Protective effect of intervention with cannabinoid type-2 receptor agonist JWH133 on pulmonary fibrosis in mice / 中华内科杂志

Objective: JWH133, a cannabinoid type 2 receptor agonist, was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis. Methods: By using a random number generator, 24 C57BL/6J male mice were randomly divided into the control group, model group, JWH133 intervention group, and JWH133+a cannabinoid type-2 receptor antagonist (AM630) inhibitor group, with 6 mice in each group. A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin (5 mg/kg). Starting from the first day after modeling, the control group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution, and the model group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution. The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg, dissolved in physiological saline), and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg) and AM630 (2.5 mg/kg). After 28 days, all mice were killed; the lung tissue was obtained, pathological changes were observed, and alveolar inflammation scores and Ashcroft scores were calculated. The content of type Ⅰ collagen in the lung tissue of the four groups of mice was measured using immunohistochemistry. The levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay (ELISA), and the content of hydroxyproline (HYP) in the lung tissue of the four groups of mice was measured. Western blotting was used to measure the protein expression levels of type Ⅲ collagen, α-smooth muscle actin (α-SMA), extracellular signal regulated kinase (ERK1/2), phosphorylated P-ERK1/2 (P-ERK1/2), and phosphorylated ribosome S6 kinase type 1 (P-p90RSK) in the lung tissue of mice in the four groups. Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagen Ⅰ, collagen Ⅲ, and α-SMA mRNA in the lung tissue of the four groups of mice. Results: Compared with the control group, the pathological changes in the lung tissue of the model group mice worsened, with an increase in alveolar inflammation score (3.833±0.408 vs. 0.833±0.408, P<0.05), an increase in Ashcroft score (7.333±0.516 vs. 2.000±0.633, P<0.05), an increase in type Ⅰ collagen absorbance value (0.065±0.008 vs. 0.018±0.006, P<0.05), an increase in inflammatory cell infiltration, and an increase in hydroxyproline levels [(1.551±0.051) μg/mg vs. (0.974±0.060) μg/mg, P<0.05]. Compared with the model group, the JWH133 intervention group showed reduced pathological changes in lung tissue, decreased alveolar inflammation score (1.833±0.408, P<0.05), decreased Ashcroft score (4.167±0.753, P<0.05), decreased type Ⅰ collagen absorbance value (0.032±0.004, P<0.05), reduced inflammatory cell infiltration, and decreased hydroxyproline levels [(1.148±0.055) μg/mg, P<0.05]. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice, increased alveolar inflammation score and Ashcroft score, increased type Ⅰ collagen absorbance value, increased inflammatory cell infiltration, and increased hydroxyproline levels. Compared with the control group, the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK proteins in the lung tissue of the model group mice increased, while the expression of type Ⅰ collagen, type Ⅲ collagen, and α-SMA mRNA increased. Compared with the model group, the protein expression of α-SMA (relative expression 0.60±0.17 vs. 1.34±0.19, P<0.05), type Ⅲ collagen (relative expression 0.52±0.09 vs. 1.35±0.14, P<0.05), P-ERK1/2 (relative expression 0.32±0.11 vs. 1.14±0.14, P<0.05), and P-p90RSK (relative expression 0.43±0.14 vs. 1.15±0.07, P<0.05) decreased in the JWH133 intervention group. The type Ⅰ collagen mRNA (2.190±0.362 vs. 5.078±0.792, P<0.05), type Ⅲ collagen mRNA (1.750±0.290 vs. 4.935±0.456, P<0.05), and α-SMA mRNA (1.588±0.060 vs. 5.192±0.506, P<0.05) decreased. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group increased the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK protein in the lung tissue of mice, and increased the expression of type Ⅲ collagen and α-SMA mRNA. Conclusion: In mice with bleomycin-induced pulmonary fibrosis, the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition, which alleviated lung fibrosis. The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.

Alteración del colágeno en la Hipertrofia Gingival en pacientes con ortodoncia: caracterización histológica e inmunohistoquímica / Collagen alteration in gingival hypertrophy in orthodontic patients: histological and immunohistochemical characterization

