Curcumin alleviated oxidation stress injury by mediating osteopontin in nephrolithiasis rats

Purpose: To explore the role and mechanism of curcumin (Cur) in reducing oxidative stress damage in rats with nephrolithiasis induced by ethylene glycol (EG). Methods: Thirty male rats were divided into normal control, model, positive (10% potassium citrate), Cur-10 (10 mg/kg curcumin) and Cur-20 (20 mg/kg curcumin) groups. Results: The results of kidney tissue section stained by hematoxylin-eosin and von Kossa showed that curcumin treatment can inhibit the formation of kidney stones. The biochemical test results showed that the urea (Ur), creatinine (Cr), uric acid (UA), inorganic phosphorus and Ca2+ concentrations in urine decreased after being treated with curcumin. There were significant differences between different doses of curcumin (P < 0.05). Compared with the Cur-10 group, Cur-20 had a more significant inhibitory effect on malondialdehyde (MDA) (P < 0.05). In addition, reverse transcription polymerase chain reaction (PCR) detection and immunohistochemical results indicated that the osteopontin (OPN) in the kidney was significantly reduced after curcumin treatment. Conclusion: Curcumin could reduce the oxidative stress damage caused by EG-induced kidney stones.

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas

ABSTRACT: Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P 0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.

RESUMO: A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P 0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas / Expressão de osteopontina e sua relação com biomarcadores prognóstico nos carcinomas mamários caninos

Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P<0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.(AU)

A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P<0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.(AU)

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas / Expressão de osteopontina e sua relação com biomarcadores prognóstico nos carcinomas mamários caninos

Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P<0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.(AU)

A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P<0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.(AU)

The effect of graft application and allopurinol treatment on calvarial bone defect in rats

Purpose:To investigate the effects of allopurinol administration on osteoinductive reaction and bone development with graft material.Methods:Thirty-six Wistar albino rats were divided into 3 groups. In the control group, calvarial bone defect was only created without any treatment. In the Defect + Graft group, allograft treatment was performed by forming 8 mm calvarial bone defect. In the Defect + Graft + Allopurinol group, alloplastic bone graft was placed in the calvarial bone defect and then, allopurinol (50 mg/kg/day) treatment was intraperitoneally applied for 28 days.Results:Histopathological examination revealed inflammation, congestion in the vessels, and an increase in osteoclast cells in the defect area. We also observed that new osteocyte cells, increase in connective tissue fibers, and new bone trabeculae. Osteopontin expression was positive in osteoblast cells and lacunated osteocyte cells were located in the periphery of the new bone trabeculae. Osteopontin expression was also positive in osteoblasts and osteocytes cells of new bone trabeculae in the graft site.Conclusion:It has been shown that allopurinol treatment in rat calvaria defects may induce osteoblastic activity, matrix development, mature bone cell formation and new bone formation when used with autogenous grafts.(AU)

The effect of graft application and simvastatin treatment on tibial bone defect in rats. A histological and immunohistochemical study

Purpose: To evaluate histologically and immunohistochemically the bone regeneration after application of simvastatin on tibial bone defects in rats. Methods: Sixty Wistar albino rats were divided into 3 groups as control (6 mm tibial bone defect), defect + graft (allograft treatment), and defect + graft + simvastatin (10 mg/kg/day) for 28 days. Results: Histopathological examination revealed inflammation in control group (defect group), congestion in blood vessels, and an increase in osteoclast cells. In defect + graft group, osteoclastic activity was observed and osteocyte cells were continued to develop. In defect + graft + simvastatin group, osteocytes and matrix formation were increased in the new bone trabeculae. Osteopontin and osteonectin expression were positive in the osteclast cells in the control group. Osteoblasts and some osteocytes showed a positive reaction of osteopontin and osteopontin. In defect + graft + simvastatin group, osteonectin and osteopontin expression were positive in osteoblast and osteocyte cells, and a positive expression in osteon formation was also seen in new bone trabeculae. Conclusion: The simvastatin application was thought to increase bone turnover by increasing the osteoinductive effect with graft and significantly affect the formation of new bone.(AU)

Effect of low power laser in biomodulation of cultured osteoblastic cells of Wistar rats

