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The extracellular matrix surrounding the tumor undergoes changes in its organization during the metastasis process. The present study aims to quantify total collagen, collagen I (Col I) and collagen III (Col III), analyze the alignment of collagen fibers and assess the basement membrane integrity in samples from patients with metastatic and non-metastatic prostate cancer. Tissue samples from 60 patients were classified into groups based on prognostic parameters: better prognosis (n = 20), worse prognosis without metastasis (n = 23) and metastatic (n = 17). Picrosirius red with further analysis under polarizing microscope was used to quantify (with validation using immunohistochemistry) and analyze collagen alignment, and Periodic Acid Schiff staining was used to analyze the basement membrane integrity. The Col I/Col III ratio was found to be higher in the metastatic group than in the groups with better prognosis (p = 0.012) and worse prognosis without metastasis (p = 0.018). Basement membrane integrity constitution in malignant tumor tissue differed from that of adjacent non-tumor tissue (p < 0.001). Moreover, the worsening in the tumor tissue integrity was positively correlated with worse prognostic parameters. All in all, absence of Col III and basement membrane integrity might be indicators of poor prognosis in prostate cancer.

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OBJECTIVES: To compare the biodegradability, mechanical behavior, and physicochemical features of the collagen-rich extracellular matrix (ECM) of artificial caries-affected dentin (ACAD), natural caries-affected dentin (NCAD) and sound dentin (SD). METHODS: Dentin specimens from human molars were prepared and assigned into groups according to the type of dentin: ACAD, NCAD, or SD. ACAD was produced by incubation of demineralized SD with Streptococcus mutans in a chemically defined medium (CDM) with 1% sucrose for 7 days at 37 °C under anaerobic conditions. Specimens were assessed to determine collagen birefringence, biodegradability, mechanical behavior, and chemical composition. Data were individually processed and analyzed by ANOVA and post-hoc tests (α = 0.05). RESULTS: CDM-based biofilm challenge reduced loss, storage, and complex moduli in ACAD (p < 0.001), while the damping capacity remained unaffected (p = 0.066). Higher red and lower green birefringence were found in ACAD and NCAD when compared with SD (p < 0.001). Differently to ACAD, SD and NCAD presented higher biodegradability to exogenous proteases (p = 0.02). Chemical analysis of the integrated areas of characteristic bands that assess mineral quality (carbonate/phosphate and crystallinity index), mineral to matrix (phosphate/amide I) and post-translational modifications (amide III/CH2, pentosidine/CH2, and pentosidine/amide III) (p<0.05) showed that NCAD was significantly different from SD while ACAD exhibited intermediate values. CONCLUSIONS: CDM-based biofilm challenge produced a dentin ECM with decreased mechanical properties and increased collagen maturity. The compositional and structural conformation of the ACAD suggested that CDM-based biofilm challenge showed potential to produce artificial lesions by revealing a transitional condition towards mimicking critical features of NCAD. CLINICAL SIGNIFICANCE: This study highlights the importance of developing a tissue that mimics the features of natural caries-affected dentin ECM for in vitro studies. Our findings suggested the potential of a modified biofilm challenge protocol to produce and simulate a relevant substrate, such as caries-affected dentin.

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Background: Malignant pleural mesotheliomas (MM) are known for their heterogenous histology and clinical behavior. MM histology reveals three major tumor cell populations: epithelioid, sarcomatoid, and biphasic. Using a dissecting approach, we showed that histochemical gradients help us better understand tumor heterogeneity and reconsider its histologic classifications. We also showed that this method to characterize MM tumor cell populations provides a better understanding of the underlying mechanisms for invasion and disease progression. Methods: In a cohort of 87 patients with surgically excised MM, we used hematoxylin and eosin to characterize tumor cell populations and Movat's pentachrome staining to dissect the ECM matrisome. Next, we developed a computerized semi-assisted protocol to quantify and reconstruct the ECM in 3D and examined the clinical association between the matricellular factors and patient outcome. Results: Epithelioid cells had a higher matrix composition of elastin and fibrin, whereas, in the sarcomatoid type, hyaluronic acid and total collagen were most prevalent. The 3D reconstruction exposed the collagen I and III that form channels surrounding the neoplastic cell blocks. The estimated volume of the two collagen fractions was 14% of the total volume, consistent with the median estimated area of total collagen (12.05 mm2) for epithelioid MM. Conclusion: Differential patterns in matricellular phenotypes in MM could be used in translational studies to improve patient outcome. More importantly, our data raise the possibility that cancer cells can use the matrisome for disease expansion and could be effectively targeted by anti-collagen, anti-elastin, and/or anti-hyaluronic acid therapies.