Resumen Introducción: La hipertrofia gingival (HG) es el aumento del volumen de la encía asociado a ciertas enfermedades sistémicas, hereditarias (idiopático), ingesta de algunos medicamentos o a factores locales como el tratamiento ortodóntico, capaz de provocar cambios histológicos en el tejido conectivo gingival. Objetivo: Describir las características histológicas e identificar el colágeno tipo I y tipo III en tejidos gingivales de sujetos con hipertrofia gingival portadores de ortodoncia. Materiales y método: Se diseñó un estudio de casos y controles que incluyó el análisis de biopsias de tejido gingival de 12 pacientes sometidos a cirugías periodontales. La muestra se dividió en dos grupos: individuos sanos (control; n= 6) y pacientes con HG portadores de ortodoncia (pacientes; n= 6). Las muestras fueron procesadas e incluidas en parafina. Las tinciones Masson-Goldner y rojo sirius/verde rápido fueron empleadas. El colágeno tipo I y tipo III fueron identificados mediante inmunohistoquímica con anticuerpos monoclonales. Resultado: En los pacientes con HG portadores de ortodoncia se observó un epitelio hiperplásico y tejido conectivo denso con abundantes fibras de colágeno distribuidos aleatoriamente. La inmunodetención de colágeno tipo I indicó la presencia de abundantes fibras desorganizadas y el colágeno tipo III fue inmunolocalizado subyacente a la membrana basal, vasos sanguíneos y toda la extensión del tejido conectivo de los pacientes con HG con tratamiento ortodóntico. Conclusión: La acumulación de fibras de colágeno, particularmente del colágeno tipo I y tipo III, son hallazgos histológicos que caracterizan la HG en pacientes portadores de ortodoncia. Futuros estudios son necesarios para dilucidar el fenotipo de los fibroblastos gingivales y la probable pérdida homeostática entre la producción y degradación de colágeno en esta patología.

Abstract Introduction: Gingival hypertrophy (GH) is the increase in the volume of the gingiva associated with certain systemic, hereditary (idiopathic) diseases, the intake of some medications or local factors such as orthodontic treatment, capable of causing histological changes in the gingival connective tissue. Objective: To describe the histological characteristics and identify type I and type III collagen in gingival tissues of subjects with gingival hypertrophy wearing orthodontics. Method: A case-control study was designed that included the analysis of gingival tissue biopsies from 12 patients submitted to periodontal surgeries. The sample was divided into two groups: healthy individuals (Control; n= 6) and patients with GH wearing orthodontics (Patients; n= 6). The samples were processed and embedded in paraffin. Masson-goldner and sirius red/fast green stains were used. Type I and type III collagen were identified by immunohistochemistry with monoclonal antibodies. Result: A hyperplastic epithelium and dense connective tissue with abundant randomly distributed collagen fibers were observed in patients with orthodontic GH. Immunodetention of type I collagen indicated the presence of abundant disorganized fibers and type III collagen was inmunolocalized underlying the basement membrane, blood vessels and the entire extension of the connective tissue of patients with GH orthodontic. Conclusion: The accumulation of collagen fibers, particularly type I and type III collagen, are histological findings that characterize GH in orthodontic wearers. Future studies are necessary to elucidate the phenotype of gingival fibroblasts and the probable homeostatic loss between collagen production and degradation in this pathology.

Identification of COL3A1 variants associated with sporadic thoracic aortic dissection: a case-control study / 医学前沿

Thoracic aortic dissection (TAD) without familial clustering or syndromic features is known as sporadic TAD (STAD). So far, the genetic basis of STAD remains unknown. Whole exome sequencing was performed in 223 STAD patients and 414 healthy controls from the Chinese Han population (N = 637). After population structure and genetic relationship and ancestry analyses, we used the optimal sequence kernel association test to identify the candidate genes or variants of STAD. We found that COL3A1 was significantly relevant to STAD (P = 7.35 × 10

Acellular dermal matrix in skin wound healing in rabbits - histological and histomorphometric analyses

OBJECTIVES: To analyze the histology and histomorphometry of healing associated with acellular dermal matrix in skin wounds in rabbits. METHODS: Twelve male rabbits were divided into two groups: the control group (CG) and the matrix group (MG). Three skin wounds with a total area of 20 × 20 mm were created on the dorsal region of each animal. Photographic records of the lesions taken over a 21-day period and use of the ImageJ program allowed calculation of the wound contraction rate. The lesions were biopsied on days 3, 14 and 21 for histomorphometric analysis to define the thicknesses of the dermis and epidermis (hematoxylin-eosin) and calculate the densities of type I and type III collagen (picrosirius). RESULTS: No significant difference in the healing rate was found between the groups (p>0.05). The MG presented greater epidermal thickness on day 3 (p<0.05) and on days 14 and 21 (p<0.001). The MG presented greater dermal thickness throughout the study period (p<0.05). The type I collagen density was higher in the MG throughout the study period (p<0.05), and the type III collagen density was higher in the MG on days 3 and 14 (p<0.05) and on day 21 (p<0.001). CONCLUSION: The use of acellular dermal matrix increased the thickness of the dermal and epidermal layers and the amount of type I and III collagen during skin wound healing and did not alter the rate of wound contraction.