Purpose:To analyze aspects of the biomodulating effect of light in biological tissues, bone cells from surgical explants of the femur of rats were irradiated with low intensity laser.Methods:Bone cells were cultured and irradiated with LASER light (GaAlAs). Growth, cell viability, mineralized matrix formation, total protein dosage, immunostimulatory properties, cytochemical analysis, gene expression of bone proteins were examined using live cell imaging and cell counting by colorimetric assay. The gene expression of: alkaline phosphatase (ALP), type 1 collagen, osteocalcin and osteopontin through the real-time polymerase chain reaction.Results:At 8 days, the viability of the irradiated culture was 82.3% and 72.4% in non-irradiated cells. At 18 days, the cellular viability (with laser) was 77.42% and 47.62% without laser. At 8 days, the total protein concentration was 21.622 mg / mol in the irradiated group and 16, 604 mg / mol in the non-irradiated group and at 18 days the concentration was 37.25 mg / mol in the irradiated group and 24, 95 mg / mol in the non-irradiated group.Conclusion:The laser interfered in the histochemical reaction, cell viability, matrix mineralization, and maintained the cellular expression of proteins.(AU)

Short-term use of resveratrol in alloplastic graft material applied with calvarial bone defects in rats

Purpose: The effects of resveratrol administration on calvarial bone defects with alloplastic graft material was investigated for osteoinductive reaction and bone development in rats. Methods: Healthy male rats were randomly divided into 3 groups consisting of 10 rats. Groups were as follows: control (defect) group, defect + graft group, and defect + graft + resveratrol group. A calvarial bone defect was created in all groups, alloplastic bone grafts were applied to the defect in the 2nd and 3rd group, resveratrol (5 mg/kg/day) was added to the drinking water of the animals following graft application for 28 days in the 3rd group. Results: Increase in osteoclasts and necrotic changes were observed histopathologically in the control group. In the 2nd group, reduction of inflammation, congestion of blood vessels, increased osteblastic activity, osteoinductive effect, progression of osteocyte development and increased collagen fibers in connective tissue were observed. In the 3rd group, osteoblasts seemed to secrete bone matrix and accelerate osteoinductive effect with increased osteopregenitor activity and positive osteopontin and osteonectin expressions. Conclusion: Resveratrol treatment was thought to be an alternative and supportive drug for implant application by inducing new bone formation in the calvaral defect region as a result of short-term treatment.(AU)

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin

ABSTRACT The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.

RESUMO O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin / Diferenciação osteogênica de células tronco mesenquimais do tecido adiposo cultivadas com diferentes concentrações de prolactina

The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.(AU)

O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.(AU)

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin / Diferenciação osteogênica de células tronco mesenquimais do tecido adiposo cultivadas com diferentes concentrações de prolactina

The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.(AU)

O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.(AU)

In vitro effects of triiodothyronine on the reduced osteogenic potential of bone marrow mesenchymal stem cells of rats with osteoporosis

Objective: Evaluate the effect of in vitro triiodothyronine (T3) on the reduced osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) of adult rats with osteoporosis compared with BMMSCs of young and adult rats without osteoporosis. Methods: groups were tested: BMMSCs of young rats; BMMSCs of adult rats without osteoporosis; BMMSCs of adult rats with osteoporosis without T3 and BMMSCs of adult rats with osteoporosis treated with T3 (0.01, 1, 100 and 1000 nM). Alkaline phosphatase activity, MTT reduction, mineralized nodules and gene expression for collagen, osteocalcin, sialoprotein, osteopontin and BMP-2 were evaluated. Results: Osteoporosis increased the alkaline phosphatase activity and reduced the formation of mineralized nodules and expression of collagen and osteopontin in at least one of the observed time points. However, the T3 treatment of BMMSCs of rats with osteoporosis altered these parameters. Conclusion: It was concluded that doses of T3, 0.01 and 1000 nM had a positive effect promoted by increased osteogenic matrix synthesis and collagen expression in at least one of the evaluated time points compared to BMMSCs of rats with osteoporosis without treatment. However, T3 was unable to reach the osteogenic potential of the MSCs of healthy young rats.(AU)

Avaliação in vitro de condrócitos da cartilagem articular e da diferenciação osteogênica das células tronco da medula óssea extraídas da prole de ratas tratadas com etanol