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The human amniotic membrane (AM) is emerging as an interesting biomaterial for regenerative medicine due to its biological and mechanical proprieties. The beneficial effects of the AM are probably related to its bioactive factors produced by local cells and stored in the stromal matrix. However, the search for a preservation method capable of preserving AM properties remains a challenge. The aim of this study was to evaluate important features of 2 anatomical regions of the human AM (reflected and placental amnion) after different preservation methods. For this purpose, human placentas were harvested and processed for AM isolation and storage at 2 different conditions: room temperature for 18 h in DMEM (fresh AM) and -80°C in DMEM/glycerol solution for 30 days (cryopreserved AM). After the storage period, the structural integrity of the membrane was assessed by histological and Picrosirius polarization analysis, cellular viability analysis was performed using the MTT assay, and the soluble proteins were quantified with the Qubit Protein Assay Kit. Both preservation protocols reduced the cell viability, mainly in the placental amnion region of the AM, but preserved the morphology of epithelial and stromal layers, as well as the organization and distribution of collagen fibers. There was a reduction in soluble proteins only in fresh AM. Importantly, the cryopreserved AM group presented the same concentration as the control group. In conclusion, the cryopreservation using DMEM/glycerol was ideal for preserving the structural integrity and soluble protein content, indicating the feasibility of this method in preserving AM for its use in regenerative medicine.

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O colágeno é sintetizado e segregado no espaço extracelular e organizados em fibrilas estriadas de acordo com o tipo de tecido. Utilizaram-se 24 coelhos brancos da raça Nova Zelândia, com idade de 12 meses e com 3,0kg de peso corporal, para avaliar a porcentagem de colágeno das feridas cutâneas tratadas com plasma rico em plaquetas de equino e pomada contendo gentamicina, sulfanilamida, sulfadiazina, ureia e vitamina A. Os animais foram separados em quatro grupos de igual número e submetidos à remoção de pele na região das linhas médias dorsal torácica (feridas tratadas) e lombar (feridas controle). As feridas torácicas foram tratadas com plasma rico em plaqueta de equino e pomada contendo gentamicina, sulfanilamida, sulfadiazina, ureia e vitamina A, e as do grupo controle somente com a pomada. Dos animais do grupo I, foi coletado tecido cutâneo, para a avaliação histológica e a ultraestrutural, com três dias de pós-operatório; dos animais do grupo II, com sete dias; do grupo III, com 14 dias; e do grupo IV, com 21 dias. Decorrido o período de avaliação de cada grupo, foi coletado fragmento de pele para avaliação da porcentagem de colágeno, bem como do diâmetro e da densidade da fibrila de colágeno por microscopia eletrônica de transmissão. O tratamento com PRP de equino associado à aplicação tópica da pomada mostrou-se eficaz na maturação das fibrilas colágenas e na antecipação do processo cicatricial.(AU)

Collagen is synthesized and secreted into the extracellular space and organized into striated fibrils according to the tissue type. This study evaluated the concentration of collagen in rabbit skin wounds treated with equine platelet-rich plasma (PRP) and ointment containing gentamicin, sulfanilamide, sulfadiazine, urea, and vitamin A. Twenty-four New Zealand white rabbits aged 2 to 12 months and weighing 3.0kg were included. The animals were allocated equally into four groups and the skin was removed from the thoracic dorsal midline (treated wound) and lumbar (control wound) regions. The thoracic wounds were treated with equine PRP and ointment containing gentamicin, sulfanilamide, sulfadiazine, urea, and vitamin A, and the control group was treated with the ointment alone. For histological and ultrastructural assessment, cutaneous tissue was collected on postoperative days 3 (group I), 7 (group II), 14 (group III), and 21 (group IV). After the evaluation period, in each group, a skin fragment was collected for analysis of the collagen concentration, as well as the collagen fibril diameter and density by transmission electron microscopy. The results indicated that treatment with equine PRP combined with topical application of the ointment was effective in facilitating the maturation of collagen fibrils and the wound healing process.(AU)

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O colágeno é sintetizado e segregado no espaço extracelular e organizados em fibrilas estriadas de acordo com o tipo de tecido. Utilizaram-se 24 coelhos brancos da raça Nova Zelândia, com idade de 12 meses e com 3,0kg de peso corporal, para avaliar a porcentagem de colágeno das feridas cutâneas tratadas com plasma rico em plaquetas de equino e pomada contendo gentamicina, sulfanilamida, sulfadiazina, ureia e vitamina A. Os animais foram separados em quatro grupos de igual número e submetidos à remoção de pele na região das linhas médias dorsal torácica (feridas tratadas) e lombar (feridas controle). As feridas torácicas foram tratadas com plasma rico em plaqueta de equino e pomada contendo gentamicina, sulfanilamida, sulfadiazina, ureia e vitamina A, e as do grupo controle somente com a pomada. Dos animais do grupo I, foi coletado tecido cutâneo, para a avaliação histológica e a ultraestrutural, com três dias de pós-operatório; dos animais do grupo II, com sete dias; do grupo III, com 14 dias; e do grupo IV, com 21 dias. Decorrido o período de avaliação de cada grupo, foi coletado fragmento de pele para avaliação da porcentagem de colágeno, bem como do diâmetro e da densidade da fibrila de colágeno por microscopia eletrônica de transmissão. O tratamento com PRP de equino associado à aplicação tópica da pomada mostrou-se eficaz na maturação das fibrilas colágenas e na antecipação do processo cicatricial.(AU)