Expression of type III collagen in hypertrophic gingival tissue of patients with orthodontic treatment: a pilot study / Expresión del colágeno tipo III en tejido con hipertrofia gingival de pacientes con tratamiento ortodóntico: un estudio piloto

ABSTRACT Introduction: gingival hypertrophy (GH) is the uncontrolled increase in gingival volume induced by different etiological factors, including orthodontic treatment. This pathology is characterized by changes in epithelial and connective tissue, including modifications in the extracellular matrix. The present study determined the presence and distribution of type III collagen in tissues of patients with GH wearing fixed orthodontic appliances. Methods: 12 samples of gingival tissue were obtained from patients undergoing periodontal surgery. They were divided into two groups, the first with healthy patients (control; n = 6) and the second with patients diagnosed with GH and orthodontic treatment (patients; n = 6). Each obtained sample was subjected to the hematoxylin-eosin stain, Masson-Goldner staining, and type III collagen immunohistochemistry. Results: the hematoxylin-eosin and Masson-Goldner histological stains showed hypertrophia of epithelial tissue and connective tissue with a marked collagen fiber increase in the gingival tissue of orthodontic wearers with GH compared to individuals in the control group. The gingival tissue of patients with GH caused by orthodontic treatment showed a distribution and location of type III collagen near the basal lamina, around the blood vessels, but unlike the control group, its location was noticeable throughout the connective tissue. Conclusion: the gingival tissues of orthodontic wearers with GH experience an increase in the number and density of collagen fibers. Type III collagen seems to lose its usual location in the gingival tissues of orthodontic wearers with GH.

RESUMEN Introducción: la hipertrofia gingival (HG) es el aumento descontrolado del volumen de la encía debido a diversos factores etiológicos, entre ellos el tratamiento ortodóntico. Esta patología se caracteriza por cambios del tejido epitelial y conectivo, incluyendo modificaciones en la matriz extracelular. El presente estudio determinó la presencia y distribución de colágeno tipo III en tejidos de pacientes con HG portadores de ortodoncia fija. Métodos: se obtuvieron 12 muestras de tejido gingival de pacientes sometidos a cirugías periodontales. Se dividieron en dos grupos, el primero, integrado por pacientes sanos (control; n=6), y el segundo por pacientes diagnosticados con HG con ortodoncia (pacientes; n=6). Cada muestra obtenida fue sometida a la coloración hematoxilina-eosina, Masson-Goldner e inmunohistoquímica del colágeno tipo III. Resultados: las tinciones histológicas hematoxilina-eosina y Masson-Goldner permitieron constatar hiperplasia del tejido epitelial y un tejido conectivo denso con notable aumento de las fibras de colágeno en el tejido gingival de los pacientes con HG portadores de ortodoncia en comparación con los individuos del grupo control. El tejido gingival de pacientes con HG por ortodoncia evidenció una distribución y localización del colágeno tipo III cerca de la lámina basal, alrededor de los vasos sanguíneos, pero a diferencia del grupo control, su localización fue notoria en toda la extensión del tejido conectivo. Conclusión: los tejidos gingivales de pacientes con HG portadores de ortodoncia experimentan aumento en número y densidad de las fibras de colágeno. El colágeno tipo III parece perder su localización habitual en los tejidos gingivales de pacientes con HG portadores de ortodoncia.

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.

Effects of Physical Training on the Myocardium of Oxariectomized LDLr Knockout Mice: MMP 2/9, Collagen I/III, Inflammation and Oxidative Stress / Efeitos do Treinamento Físico sobre o Miocárdio de Camundongos LDLr Knockout Ovariectomizadas: MMP-2 e -9, Colágeno I/III, Inflamação e Estresse Oxidativo

Abstract Background: The emergence of coronary heart disease is increased with menopause, physical inactivity and with dyslipidemia. Physical training is known to promote the improvement of cardiovascular functions. Objective: To investigate the effects of aerobic physical training on the left ventricle in ovariectomized LDL knockout mice. Methods: Thirty animals were divided into 6 groups (n = 5): Sedentary non-ovariectomized control; Sedentary ovariectomized control; Trained ovariectomized control; Sedentary non-ovariectomized LDL-knockout, sedentary ovariectomized LDL-knockout and trained ovariectomized LDL-knockout. We analyzed the average parameters of apparent density of collagen fibers types I and III, and metalloproteinase type 2 and type 9, were considered significant p < 0.05. Results: The results showed that the proposed exercise protocol altered the volume of type I collagen fibers, altered collagen remodeling parameters (MMP-2), and also reduced the 8-hydroxy-2'-deoxyguanosine (8OHdG) oxidative stress parameter. Conclusion: Moderate intensity aerobic training acts on collagen fiber volume, on collagen remodeling with the reduction of oxidative stress in the left ventricles of ovariectomized LDL-knockout mice.