Foram realizados dois estudos para avaliar os efeitos do consumo materno de etanol sobre os condrócitos da cartilagem articular e na diferenciação osteogênica das células tronco mesenquimais da medula óssea (CTM-MO) da prole. Foram utilizadas 13 ratas Wistar adultas distribuídas em dois grupos experimentais, grupo controle e o grupo tratado com etanol. As ratas do grupo etanol e controle, receberam por gavagem diária, a partir do nono dia de gestação, solução alcoólica 40% e água destilada, respectivamente até o trigésimo dia de lactação. No dia do parto, foram eutanasiados quatro neonatos por fêmea e, aos 30 dias de lactação, três filhotes por fêmea. O primeiro estudo avaliou, in vitro, o efeito do consumo materno de etanol sobre os condrócitos da cartilagem articular. Os condrócitos foram extraídos das cartilagens articulares e cultivados em meio condrogênico por sete, 14 e 21 dias. Foram realizados os testes de conversão do MTT, atividade de fosfatase alcalina, porcentagem de células por campo e porcentagem de áreas PAS+ em cultura 3D. Aos 21 dias, foi realizada a quantificação dos transcritos gênicos para agrecano, Sox-9, colágeno tipo II, colágeno X, Runx-2 e VEGF pelo RT-PCR em tempo real. O segundo estudo avaliou o efeito do consumo materno de etanol durante a gestação e lactação sobre a diferenciação osteogênica CTM-MO dos filhotes ao desmame. As CTM-MO foram extraídas dos membros pélvicos e cultivadas em meio osteogênico por sete, 14 e 21 dias. Foram realizados os testes de conversão do MTT, atividade de fosfatase alcalina e porcentagem de células por campo. Aos 21 dias, foram realizados a quantificação do tamanho médio dos nódulos mineralizados e a quantificação dos transcritos gênicos para osteopontina, osteocalcina, BMP-2, Runx-2 e colágeno tipo I por RT-PCR em tempo real. As médias foram comparadas pelo teste t de student e as diferenças foram consideradas significativas se p<0,05. Os neonatos das mães que receberam etanol foram menos pesados quando comparados aos neonatos controle. A cultura dos condrócitos do grupo etanol apresentou maior conversão de MTT, maior atividade de fosfatase alcalina e maior porcentagem de células relação ao grupo controle em todos os períodos. Não houve diferença entre ambos os grupos na porcentagem de áreas PAS+ em cultura 3D. Houve maior expressão de colágeno tipo II e menor expressão de Sox-9 no grupo etanol 15 em relação ao grupo controle. O peso dos filhotes, ao desmame, das mães que receberam etanol foi significativamente menor quando comparado ao controle. As CTM-MO do grupo etanol apresentaram menor conversão de MTT aos sete dias de diferenciação osteogênica porém maior atividade de fosfatase alcalina, maior porcentagem de células, maior tamanho médio dos nódulos mineralizados bem como maior expressão de BMP-2, osteopontina e osteocalcina. Conclui-se que o consumo materno de etanol durante a gestação e lactação altera, in vitro, o fenótipo e a atividade de síntese dos condrócitos dos neonatos bem como aumenta a diferenciação osteogênica das CTM-MO da prole ao desmame