Collagen is synthesized and secreted into the extracellular space and organized into striated fibrils according to the tissue type. This study evaluated the concentration of collagen in rabbit skin wounds treated with equine platelet-rich plasma (PRP) and ointment containing gentamicin, sulfanilamide, sulfadiazine, urea, and vitamin A. Twenty-four New Zealand white rabbits aged 2 to 12 months and weighing 3.0kg were included. The animals were allocated equally into four groups and the skin was removed from the thoracic dorsal midline (treated wound) and lumbar (control wound) regions. The thoracic wounds were treated with equine PRP and ointment containing gentamicin, sulfanilamide, sulfadiazine, urea, and vitamin A, and the control group was treated with the ointment alone. For histological and ultrastructural assessment, cutaneous tissue was collected on postoperative days 3 (group I), 7 (group II), 14 (group III), and 21 (group IV). After the evaluation period, in each group, a skin fragment was collected for analysis of the collagen concentration, as well as the collagen fibril diameter and density by transmission electron microscopy. The results indicated that treatment with equine PRP combined with topical application of the ointment was effective in facilitating the maturation of collagen fibrils and the wound healing process.(AU)

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The reconstructive techniques have been widely used in Veterinary Medicine. The post-operative adjuvants therapies like the low-level laser therapy (LLLT) are used to decrease inherent complications to reconstructive surgeries. This article purposed to define the LLLT effects on the healing, inflammation, and vascularization of the skin grafts in applicable time intervals to veterinary surgical routine. Forty rats (Rattus norvegicus albinus wistar) were used and each one was submitted to autogenous cutaneous mesh grafting in the interescapular region. The rats were randomly distributed in five groups (G1, G2, G3, G4, and G5) in accordance with the 6 J/cm2 or 10 J/cm2 dose every 3 or 5 days. These treatments were applied on the skin graft for 15 days. The histochemical evaluation with Picrosirius showed greater expression of collagen type 1 - red in grafts of G5 (p < 0.05), while in G1 did not; the expression of collagen type III - green was not induced by LLLT. The histochemical evaluation with hematoxylin-eosin showed greater numbers of fibroblasts in grafts of G4 (p < 0.05) and less hemorrhage in grafts of G5 (p < 0.05). There was modulation of the inflammatory response in irradiated skin grafts. It is concluded the exhibition of the skin grafts to 6 J/cm2 or 10 J/cm2 dose every 5 days improved the healing and the modulation of the local inflammation.

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Although studies have focused on extracellular matrix (ECM) remodeling during the formation and functioning of adult ovaries, there is no comprehensive understanding of the mechanisms controlling preantral follicle development in fetal bovine ovaries during gestation. Thus, to gain insights into ECM remodeling during initial ovarian development, we used fetal ovaries to quantify the fractal dimension (FD), total collagen, and relative mRNA abundance of genes related to ECM remodeling (COL1A1, COL1A2, COL4A1, MMP2, MMP9, MMP14, TIMP1, and TIMP2). For this, pairs of fetal ovaries were obtained from cows in a local abattoir at days 60, 90, 120, and 150 of gestation; one of each pair was submitted to RNA extraction for target transcript analysis, and the other was used for total collagen and FD evaluation. From day 120 total collagen appeared to occupy a greater area in the fetal ovary. The fractal analysis with picrosirius red staining shows higher at day 150 when compared with that on day 60. On the contrary, we found an inverse pattern when we used the hematoxylin and eosin staining approach. Concerning target gene expression, the relative abundances of COL1A1, COL4A1, MMP2, MMP14, TIMP1, and TIMP2 mRNA were higher on day 150 when compared with that on day 60. We conclude that fractal analysis reflects the morphological changes occurring during structural organization of the fetal ovary and that the expression of genes related to ECM remodeling is modulated throughout gestation in bovine fetal ovaries.

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Abstract Angiostrongylus vasorum is a pulmonary artery parasite of domestic and wild canid. On molluscs, intermediate host, first stage larvae (L1) are found after the first day of infection, in the 8th L2 and in the 30 th L3. It was evaluated L1, L2 and L3 recovered by Baermann technique from Achatina fulica infected with 1000 L1. Fifty larvae/stage were incubated with antibodies anti-β-tubulin, anti-α-tubulin, anti- α-actin, anti-β-actin and anti-collagen, and then with Alexa 633. Fifty larvae/stage were observed with picrosirius red and Oil Red O. It was also observed in the anterior region of L1 the beginning of the chitinous stems development, in the initial portion of the intestine and genital primordium. In L2 anterior region, the papillae, chitinous canes juxtaposed to the mouth and intestines bigger than L1. The L3 musculature is well defined, next to the chitinous stems, there are two round distally arranged from each other. It was observed the whole extension of the intestine genital primordium and intense cellularity in the L3 distal portion. With the picrosirius red the L1, L2 and L3 musculature could be observed, as the nerve ganglia on L3. Oil Red O revealed that L1, L2 and L3 store energy on lipid droplets.