Resumo Fundamento: O surgimento da doença cardíaca coronariana aumenta com a menopausa, inatividade física e dislipidemia. Sabe-se que o treinamento físico promove a melhora das funções cardiovasculares Objectivo: Investigar os efeitos do treinamento físico aeróbico sobre o ventrículo esquerdo em camundongos LDL knockout ovariectomizadas. Métodos: Trinta animais foram divididos em 6 grupos (n = 5): controle sedentário não ovariectomizado, controle sedentário ovariectomizado, controle treinado ovariectomizado, sedentário LDL-knockout não ovariectomizado, sedentário LDL-knockout ovariectomizado e treinado LDL-knockout ovariectomizado. Analisamos os parâmetros médios da densidade de volume de fibras colágenas tipo I e III, e metaloproteinases 2 e 9. Valores de p < 0,05 foram considerados significativos. Resultados: Os resultados mostram que o protocolo de exercício proposto alterou o volume de fibras colágenas do tipo I e os parâmetros de remodelamento do colágeno (MMP-2), e ainda reduziu o parâmetro de estresse oxidativo do 8-hidroxi-2'-deoxiganosina (8-OhdG). Conclusão: O treinamento aeróbico de intensidade moderada age sobre o volume das fibras colágenas e sobre o remodelamento de colágeno, com redução do estresse oxidativo em ventrículos esquerdos de camundongos ovariectomizados LDLr Knockout.

Two cases of Type Ⅲ collagen glomerulopathy and literature review / 中南大学学报(医学版)

In this paper, 2 cases of collagen Type Ⅲ glomerulopathy were analyzed. The clinical manifestations mainly included nephrotic syndrome, proteinuria, hypertension and renal dysfunction. One patient showed that the complement factor H-related protein 5 (CFHR5) gene was likely a disease-causing mutation. The pathological examination of renal tissues showed hyperplasia of mesangial matrix, sub-endothelial insertion, and double-track formation. Immunohistochemistry of Type III collagen was positive. Electron microscopy revealed that massive collagen fibers (40-70 nm in diameter) deposited in the mesangial matrix and basement membrane. As for the follow-up results, the normal renal function had kept steady and the proteinuria was moderate in 1 case treated with angiotensin Ⅱ receptor blocker. Due to other system disease, another case developed into acute kidney injury and then received hemodialysis. The clinical manifestations of collagen Type Ⅲ glomerulopathy was atypical, the light microscope pathological features were various, and the disease was mainly diagnosed by electron microscopy and immunohistochemistry.

Morphometric and molecular muscle remodeling after passive stretching in elderly female rats

OBJECTIVES: To determine the effects of three sessions of a passive stretching exercise protocol on the muscles of elderly female rats. METHODS: The effects of the stretching exercises on the soleus muscle were analyzed using immunohistochemistry [tissue inhibitors of matrix metalloproteinases (TIMP), the tumor necrosis factor-alpha (TNF-α), and the gene expression levels using real-time PCR of the transforming growth factor-beta 1 (TGF-β1), collagen type 1 (COL1), and collagen type 3 (COL3)]. Fifteen 26-month-old female Wistar rats were randomly divided into two groups, namely, Stretching (SG, n=8) and Control (CG, n=7). The passive mechanical stretching protocol consisted of a set of 4 1-minute repetitions, with 30 seconds between each repetition (total treatment of 4 minutes), three times a week for 1 week. RESULTS: Immunohistochemical analysis revealed an increase of 71.4% in the TNF-α (p=0.04) gene expression levels for the SG and a 58% decrease in the TGF-β1 gene expression levels (p=0.005) in the SG compared to that in the CG. No significant differences were observed between the groups for the immunostaining of TIMP-1 or the gene expression levels of COL1 and COL3. CONCLUSION: Three sessions of static stretching reduced the gene expression level of TGF-β1, which, owing to its anti-fibrotic role, might contribute to the remodeling of the intramuscular connective tissue of the aging muscle. In addition, immunostaining revealed that TNF-α levels increased in the aging muscle tissue in response to stretching, indicating its effect on stimulating extracellular matrix degradation. These outcomes have important clinical implications in reinforcing the use of stretching exercises in the elderly, considering that the aging muscle presents an infiltration of connective tissue.