Two studies were performed to evaluate the effects of maternal ethanol consumption on articular cartilage chondrocytes and on osteogenic differentiation of offspring bone marrow mesenchymal stem cells (BMMSCs). Thirteen adult female Wistar rats were divided into two experimental groups, the control group and the ethanol-treated group. The rats of the ethanol and control group received by daily gavage, from the ninth day of gestation, 40% alcohol solution and distilled water, respectively until the thirtieth day of lactation. On the day of delivery, four newborns were euthanized per female and, at 30 days of lactation, three pups per female. The first study evaluated in vitro the effect of maternal ethanol consumption on articular cartilage chondrocytes. Chondrocytes were extracted from articular cartilage and cultivated in chondrogenic medium for seven, 14 and 21 days. MTT conversion tests, alkaline phosphatase activity, percentage of cells per field and percentage of PAS+ areas in 3D culture were performed. At 21 days, the quantification of gene transcripts for agrecan, Sox-9, collagen type II, collagen X, Runx-2 and VEGF was performed by real-time RT-PCR. The second study evaluated the effect of maternal ethanol consumption during pregnancy and lactation on BMMSCs osteogenic differentiation of pups at weaning. The BMMSCs were extracted from the pelvic limbs and cultured in osteogenic medium for seven, 14 and 21 days. MTT conversion, alkaline phosphatase activity and cell percentage per field tests were performed. At 21 days, the average size of the mineralized nodules was quantified and the gene transcripts were quantified for osteopontin, osteocalcin, BMP-2, Runx-2 and collagen I by real-time RT-PCR. Means were compared by Student's t-test and differences were considered significant if p <0.05. Neonates of mothers receiving ethanol were less heavy when compared to control neonates. The chondrocyte culture of the ethanol group showed higher MTT conversion, higher alkaline phosphatase activity and higher percentage of cells compared to the control group in all periods. There was no difference between both groups in the percentage of PAS+ areas in 3D culture. There was higher expression of collagen type II and lower expression of Sox-9 in the ethanol group compared to the control group. The weight of pups at weaning of mothers receiving ethanol was significantly lower when compared to control. BMMSCs ethanol group showed lower MTT conversion after seven days of osteogenic differentiation but 17 higher alkaline phosphatase activity, increased percentage of cells larger average size of mineralized nodules as well as increased expression of BMP-2, osteopontin and osteocalcin. In conclusion, maternal ethanol consumption during pregnancy and lactation alters, in vitro, the phenotype and chondrocyte synthesis activity of neonates, as well as increases the osteogenic differentiation of BMMSCs from weaning offspring

In vitro effects of triiodothyronine on the reduced osteogenic potential of bone marrow mesenchymal stem cells of rats with osteoporosis

Objective: Evaluate the effect of in vitro triiodothyronine (T3) on the reduced osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) of adult rats with osteoporosis compared with BMMSCs of young and adult rats without osteoporosis. Methods: groups were tested: BMMSCs of young rats; BMMSCs of adult rats without osteoporosis; BMMSCs of adult rats with osteoporosis without T3 and BMMSCs of adult rats with osteoporosis treated with T3 (0.01, 1, 100 and 1000 nM). Alkaline phosphatase activity, MTT reduction, mineralized nodules and gene expression for collagen, osteocalcin, sialoprotein, osteopontin and BMP-2 were evaluated. Results: Osteoporosis increased the alkaline phosphatase activity and reduced the formation of mineralized nodules and expression of collagen and osteopontin in at least one of the observed time points. However, the T3 treatment of BMMSCs of rats with osteoporosis altered these parameters. Conclusion: It was concluded that doses of T3, 0.01 and 1000 nM had a positive effect promoted by increased osteogenic matrix synthesis and collagen expression in at least one of the evaluated time points compared to BMMSCs of rats with osteoporosis without treatment. However, T3 was unable to reach the osteogenic potential of the MSCs of healthy young rats.

Efeito do consumo de etanol durante a gestação e lactação sobre a histomorfometria óssea e a diferenciação osteogênica, in vitro, das células tronco mesenquimais maternas da medula óssea de ratas

O objetivo do estudo foi avaliar o efeito do consumo materno de etanol durante a gestação e lactação sobre a massa óssea e sobre a diferenciação osteogênica in vitro das células tronco mesenquimais da medula óssea (CTM-MO) de ratas. Foram utilizadas 13 ratas Wistar adultas distribuídas em dois grupos: 1) controle e 2) tratado com etanol. As ratas do grupo etanol e controle, receberam diariamente, a partir do nono dia de gestação, solução alcoólica 40% (4g de etanol/kg) e água destilada, respectivamente até o trigésimo dia de lactação. As CTM-MO foram extraídas dos fêmures e tíbias direitas e cultivadas em meio osteogênico por sete, 14 e 21 dias. Foram realizados os testes de conversão do MTT em cristais de formazan, atividade de fosfatase alcalina e avaliação da porcentagem de células por campo. Aos 21 dias, foram realizadas as quantificações dos nódulos mineralizados por campo e dos transcritos gênicos para osteopontina, osteocalcina e BMP-2 por RT-PCR tempo real. As avaliações morfológicas e morfométricas da porcentagem de osso trabecular e espessura do osso cortical foram realizadas nos fêmures e tíbias esquerdas. As médias foram comparadas pelo teste t de student e as diferenças foram consideradas significativas se p<0,05. As CTM-MO das ratas que consumiram etanol durante a gestação e lactação, quando submetidas a diferenciação osteogênica, in vitro, apresentaram maior conversão de MTT em formazan, maior atividade de fosfatase alcalina, maior porcentagem de células por campo, maior expressão de BMP-2 e maior síntese de nódulos mineralizados quando comparadas às células das ratas controle. Entretanto, não houve diferença significativa entre grupos, na porcentagem de tecido ósseo trabecular e na espessura da cortical. Conclui-se que, o consumo de etanol durante a gestação e lactação não altera os tecidos ósseos trabecular e cortical do fêmur e tíbia em comparação ao de ratas gestantes e lactantes controles, apesar das CTM-MO apresentarem, in vitro, maior diferenciação osteogênica caracterizada pela maior síntese de matriz mineralizada.