Resumo Angiostrongylus vasorum é um parasito de artérias pulmonares dos canídeos domésticos e silvestres. Nos moluscos, hospedeiros intermediários, encontram-se no primeiro dia após a infecção, larvas de primeiro estágio (L1), ao 8° L2 e ao 30° L3. Avaliou-se L1, L2 e L3 recuperadas pela técnica de Baermann de Achatina fulica infectada com 1.000 L1. Incubou-se 50 larvas/estádio com anticorpos anti-β-tubulina, anti-α-tubulina, anti-β-actina e anti-colágeno e, em seguida, com anticorpo Alexa 633. Observaram-se também 50 larvas/estádio com picrosirius red e Oil Red O, na região anterior da L1, o início do desenvolvimento de hastes quitinosas, a porção inicial do intestino e o primórdio genital. Na região anterior de L2, papilas, bastões quitinosos justapostos à boca e ao intestino maior que em L1. A musculatura de L3 é bem desenvolvida, próximo às hastes quitinosas, há duas estruturas redondas dispostas distalmente uma da outra. Observaram-se também toda a extensão do intestino, o primórdio genital e a intensa celularidade na porção distal da L3. Com o picrosirius red observou-se a musculatura de L1, L2 e L3, assim como, gânglios nervosos na L3. Oil Red O revelou que L1, L2 e L3 armazenam energia em gotículas lipídicas.

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RESUMO

Angiostrongylus vasorum is a pulmonary artery parasite of domestic and wild canid. On molluscs, intermediate host, first stage larvae (L1) are found after the first day of infection, in the 8th L2 and in the 30 th L3. It was evaluated L1, L2 and L3 recovered by Baermann technique from Achatina fulica infected with 1000 L1. Fifty larvae/stage were incubated with antibodies anti-β-tubulin, anti-α-tubulin, anti- α-actin, anti-β-actin and anti-collagen, and then with Alexa 633. Fifty larvae/stage were observed with picrosirius red and Oil Red O. It was also observed in the anterior region of L1 the beginning of the chitinous stems development, in the initial portion of the intestine and genital primordium. In L2 anterior region, the papillae, chitinous canes juxtaposed to the mouth and intestines bigger than L1. The L3 musculature is well defined, next to the chitinous stems, there are two round distally arranged from each other. It was observed the whole extension of the intestine genital primordium and intense cellularity in the L3 distal portion. With the picrosirius red the L1, L2 and L3 musculature could be observed, as the nerve ganglia on L3. Oil Red O revealed that L1, L2 and L3 store energy on lipid droplets.(AU)

Angiostrongylus vasorum é um parasito de artérias pulmonares dos canídeos domésticos e silvestres. Nos moluscos, hospedeiros intermediários, encontram-se no primeiro dia após a infecção, larvas de primeiro estágio (L1), ao 8° L2 e ao 30° L3. Avaliou-se L1, L2 e L3 recuperadas pela técnica de Baermann de Achatina fulica infectada com 1.000 L1. Incubou-se 50 larvas/estádio com anticorpos anti-β-tubulina, anti-α-tubulina, anti-β-actina e anti-colágeno e, em seguida, com anticorpo Alexa 633. Observaram-se também 50 larvas/estádio com picrosirius red e Oil Red O, na região anterior da L1, o início do desenvolvimento de hastes quitinosas, a porção inicial do intestino e o primórdio genital. Na região anterior de L2, papilas, bastões quitinosos justapostos à boca e ao intestino maior que em L1. A musculatura de L3 é bem desenvolvida, próximo às hastes quitinosas, há duas estruturas redondas dispostas distalmente uma da outra. Observaram-se também toda a extensão do intestino, o primórdio genital e a intensa celularidade na porção distal da L3. Com o picrosirius red observou-se a musculatura de L1, L2 e L3, assim como, gânglios nervosos na L3. Oil Red O revelou que L1, L2 e L3 armazenam energia em gotículas lipídicas.(AU)