Effect of low intensity photobiomodulation associated with norbixin-based poly (hydroxybutyrate) membrane on post-tenotomy tendon repair. In vivo study

Abstract Purpose: To evaluate the in vivo response of photobiomodulation therapy associated with norbixin-based poly(hydroxybutyrate) membrane (PHB) in tenotomized calcaneal tendon. Methods: Thirty rats were randomly allocated to six groups (n=5 each): LED groups (L1, L2 and L3) and membrane + LED groups (ML1, ML2 and ML3). The right calcaneal tendons of all animals were sectioned transversely and were irradiated with LED daily, one hour after surgery every 24 hours, until the day of euthanasia. At the end of the experiments the tendons were removed for histological analysis. Results: The histological analysis showed a significant reduction in inflammatory cells in the ML1, ML2 and ML3 groups (p=0.0056, p=0.0018 and p<0.0001, respectively) compared to those in the LED group. There was greater proliferation of fibroblasts in the ML1 (p<0.0001) and L3 (p<0.0001) groups. A higher concentration of type I collagen was also observed in the ML1 group (p=0.0043) replacing type III collagen. Conclusion: Photobiomodulation in association with norbixin-based PHB membrane led to control of the inflammatory process. However, it did not favor fibroblast proliferation and did not optimize type I collagen formation in the expected stage of the repair process.

Effect of norbixin-based poly(hydroxybutyrate) membranes on the tendon repair process after tenotomy in rats

Abstract Purpose: To determine the efficacy of norbixin-based poly(hydroxybutyrate) (PHB) membranes for Achilles tendon repair. Methods: Thirty rats were submitted to total tenotomy surgery of the right Achilles tendon and divided into two groups (control and membrane; n = 15 each), which were further subdivided into three subgroups (days 7, 14, and 21; n = 5 each). Samples were analyzed histologically. Results: Histological analysis showed a significant reduction in inflammatory infiltrates on days 7, 14 (p < 0.0001 for both), and 21 (p = 0.0004) in the membrane group compared to that in the control group. There was also a significant decrease in the number of fibroblasts in the control group on days 7, 14 (p < 0.0001), and 21 (p = 0.0032). Further, an increase in type I collagen deposition was observed in the membrane group compared to that in the control group on days 7 (p = 0.0133) and 14 (p = 0.0107). Conclusion: Treatment with norbixin-based PHB membranes reduces the inflammatory response, increases fibroblast proliferation, and improves collagen production in the tendon repair region, especially between days 7 and 14.

Quantificação do colágeno dérmico equino por duas técnicas morfométricas: contagem de pontos e segmentação de cor / Quantification of equine dermal collagen by two morphometric techniques: point counting and color segmentation

Os colágenos tipos I e III apresentam diferentes tonalidades de birrefringência em cortes histológicos corados com Picrosirius red e analisados em microscópio sob luz polarizada. Com base nessa propriedade, os colágenos podem ser quantificados por histomorfometria. Entretanto, são muitas as variáveis que podem afetar a distribuição das cores na imagem histológica, e a escolha adequada dos parâmetros de análise têm grande influência no resultado final. O objetivo deste trabalho foi comparar a quantificação histomorfométrica de colágeno em pele equina pela morfometria por contagem de pontos e pela segmentação de cor com diversas configurações, a fim de se determinar o melhor método de avaliação. Para a morfometria por contagem de pontos, foram utilizadas três gratículas diferentes (391, 588 e 792 pontos de interseções) e, para a segmentação de cor, seis combinações de hue e brightness no software ImageJ. Os valores foram submetidos ao teste de Friedman, seguido pelo teste de Tukey com 5% de significância. Os resultados demonstraram que a quantificação dos colágenos na gratícula de 792 pontos foi equivalente aos resultados da segmentação de cor com brightness de 1-255 e hue de 0-42 e 43-120 para os colágenos tipos I e III, respectivamente. Dessa forma, conclui-se que a análise automática da segmentação de cor, utilizando configuração adequada para brightness e hue, pode substituir a morfometria por contagem de pontos de forma confiável e segura.(AU)

The types I and III collagens present different tonalities of birefringence in histological sections stained with Picrosirius red, that can be analyzed under a polarized light microscope. Based on this property, collagens can be quantified by histomorphometry. However, many variables can affect the color distribution in the histological image, and the appropriate choice of the analysis parameters have a significant influence on the final result. The objective of this study was to compare the histomorphometric quantification of collagen in the equine skin by counting points planimetry and color segmentation with different configurations to determine the best method of evaluation. For planimetry, three different graticules (391, 588 and 792 intersections) were used and, for color segmentation, six combinations of hue and brightness in ImageJ software. The values were submitted to the Friedman test followed by Tukey with 5% significance. The results showed that the quantification of collagens in the graticule of 792 intersections was equivalent to the results of color segmentation with a brightness of 1-255 and hue of 0-42 and 43-120 for collagens type I and III, respectively. Automatic analysis of the color segmentation, using suitable configuration for brightness and hue can replace the counting points planimetry reliably and safely.(AU)

The Effect of Trigonella foenum-graceum L. (Fenugreek) Towards Collagen Type I Alpha 1 (COL1A1) and Collagen Type III Alpha 1 (COL3A1) on Postmenopausal Woman's Fibroblast

