua, as fibras de colágeno, o perímetro dos vasos sanguíneos, em idades gestacionais e de acordo com o peso fetal. Materiais e métodos: Línguas (n = 55) de natimortos autopsiados com 23-40 semanas de idade gestacional foram analisadas macroscopicamente, medidas em comprimento e perímetro; 55 fragmentos das línguas foram coletados por meio de uma secção longitudinal na região que acompanha o sulco lingual médio e processados histologicamente. As lâminas foram coloridas com picrosirius e imunomarcadas com o anticorpo CD31. A quantificação foi feita em fibras de colágeno examinadas com microscópio de luz polarizada e o perímetro dos vasos com o CD31. Resultados: Foi encontrada uma correlação positiva e significativa da idade gestacional com o perímetro e o comprimento da língua. Houve uma correlação positiva e significativa entre as fibras de colágeno e a idade gestacional; bem como entre a idade gestacional e o perímetro dos vasos sanguíneos; e houve um aumento positivo e significativo entre as fibras de colágeno e o peso fetal. No que diz respeito à correlação entre o perímetro dos vasos sanguíneos e o peso fetal, houve um aumento. Conclusão: Conforme a idade gestacional avança, há um aumento no perímetro e no comprimento da língua, um aumento no percentual de fibras de colágeno e um aumento no perímetro vascular, demonstra que a formação da língua está diretamente relacionada ao crescimento e ao desenvolvimento da língua.

Color-based segmentation vs. stereology: a simple comparison between two semi-automated methods of image analysis for the quantification of collagen / Segmentación basada en colores vs. estereología: una comparación simple entre dos métodos semiautomáticos de análisis de imágenes para la cuantificación del colágeno

Image processing techniques are being widely developed for helping specialists in analysis of histological images and its application is especially useful in obtaining numerical data for the realization of the subsequent statistical analysis. The use of these methods makes the histological analysis of experts more objective and less time-consuming. In this paper we evaluate how well the quantitative methods - color-based image segmentation and stereology - agree on average, and how well they agree for the individuals when they are used to quantify type I and III collagen. Digital images of sections of Salmo salar jaws (5 µm, SiriusRed staining) were analyzed. Collagen quantification was performed by two methods in the same group of images: i) Color Based-Segmentation (K-means algorithm; pixel cluster; ImageJ32 v1.51p) and ii) Stereology (VV; M36; STEPanizer Stereological Tools). They were evaluated 200 images per group. The difference between groups and concordance was analyzed using t-Student (p<0.05) and Blant Altman Comparison Method, respectively. The data analysis of average and individual assessments showed that there is concordance between two methods. In conclusion, stereology and color-based image segmentation are powerful tools which quantify collagen in histological sections.

Las técnicas de procesamiento de imágenes han sido ampliamente desarrolladas para ayudar a los especialistas en el análisis de imágenes histológicas y su aplicación es especialmente útil para obtener datos numéricos para la realización del posterior análisis estadístico. El uso de estos métodos hace que el análisis histológico realizado por expertos sea más objetivo. En este estudio evaluamos qué tan bien coinciden o concuerdan en promedio y entre evaluaciones individuales los métodos cuantitativos "segmentación basada en color" y la "estereología" cuando son utilizados para cuantificar el colágeno tipo I y III. Se analizó imágenes digitales de tejido mandibular de Salmo salar (5 µm, tinción SiriusRed). La cuantificación del colágeno se realizó mediante dos métodos en el mismo grupo de imágenes: i) Segmentación basada en color (algoritmo K-mean, cluster de píxeles, ImageJ32 v1.51p) y ii) Estereología (VV; M36; STEPanizer Stereological Tools). Fueron evaluados 200 imágenes por grupo. Se analizó la diferencia de medias y concordancia entre métodos mediante t-Students (p>0,05) y el Método de Comparación de Blant-Altman. El análisis de los datos de las evaluaciones promedio e individuales demostró que hay concordancia entre los dos métodos. En conclusión, la estereología y la segmentación de imágenes basadas en color son poderosas herramientas útiles para cuantificar colágeno en secciones histológicas.

Pro - inflammatory cytokines and metalloproteinase activation in polypropylene mesh implant in rat subcutaneous tissue

ABSTRACT Aims and Objectives: Polypropylene meshes have been increasingly adopted for correction of pelvic organ prolapse due to its lower recurrence rate when compared to surgeries without meshes. The study of the interaction of these materials with the host tissue may contribute to the development of materials with best biocompatibility and, consequently, less complication rates. Materials and Methods: The present study compares the inflammatory reaction of standard-weight (SW) and lightweight (LW) meshes (72 g/m216g/m2 respectively), implanted in the abdomen of 20 adult rats, which were euthanized in four or 30 days. Quantification of pro-inflammatory markers, IL-1 and TNF-α, and of metalloproteinases, MMP2 and MMP3, were carried out through immunohistochemistry with AxioVision® software. Results: There were no significant differences in the quantification of IL-1 and TNF-α in LW versus SW meshes. However, IL-1 quantification increased along time (30 days >4 days, p=0.0269). Also, MMP-2 quantification was similar to SW and LW and both presented a significant increase along time (30 days >4 days, p <0.0001). MMP-3 quantification also showed no difference between the SW and LW groups, but increased along time (30 days >4 days, p=0.02). Conclusions: Mesh's density did not influence the quantification of pro-inflammatory cytokines IL-1 and TNF-α and metalloproteinases 2 and 3. The increased expression of IL-1, MMP-2 and MMP-3 over time could represent a longstanding inflammatory response after PP mesh implantation. Possibly, the occurrence of adverse events following PP prosthetic implants can be influenced by other factors, not solely related to the amount of implanted material.

Rectal application of argan oil improves healing of colorectal anastomosis in rats

Abstract Purpose: To investigate the possible effects of argan oil on the healing of colorectal anastomoses. Methods: I n Group 1 (sham), laparotomy was performed and the colon was mobilized. In the control (Group 2) and argan oil (Group 3) groups, colonic resection and anastomosis were applied. To the control and sham groups, 2 mL of 0.9% NaCl was administred rectally, and in the argan oil group, 2 mL/day argan oil was applied rectally for 7 days. Results: The mean bursting pressures of the argan oil and sham groups were significantly higher than the values in the control group. A significant difference was determined between the tissue hydroxyproline and prolidase levels of control group and other groups. Histopathologically, argan oil showed significant beneficial effects on colonic wound healing. In the argan oil and sham groups, the tissue malondialdehyde and fluorescent oxidation product levels were found to be lower and total sulfhydryl levels were higher than the control group. Conclusions: The rectally administered argan oil was observed to have significantly ameliorated wound healing parameters and exerted a significant antioxidant effect. This is the first study in the literature about the beneficial effects of argan oil on colorectal anastomoses.