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Canine endocrinopathies, such as hypothyroidism and hyperadrenocortism,induce typical dermatological alterations. Collagen fibers are significant for the maintenance of structural integrity,as well as in the determination of tissue function. This study aimed at assessing the coloration caused by Picrosirius Red staining under circular polarization and Masson Trichrome staining, as tools to quantify the total collagen in the skin of dogs exhibiting endocrine dermatopathies. Skin samples taken from dogs with hypothyroidism and hyperadrenocorticism were stained using Hematoxylin and Eosin (HE), Massons Trichrome (MT) and Picrosirius Red (PSR). The animals with hyperadrenocorticism revealed a higher percentage of collagen area than did the animals with hypothyroidism PSRp (hypothyroidism = 43.76 ± 0.8255 and hyperadrenocorticism = 47.08 ± 0.8584). The percentage of the collagen area using MT between the groups is given in parenthesis (hypothyroidism = 52.06 ± 0.9371, hyperadrenocorticism = 61 ± 0.7529 and control = 56.88 ± 0.64) (p <0.05). It is therefore, evident that the special stains employed are useful in estimating the percentage of collagen area in the skin, as they revealed that dogs with hypothyroidism had lower collagen deposition whereas those animals with hyperadrenocorticism showed higher quantity of collagen in the dermis.(AU)

Endocrinopatias caninas como hipotireoidismo e o hiperadrenocortismo causam alterações dermatológicas características. As fibras de colágeno desempenham um papel vital na manutenção da integridade estrutural e também na determinação da função dos tecidos. O objetivo deste estudo foi avaliar as colorações de picrosirius red sob polarização circular e coloração tricômio de Masson como ferramentas para quantificar o colágeno total da pele de cães com dermatopatias endócrinas. Amostras de pele obtidas de cães com hipotireoidismo e hiperadrenocorticismo foram coradas por Hematoxilina e Eosina (HE), Tricrômio de Masson (MT) e Picrosirius red (PSR). A porcentagem de área de colágeno foi maior nos animais com hiperadrenocorticismo e menor nos animais com hipotireoidismo PSRp (hipotireoidismo = 43,76 ± 0.8255 e hiperadrenocorticismo = 47,08 ± 0.8584). Já a porcentagem de área de colágeno no MT entre os grupos (hipotireoidismo = 52,06 ± 0.9371, hiperadrenocorticismo = 61 ± 0,7529 e controle = 56,88 ± 0.64) (p <0.05). Concluiu-se que as colorações especiais utilizadas são úteis para avaliar a porcentagem de área de colágeno na pele, pois demonstraram que cães com hipotireoidismo demonstraram uma diminuição na deposição de colágeno e os animais com hiperadrenocorticismo apresentaram maior quantidade de colágeno na derme.(AU)

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This study aimed to investigate collagen (Coll-I, III, IV) and elastin in canine normal prostate and prostate cancer (PC) using Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Coll-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle, and 5% surrounding blood vessels, in both normal prostate and PC. There was a higher median area of Coll-III in PC when compared to normal prostatic tissue (p = 0.001 for PSR and p = 0.05 for IHC). Immunostaining for Coll-IV was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and nerve fibers of normal and PC samples. Although there was no difference in Coll-IV area between normal tissue and PC, tumors with Gleason score 10 showed absence of Coll-IV, when compared to scores 6 and 8 (p = 0.0095). Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples and were statistically higher in PC compared to normal tissue (p = 0.00229). Investigation of ECM components brings new information and should be correlated with prognosis in future studies.

RESUMO

OBJECTIVE: The aim of the study was to analyze bone matrix (BMX) organization after bone grafting and repair using a new bioactive glass-ceramic (Biosilicate®) associated or not with particulate autogenous bone graft. MATERIAL AND METHODS: Thirty rabbits underwent surgical bilateral parietal defects and divided into groups according to the materials used: (C) control-blood clot, (BG) particulate autogenous bone, (BS) bioactive glass-ceramic, and BG + BS. After 7, 14, and 30 days post-surgery, a fragment of each specimen was fixed in - 80 °C liquid nitrogen for zymographic evaluation, while the remaining was fixed in 10% formalin for histological birefringence analysis. RESULTS: The results of this study demonstrated that matrix organization in experimental groups was significantly improved compared to C considering collagenous organization. Zymographic analysis revealed pro-MMP-2, pro-MMP-9, and active (a)-MMP-2 in all groups, showing gradual decrease of total gelatinolytic activity during the periods. At day 7, BG presented more prominent gelatinolytic activity for pro-MMP-2 and 9 and a-MMP-2, when compared to the other groups. In addition, at day 7, a 53% activation ratio (active form/[active form + latent form]) was evident in C group, 33% in BS group, and 31% in BG group. CONCLUSION: In general, BS allowed the production of a BMX similar to BG, with organized collagen deposition and MMP-2 and MMP-9 disponibility, permitting satisfactory bone remodeling at the late period. CLINICAL RELEVANCE: The evaluation of new bone substitute, with favorable biological properties, opens the possibility for its use as a viable and efficient alternative to autologous bone graft.