Trigonella foenum-graceum L. (fenugreek) is a phytoestrogen, a nonsteroidal organic chemical compound from plants which has similar mechanism of action to sex hormone estradiol-17β. This study aims to assess the effectivity of fenugreek seeds extract on collagen type I alpha 1 (COL1A1) and collagen type III alpha 1 (COL3A1) which are both decreased in aging skin and become worsen after menopause. This in vitro experimental study used old human dermal fibroblast from leftover tissue of blepharoplasty on a postmenopausal woman (old HDF). As a control of the fenugreek's ability to trigger collagen production, we used fibroblast from preputium (young HDF). Subsequent to fibroblast isolation and culture, toxicity test was conducted on both old and young HDF by measuring cell viability on fenugreek extract with the concentration of 5 mg/mL to 1.2 µg/mL which will be tested on both HDF to examine COL1A1 and COL3A1 using ELISA, compared to no treatment and 5 nM estradiol. Old HDF showed a 4 times slower proliferation compared to young HDF (p<0.05). Toxicity test revealed fenugreek concentration of 0.5 – 2 µg/mL was non-toxic to both old and young HDF. The most significant fenugreek concentration to increase COL1A1 and COL3A1 secretion was 2 µg/mL (p<0.05).

Characterization of Tendon-Specific Markers in Various Human Tissues, Tenocytes and Mesenchymal Stem Cells

BACKGROUND: Unlike bone, cartilage, or muscle, tendon-specific markers are not well established. The purpose of the study was to investigate expression pattern and level of 6 well-known tendon-specific markers, in various human musculoskeletal tissues, tenocytes, and mesenchymal stem cells (MSCs). METHODS: Musculoskeletal tissue samples of tendon, bone, cartilage, nerve, muscle, and fat were obtained from patients undergoing orthopedic surgery. Tenocytes, MSCs from bone marrow, adipose tissue, and umbilical cord were isolated from each tissue and cultured. Six tendon-specific markers, scleraxis (Scx), tenomodulin (TNMD), thrombospondin-4 (TSP-4), tenascin-C (TNC), type I collagen (Col I), and type III collagen (Col III) were investigated in tendon tissue, tenocytes, and MSCs. RESULTS: mRNA levels of 6 tendon-specific markers were significantly higher in tendon tissue that in other connective tissues levels of Scx, TNMD, TSP-4, and Col III immediately decreased after plating tenocytes in culture dishes whereas those of TNC and Col I did not. In comparison with tendon tissue, mRNA levels pattern of Scx, TNMD, and TSP-4 in tenocytes were significantly higher than that in MSCs, but lower than in tendon tissue whereas expression pattern of TNC, Col I and III showed different pattern with each other. CONCLUSION: This study demonstrated that 6 commonly used tendon-specific markers were mainly expressed in tendon tissue, but that expression level and pattern of the tendon-specific markers with respect to kinds of tissues, culture duration of tenocytes and sources of MSCs.

Healing effect of andiroba-based emulsion in cutaneous wound healing via modulation of inflammation and transforming growth factor beta 3

Abstract Purpose: To evaluate the effects and mechanisms of andiroba-based emulsion (ABE) topical treatment on full-thickness cutaneous wounds in rats. Methods: The wounds were harvested on days 3, 7, 15, and 20 post-surgery. Wound contraction rate, quantitative immunohistochemistry [macrophages, myofibroblasts, capillaries, collagens (col) I and III, transforming growth factor β3β (TGFβ3)], and tensile strength were assessed. Results: Treated wounds were smaller, contracted earlier and had increased angiogenesis, fewer CD68+ and M2 macrophages on days 7 and 15, but higher on day 20. Myofibroblasts appeared on days 3 to 7 in untreated wounds and on days 7 to 15 in treated wounds. TGFβ3 levels were higher in the treated wounds, less dense collagen fibers, lower col I/III ratios and a higher tensile strength. Conclusion: These results demonstrate the important anti-inflammatory role of treatment and the associated modulation of macrophages, myofibroblasts, and TGFβ3 levels. Collagen fibers in the treated wounds were more organized and less dense, similar to unwounded skin, which likely contributed to the higher tensile strength.

Effects of the application of the amniotic membrane in the healing process of skin wounds in rats

Abstract Purpose: To evaluate the efficacy of the application of the human amniotic membrane (HAM) on the inflammatory process, fibroblast proliferation, formation of collagenand reduction of skin wound areas in rats. Methods: Thirty six rats were submitted to a surgical injury induction and divided into two groups (n = 18): group C (control) and T (treated with the HAM). The macroscopic evolution in the wound area and the histological characteristics of the skin samples were evaluated. Results: The regression of the wound area was greater in group T. The histological analysis revealed a significant reduction (p < 0.05) in the inflammatory infiltrate in group T at all experimental periods compared with that in the control group. Furthermore, the group T presented a significant increase in the proliferation of fibroblasts at 14 and 21 days compared with group C (p < 0.05). Regarding the deposition of mature collagen fibers, there was an increase in the replacement of type III collagen by type I collagen in group T (p < 0.05). Conclusion: Treatment with the HAM reduced the healing time as well as the inflammatory responses, increased the proliferation of fibroblasts, and induced a higher concentration of mature collagen fibers.