The aim of this study was to evaluate the effect of maternal ethanol consumption during gestation and lactation on bone mass and the osteogenic differentiation of mesenchymal stem cells of the bone marrow (BMMSCs) of rats. Thirteen adult Wistar rats were used. The rats were distributed in two groups: 1) control and 2) ethanol treated. The rats of the ethanol and control groups received daily, from the ninth day of gestation, 40% alcoholic solution and distilled water, respectively, until the thirtieth day of lactation. The BMMSCs were extracted from the right femurs and tibiae and cultured on osteogenic medium for 7, 14 and 21 days. The conversion of MTT to formazan crystals, alkaline phosphatase activity, and porcentage of cells per field were performed. The number of mineralized nodules per field and the quantification of the gene transcripts for osteopontin, osteocalcin and BMP-2 by real-time RT-PCR were performed at day 21. Morphometric evaluations of the percentage of trabecular bone and cortical thickness were performed in the left femur and tibia. The means were compared by the Students t test and the differences were considered significant if p < 0.05. The BMMSCs of the rats that consumed ethanol during gestation and lactation, when submitted to osteogenic differentiation in vitro, presented higher conversion of MTT to formazan, higher alkaline phosphatase activity, higher percentage of cells per field, higher expression of BMP-2 and higher synthesis of mineralized nodules when compared to that of control rat cells. However, there was no significant difference in the percentage of trabecular bone or cortical thickness between both groups. It is concluded that the consumption of ethanol during pregnancy and lactation did not alter the trabecular and cortical bone tissues of the femur and tibia compared with that of pregnant and lactating control rats that did not consume alcohol, despite in vitro BMMSCs showing greater osteogenic differentiation characterized by greater synthesis of mineralized matrix.

IMUNOLOCALIZAÇÃO DA OSTEOPONTINA E ASSINATURAS PROTEÔMICAS DO TRATO REPRODUTOR DE CÃES

Os estudos com a tecnologia ômica auxiliam a investigar o papel da sinalização proteica na maturação espermática, capacitação e fertilização. Apesar da importância de estudos sobre moléculas envolvidas na fertilidade, até o momento, na espécie canina, a proteômica do tecido reprodutor não foi descrita. Dessa forma, o objetivo desse estudo e a imunolocalização da osteopontina em testículo, cabeça, corpo e cauda do epidídimo (Artigo I) e descrever o proteoma destes tecidos (Artigo II). A escolha desta proteína foi devida a importância relacionada a fertilidade já demonstrada em outras espécies. Para realização do artigo I, foram colhidos de 3 cães adultos jovens entre 2 a 5 anos, testículos e epidídimos (cabeça, corpo e cauda), sem raça definida, após a orquiectomia eletiva, totalizando 24 amostras. Para realização do artigo II, testículos e epidídimos (cabeça, corpo e cauda) foram colhidos de 3 cães, adultos jovens entre 2 a 5 anos, amostras armazenadas em formalina tamponada durante 48 horas e transferidas para álcool 70%, as amostras passaram por desidratação e reidratação. Foi utilizado anticorpo anti-OPN, as amostras foram coradas com DAB. Todos os tecidos foram marcados com anticorpo, em células próximas a parte luminal do epidídimo e citoplasma, sem diferença significativa (P>0,05). As amostras foram marcadas em célula próximas a parte luminal do epidídimo e citoplasma. Apesar da imunomarcação da OPN no citoplasma das células, em todos os tecidos, esta proteína não foi identificada na abordagem shotgun. Também não houve diferença na imunomarcação da OPN entre os tecidos. No artigo II, foram encontradas 1.918 proteínas, sendo 132 utilizadas na análise multivariada, a qual incluiu a principal component analysis (PCA), variable importance in projection (VIP score) determinada pela partial least squares-discriminant analysis (PLS-DA), ANOVA, dendrograma e diagrama de Venn. Na PCA foram formados 4 clusters distintos, com proximidade dos clusters das amostras da cabeça e corpo do epidídimo. O VIP score indicou 20 proteínas ( > 1,5) e 18 foram confirmadas pelo ANOVA como mais relevantes nos clusters. Seis proteínas foram mais abundantes no corpo, 4 na cabeça do epidídimo e 10 no testículo. As proteínas inositol-3-phosphate synthase 1, equilibrative nucleoside transporter 1, tubulin alpha chain, heat shock protein 90 alpha family class A member 1, heat shock protein family A member 8, heat shock protein family A (HSP70) member 2 e cytochrome P450 family 17 subfamily A member 1, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta foram mais abundantes no testículo e reduziram gradativamente em direção a cauda do epidídimo. Concluímos que a abundância das proteínas nos tecidos do trato reprodutor está relacionada com a função dos compartimentos de cada compartimento, como o testículo, cabeça, corpo e cauda do epidídimo de cão pode possuir alguma proteína similiar a OPN que se ligue ao anticorpo, porém é necessários maiores investigações.