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Canine endocrinopathies, such as hypothyroidism and hyperadrenocortism,induce typical dermatological alterations. Collagen fibers are significant for the maintenance of structural integrity,as well as in the determination of tissue function. This study aimed at assessing the coloration caused by Picrosirius Red staining under circular polarization and Masson Trichrome staining, as tools to quantify the total collagen in the skin of dogs exhibiting endocrine dermatopathies. Skin samples taken from dogs with hypothyroidism and hyperadrenocorticism were stained using Hematoxylin and Eosin (HE), Masson’s Trichrome (MT) and Picrosirius Red (PSR). The animals with hyperadrenocorticism revealed a higher percentage of collagen area than did the animals with hypothyroidism PSRp (hypothyroidism = 43.76 ± 0.8255 and hyperadrenocorticism = 47.08 ± 0.8584). The percentage of the collagen area using MT between the groups is given in parenthesis (hypothyroidism = 52.06 ± 0.9371, hyperadrenocorticism = 61 ± 0.7529 and control = 56.88 ± 0.64) (p <0.05). It is therefore, evident that the special stains employed are useful in estimating the percentage of collagen area in the skin, as they revealed that dogs with hypothyroidism had lower collagen deposition whereas those animals with hyperadrenocorticism showed higher quantity of collagen in the dermis.

Endocrinopatias caninas como hipotireoidismo e o hiperadrenocortismo causam alterações dermatológicas características. As fibras de colágeno desempenham um papel vital na manutenção da integridade estrutural e também na determinação da função dos tecidos. O objetivo deste estudo foi avaliar as colorações de picrosirius red sob polarização circular e coloração tricômio de Masson como ferramentas para quantificar o colágeno total da pele de cães com dermatopatias endócrinas. Amostras de pele obtidas de cães com hipotireoidismo e hiperadrenocorticismo foram coradas por Hematoxilina e Eosina (HE), Tricrômio de Masson (MT) e Picrosirius red (PSR). A porcentagem de área de colágeno foi maior nos animais com hiperadrenocorticismo e menor nos animais com hipotireoidismo PSRp (hipotireoidismo = 43,76 ± 0.8255 e hiperadrenocorticismo = 47,08 ± 0.8584). Já a porcentagem de área de colágeno no MT entre os grupos (hipotireoidismo = 52,06 ± 0.9371, hiperadrenocorticismo = 61 ± 0,7529 e controle = 56,88 ± 0.64) (p <0.05). Concluiu-se que as colorações especiais utilizadas são úteis para avaliar a porcentagem de área de colágeno na pele, pois demonstraram que cães com hipotireoidismo demonstraram uma diminuição na deposição de colágeno e os animais com hiperadrenocorticismo apresentaram maior quantidade de colágeno na derme.

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RESUMO

Phytoterapic compounds have been used in wound healing for many centuries. Nowadays, scientific evidences of phytotherapeutics is a requirement of the legislation. The scientific literature notes the need for healing topics yielding scars that are both aesthetically appealing and resistant. We aimed to evaluate the cytotoxicity of several doses of T. aestivum extract (2 mg mL-1, 4 mg mL-1, 6 mg mL-1, 8 mg mL-1 and 10 mg mL-1) in a fibroblast cell line and the healing process in an in vivo experimental model (New Zealand rabbits). For this, MTT test in 3T6 cells was performed in duplicates using MEM (0 mg ml-1) as negative control. Cell viability was calculated as: absorbance average in treatments/absorbance average in controls x 100. In vivo test was performed in 78 skin wounds in rabbits that were treated with 2 mg ml-1and 10 mg ml-1 of T. aestivum and non-ionic cream for 21 days. After this period, it was evaluated the histology using picrosorius and Gomoris trichrome staining. Statistical analysis was evaluated using T test (Graphpad) for cytotoxicity assay, Fischer test for the gomori trichrome test (Grahpad) and Kruskal-Wallis (Statistic 9.0) for picrosirius test. The in vitro test resulted in cytotoxicity observed at 2mg mL-1 whereas cells were viable at higher doses. On the other hand, it was observed that collagen formation of wounds was more uniform with this dose than with 10mg mL-1 extract in the in vivo study. Thus, we conclude that the 2mg mL-1 T. aestivum aqueous extract dose was more efficient in the in vivo wound healing study, despite its cytotoxic effects in vitro.(AU)