Mechanism of thermosensitive moxibustion on knee osteoarthritis in rabbit models / 中国针灸

<p><b>OBJECTIVE</b>To observe the impacts of thermosensitive moxibustion (TSM) on the expressions of nitric oxide (NO), typeⅠdisintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), typeⅡcollagen and proteoglycan (PG) in the rabbit models of knee osteoarthritis (KOA) and explore the mechanism of TSM on KOA.</p><p><b>METHODS</b>A total of 42 Japanese long-eared male rabbits were divided into a blank group (6 rabbits), a model group (6 rabbits), a moxibustion group (24 rabbits) and a sham-operation group (6 rabbits) according to the random number table. In the blank group, the rabbits were fed normally. In the model and moxibustion groups, the papain injection was given to establish KOA models. The rabbits in the sham-operation group were treated with the intracavity injection of 0.9% NaCl solution. The rabbits were forced to move for 30 min every day, continuously for 15 days during modeling. At the end of modeling, in the moxibustion group, moxibusiton was applied at "Dubi" (ST 35), once a day, 40 min each time, for 14 days totally. According to the temperature changes during moxibustion, the rabbits were divided into a TSM group and a non-TSM group. 6 rabbits were collected randomly from the two groups. The usual feeding was given in the blank group, the model group and the sham-operation group, without any intervention. The body mass and behavioristics changes were observed in each group. At the end of treatment, the nitrate reduction method was adopted to determine NO expression in the serum. The real-time PCR was adopted to determine the expressions of ADAMTS-4, typeⅡcollagen and PG in the cartilage.</p><p><b>RESULTS</b>① After modeling, compared with the blank group, the body mass was all reduced and the Lequesne MG score was increased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). After intervention, compared with the blank group, the body mass was decreased and the Lequesne MG score was increased in the model and sham-operation groups (<0.05, <0.01). Compared with the model group, the body mass was increased and the lequesne MG score was decreased in the TSM, non-TSM, and sham-operation groups (<0.05, <0.01). Compared with the non-TSM group, the body mass in the TSM group was increased remarkably (<0.05), but the difference in Lequesne MG score was not statistically significant (>0.05). ② After intervention, compared with the blank group, the expressions of NO and ADAMTS-4 were all increased and the expressions of typeⅡcollagen and PG were decreased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the model group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the non-TSM group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group after intervention (all <0.05).</p><p><b>CONCLUSION</b>The thermosensitive moxibustion alleviates the inflammatory reactions and protects the joint cartilage through inhibiting the expressions of NO and ADAMTS-4 to achieve the effects in the treatment of KOA.</p>

Effects of renal dysfunction on healing of colonic anastomosis: experimental study in wistar rats / Efeitos da disfunção renal na cicatrização de anastomoses colônicas: estudo experimental em ratos wistar

ABSTRACT Background: Chronic kidney disease affects more than 500 million people worldwide. In this context, the uremic toxins present are related to worsening in tissue healing. Aim: Evaluate on healing of colonic anastomosis in uremic rats, serum and anatomopathological indicators, which may be related to the change tissue repair process. Methods: Twenty Wistar rats, were randomly separated into two groups. In the sham group they were submitted to 5/6 nephrectomy simulation in left kidney, simulation right nephrectomy, median laparotomy, colotomy and colorraphy. In the uremia group, they were submitted to 5/6 nephrectomy of the left kidney, total nephrectomy of the right kidney and median laparotomy, colotomy and colorraphy. Were collected for serum urea, creatinine and CRP dosages and the colonic segments were studied for evaluation of granulation tissue, collagen maturation, microvascular and myofibroblasts density, and cell viability. Through histochemical processing, microvascular density was evaluated by anti-CD34 monoclonal antibody marking, cell viability by cell proliferation nuclear antigen screening and myofibroblasts density with monoclonal anti-α-actin antibody. Computerized histometry was used for evaluations of collagens type I and III by the coloration of picrosirius. Results: The group submitted to nephrectomy 5/6, compared to the sham group, show urea increase (p<0.0000) and higher C reactive protein (p=0.0142). Decrease of granulation tissue formation (border reepithelialization p=0,0196, angiofibroblast proliferation p=0.0379), mean collagen I (p=0,0009) and collagen III (p=0,016), microvascular density (p=0,0074), cell proliferation nuclear antigen (p<0,0000) and myofibroblasts (p<0,0001). Conclusion: The uremia induced by nephrectomy 5/6 model establishes negative impact in the colonic wound healing.