Os extratos vegetais têm sido utilizados na cicatrização de feridas a muitos séculos. No entanto nos dias atuais a comprovação científica dos fitoterápicos é uma exigência da legislação. Na literatura científica se observa a necessidade de cicatrizantes tópicos que proporcionem uma cicatriz estética e resistente. Devido a isso objetivou-se avaliar a citotoxicidade de diversas doses de T. aestivum (2 mg mL1, 4 mg mL-1, 6 mg mL-1, 8 mg mL-1 e 10 mg mL-1) em linhagem celular de fibroblasto, e o processo cicatricial em modelo experimental (New Zealand rabbits) in vivo. Para isso foi realizado o teste de MTT em linhagem celular 3T. Tests were performed in duplicates, using MEM (0 mg mL-1) as negative control. Cell viability was calculated as: absorbance average in treatments/absorbance average in controls x 100. No ensaio in vivo foram realizadas 78 feridas experimentais em coelhos que foram tratadas T. aestivum 2mg mL-1, T. aestivum 10 mg mL-1e creme não iônico por 21 dias, após foi avaliado a histologia do tricrômico de golmori e de picrosirius. Na análise estatística do ensaio de citotoxicidade foi realizado o teste de t (Graphpad), para avaliação do tricomico de golmori foi realizado o teste de fischer (Graphpad) e no picrosirius foi avaliado através de Kruskal - Wallis (Statistic 9.0). O resultado in vitro demonstrou que a dose de 2mg mL-1 foi citotóxica para as células e que doses maiores a essa apresentavam viabilidade celular. No entanto no estudo in vivo foi constatado que as feridas tratadas com essa dose apresentaram a formação de colágeno mais uniforme que as tratadas com 10 mg mL-1. Concluí-se que a dose de 2mg mL-1 do extrato aquoso de T. aestivum é eficiente no ensaio in vivo com as feridas experimentais, o que não foi observado no estudo in vitro.(AU)

Assuntos

RESUMO

Phytoterapic compounds have been used in wound healing for many centuries. Nowadays, scientific evidences of phytotherapeutics is a requirement of the legislation. The scientific literature notes the need for healing topics yielding scars that are both aesthetically appealing and resistant. We aimed to evaluate the cytotoxicity of several doses of T. aestivum extract (2 mg mL-1, 4 mg mL-1, 6 mg mL-1, 8 mg mL-1 and 10 mg mL-1) in a fibroblast cell line and the healing process in an in vivo experimental model (New Zealand rabbits). For this, MTT test in 3T6 cells was performed in duplicates using MEM (0 mg ml-1) as negative control. Cell viability was calculated as: absorbance average in treatments/absorbance average in controls x 100. In vivo test was performed in 78 skin wounds in rabbits that were treated with 2 mg ml-1and 10 mg ml-1 of T. aestivum and non-ionic cream for 21 days. After this period, it was evaluated the histology using picrosorius and Gomori’s trichrome staining. Statistical analysis was evaluated using T test (Graphpad) for cytotoxicity assay, Fischer test for the gomori trichrome test (Grahpad) and Kruskal-Wallis (Statistic 9.0) for picrosirius test. The in vitro test resulted in cytotoxicity observed at 2mg mL-1 whereas cells were viable at higher doses. On the other hand, it was observed that collagen formation of wounds was more uniform with this dose than with 10mg mL-1 extract in the in vivo study. Thus, we conclude that the 2mg mL-1 T. aestivum aqueous extract dose was more efficient in the in vivo wound healing study, despite its cytotoxic effects in vitro.

Os extratos vegetais têm sido utilizados na cicatrização de feridas a muitos séculos. No entanto nos dias atuais a comprovação científica dos fitoterápicos é uma exigência da legislação. Na literatura científica se observa a necessidade de cicatrizantes tópicos que proporcionem uma cicatriz estética e resistente. Devido a isso objetivou-se avaliar a citotoxicidade de diversas doses de T. aestivum (2 mg mL1, 4 mg mL-1, 6 mg mL-1, 8 mg mL-1 e 10 mg mL-1) em linhagem celular de fibroblasto, e o processo cicatricial em modelo experimental (New Zealand rabbits) in vivo. Para isso foi realizado o teste de MTT em linhagem celular 3T. Tests were performed in duplicates, using MEM (0 mg mL-1) as negative control. Cell viability was calculated as: absorbance average in treatments/absorbance average in controls x 100. No ensaio in vivo foram realizadas 78 feridas experimentais em coelhos que foram tratadas T. aestivum 2mg mL-1, T. aestivum 10 mg mL-1e creme não iônico por 21 dias, após foi avaliado a histologia do tricrômico de golmori e de picrosirius. Na análise estatística do ensaio de citotoxicidade foi realizado o teste de t (Graphpad), para avaliação do tricomico de golmori foi realizado o teste de fischer (Graphpad) e no picrosirius foi avaliado através de Kruskal - Wallis (Statistic 9.0). O resultado in vitro demonstrou que a dose de 2mg mL-1 foi citotóxica para as células e que doses maiores a essa apresentavam viabilidade celular. No entanto no estudo in vivo foi constatado que as feridas tratadas com essa dose apresentaram a formação de colágeno mais uniforme que as tratadas com 10 mg mL-1. Concluí-se que a dose de 2mg mL-1 do extrato aquoso de T. aestivum é eficiente no ensaio in vivo com as feridas experimentais, o que não foi observado no estudo in vitro.