RESUMO Racional: A doença renal crônica atinge mais de 500 milhões de pessoas em todo o mundo. Neste contexto, as toxinas urêmicas estão relacionadas ao comprometimento da cicatrização tecidual. Objetivo: Avaliar, na cicatrização de anastomoses colônicas de ratos urêmicos indicadores séricos e anatomopatológicos que possam estar relacionados com alteração do processo de reparação tissular. Métodos: Utilizaram-se 20 ratos Wistar divididos aleatoriamente em dois grupos. No grupo simulação eles foram submetidos à simulação da nefrectomia 5/6 do rim esquerdo, simulação de nefrectomia total do rim direito, laparotomia mediana, colotomia e colorrafia. No grupo uremia, eles foram submetidos à nefrectomia 5/6 do rim esquerdo, nefrectomia total do rim direito, laparotomia mediana, colotomia e colorrafia. Coletaram-se amostras de sangue para dosagens séricas da ureia, creatinina e proteína C reativa, e do cólon para processamentos histológicos e histoquímicos na avaliação do tecido de granulação, maturação de colágeno, densidade microvascular e de miofibroblastos, viabilidade celular cicatricial. Empregou-se a histometria computadorizada para as avaliações de colágenos tipos I e III, densidade microvascular pela marcação com anticorpo monoclonal anti-CD34, viabilidade celular pela pesquisa do antígeno nuclear de proliferação celular e a densidade de miofibroblastos com anticorpo monoclonal anti-α-actina. Resultados: O grupo submetido à nefrectomia 5/6, em comparação ao grupo simulação, demonstraram aumentos da ureia sérica (p<0,0000) e proteína C reativa (p=0,0142), redução da formação de tecido de granulação (reepitelização de bordas p=0,0196, proliferação angiofibroblástica p=0,0379), porcentagens de colágeno I (p=0,0009) e colágeno III (p=0,016), densidade microvascular (p=0,0074) e miofibroblastos (p<0,0001) e antígeno nuclear de proliferação celular (p<0,0000). Conclusão: A uremia induzida pelo modelo de nefrectomia 5/6 determina impacto negativo no processo de cicatrização colônico.

Collagen I and III in women with diastasis recti

OBJECTIVES: Interest in elucidating the etiology of hernias has encouraged countless studies of musculoaponeurotic structures in individuals with and without hernias. Studies of hernia patients have firmly demonstrated a correlation between hernias and collagen alterations in their fascia. Diastasis recti is an increased width of the abdominal midline that is exclusively composed of interlacing aponeurotic expansions of the anterolateral abdominal muscles. The condition is common among women undergoing abdominoplasty, and many factors, not only mechanical, play a role. The goal of this study is to evaluate and compare collagen type I and III levels in the midline fascia of women with and without diastasis recti to report their possible influence on this condition. METHODS: This is a case-control study nested within a surgical cohort of 18 women with diastasis recti and 18 women without the condition (cases and controls, respectively). Fascia from the midline of the abdominal wall was collected and analyzed through immunohistochemistry using polyclonal antibodies to collagen type I and III. RESULTS: Both type I and type III collagen were less abundant in women with diastasis recti than in those without the condition, and the difference was statistically significant (p<0.001). CONCLUSION: Low collagen type I and type III levels in the midline of the abdominal wall may play a key role in the development of diastasis recti.

Biomechanic and histologic analysis of fibroblastic effects of tendon to bone healing by transforming growth factor ß1 (TGF-ß1) in rotator cuff tears

Abstract Purpose: To evaluate the effect of transforming growth factor β1 (TGF-β1) on tendon-to-bone reconstruction of rotator cuff tears. Methods: Seventy-two rat supraspinatus tendons were transected and reconstructed in situ. At 8 and 16 weeks, specimens of three groups; that is control, L-dose (low dose), and H-dose (high dose) were harvested and underwent a biomechanical test to evaluate the maximum load and stiffness values. Histology sections of the tendon-to-bone interface were identified by hematoxylin-eosin or Masson trichrome stain. Collagen type III was observed by picric acid sirius red staining under polarized light. The level of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) was measured by the enzyme-linked immunosorbent assay (ELISA) method. Results: Collagen type III of the H-dose group had a significant difference in histology structure compared with the L-dose group (P<0.05). The maximum load and stiffness decreased significantly in the control group compared with the values of the L-dose and H-dose groups. The stiffness among the three groups differed significantly at the same postoperative time (P<0.05). Interestingly, progressive reestablishment of collagen type III affected tendon-to-bone healing significantly in the later stages. Conclusion: The H-dose was associated with an increased collagen type III morphology stimulated by TGF-β1.