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RESUMO

Chronic kidney disease (CKD) is a relevant disease in feline clinic. The tubulointerstitial damage, with collagen deposition and fibrosis, is an important result of this process. The aim of this study was to quantify and correlate the deposition of collagen and severity of interstitial fibrosis (IF) in the kidney from cats in different stages of CKD. Kidney fragments from 10 adult cats with CKD were analyzed and stained by Masson's trichrome (MT) and Picrosirius red (PSR) for circular polarized microscopy. Random quantitative analysis was performed on MT sections to classify the degree of IF, per field area, with and without circular polarization. Statistics correlations were performed by Spearman's (ρ; p < .05). There was a significant correlation of IF quantification with the area of interstitial collagen deposition by polarized PSR (PSRp) (r = .7939, p = .0098) and nonpolarized PSR (PSRn) (r = .7781, p = .0080). There was a positive correlation of serum creatinine (sCr) at different stages of CKD with PSRp (r = .7939, p = .0098), PSRn (r = .8667, p = .0027) and MT (r = .7818, p = .0117). Correlations between the percentage of quantified area was also positive from PSRp to PSRn (r = .9030, p = .0009) and PSRp to MT (r = .7939, p = .0098). The PSRN was also correlated with MT (r = .9273, p = .0001). The correlation with IF and sCr follows the disease evolution and the quantification of collagen by PSR is an excellent tool for analyzing the disease severity at different stages.

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RESUMO

Collagen plays essential roles in remodeling uterine tissue during decidualization, implantation, pregnancy and involution. To understand whether the progestational agent medroxyprogesterone acetate (MPA) can modify the organization and deposit of collagen in the uteri of normal bitches (Canis Tlupus familiaris), we assessed uterine tissues by histochemistry. Uteri were grouped as: nulliparous (n=11), multiparous (n=11) and treated with MPA (n=11; nulliparous with two treatments; 5mg/kg; i.m.). The amount, location and birefringence of interstitial collagen types I and III in the fold and base of the endometrial stroma and the myometrial muscular layers were studied on sections stained with Picrosirius Red by polarized light microscopy and evaluated by ANOVA. No differences were observed in the endometrium. In the myometrium, differences were observed in collagen type I between MPA-treated and nulliparous uteri vs. multiparous (p<0.05), and differences in collagen type III between nulliparous and multiparous uteri vs. MPA-treated (p=0.0001). In conclusion, two doses of MPA had no significant effect on the investigated collagens in the extracellular matrix.(AU)

Assuntos

RESUMO

Collagen plays essential roles in remodeling uterine tissue during decidualization, implantation, pregnancy and involution. To understand whether the progestational agent medroxyprogesterone acetate (MPA) can modify the organization and deposit of collagen in the uteri of normal bitches (Canis Tlupus familiaris), we assessed uterine tissues by histochemistry. Uteri were grouped as: nulliparous (n=11), multiparous (n=11) and treated with MPA (n=11; nulliparous with two treatments; 5mg/kg; i.m.). The amount, location and birefringence of interstitial collagen types I and III in the fold and base of the endometrial stroma and the myometrial muscular layers were studied on sections stained with Picrosirius Red by polarized light microscopy and evaluated by ANOVA. No differences were observed in the endometrium. In the myometrium, differences were observed in collagen type I between MPA-treated and nulliparous uteri vs. multiparous (p<0.05), and differences in collagen type III between nulliparous and multiparous uteri vs. MPA-treated (p=0.0001). In conclusion, two doses of MPA had no significant effect on the investigated collagens in the extracellular matrix.(AU)

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RESUMO

CONTEXT: The quantification of total collagen is of major importance in a wide range of research areas, including the study of cutaneous wound healing and new drugs trials. OBJECTIVE: The total collagen content in skin biopsies was compared by biochemical hydroxyproline assay and by two computer-aided histomorphometric analyses of histological sections. MATERIALS AND METHODS: Two methods were used to evaluate collagen formation: the hydroxyproline assay, as the gold standard and histomorphometric image analysis of the filled areas by corresponding stained collagen fibres, using picrosirius and Gomori's trichrome staining. The image analyses were determined by digital densitometry recognition using computer-aided ImageJ software. One-way ANOVA, simple linear regression and ANCOVA were applied for the statistical analysis and correlation. RESULTS: In a simple linear regression analysis carried out on the 14th day period after the induction of skin injury, three techniques, picrosirius red (F = 33.57, p = 0.00), Gomori's trichrome (F = 81.61, p = 0.00) and hydroxyproline content (F = 16.85, p = 0.00) were able to detect collagen production. After scale adjustment, there were no significant differences among either the slopes (F = 1.17, p = 0.32) or the intercepts (F = 0.69, p = 0.51) of the estimated regression lines. It seems that a highly significant correlation exists between the histomorphometrical analysis and hydroxyproline assay. DISCUSSION AND CONCLUSION: The morphometric analysis proved to be adequate and can be used as a simple, rapid, low-cost technology for evaluating total collagen in cutaneous wound specimens, compared with the gold standard hydroxyproline assay.

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