Studies with omic technology help to investigate the role of protein signaling in sperm maturation, capacitation and fertilization. Thus, the objective of this study is the immunolocation of osteopontin in the testis, head, body and cauda of the epididymis (Article I) and to describe the proteome of these tissues (Article II). The choice of this protein was due to the importance related to fertility already demonstrated in other species. Article I, 3 young adult dogs aged 2 to 5 years, testicles and epididymis (caput, corpus and cauda), of mixed breed, were collected after elective orchiectomy, totaling 24 samples. For the realization of article II, testicles and epididymis (caput, corpus and tail) were collected from 3 dogs, young adults aged 2 to 5 years, samples stored in buffered formalin for 48 hours and transferred to 70% alcohol, the samples were dehydrated and rehydration. Anti-OPN antibody was used, the samples were stained with DAB. All tissues were labeled with antibody in cells close to the luminal part of the epididymis and cytoplasm, with no significant difference (P> 0.05). Despite the immunostaining of OPN in the cytoplasm of cells, in all tissues, this protein was not identified in the shotgun approach. There was also no difference in the immunostaining of OPN between tissues. In article II, 1,918 proteins were found, 132 of which were used in multivariate analysis, which included the main component analysis (PCA), variable importance in projection (VIP score) determined by the partial least squares-discriminant analysis (PLS-DA), ANOVA , dendrogram and Venn diagram. In the PCA, 4 distinct clusters were formed, with proximity to the clusters of the head and body samples of the epididymis. The VIP score indicated 20 proteins (> 1.5) and 18 were confirmed by ANOVA as the most relevant in the clusters. Six proteins were more abundant in the body, 4 in the epididymis head and 10 in the testis. Inositol-3-phosphate synthase 1, equilibrative nucleoside transporter 1, tubulin alpha chain, heat shock protein 90 alpha family class A member 1, heat shock protein family A member 8, heat shock protein family A (HSP70) member 2 and cytochrome P450 family 17 subfamily A member 1, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein zeta were more abundant in the testis and gradually reduced towards the tail of the epididymis. We conclude that the abundance of proteins in the tissues of the reproductive tract is related to the function of the compartments of each compartment, as the testis, caput, corpus and cauda of the dog epididymis may have some protein similar to OPN that binds to the antibody, however it is urther investigation is needed.

Proteínas do plasma seminal canino: revisão / Protein patterns of canine seminal plasma: review

Nas últimas décadas, o crescente interesse pela cinofilia motivou o crescimento de uma atividadecomercial lucrativa. Assim, há um interesse em aumentar a performance reprodutiva por meio de biotécnicas dareprodução, como a criopreservação de gametas e a inseminação artificial. A identificação de proteínas presentesno plasma seminal canino dá início a novas discussões sobre o uso de proteínas em aditivos que melhorem aviabilidade espermática e possam ser usados como método contraceptivo em cadelas. Dessa forma, este trabalhoobjetivou descrever as proteínas do plasma seminal canino verificadas na literatura científica, como alactoferrina, a arginina esterase, as proteínas ligadoras de heparina, a osteopontina e as proteínas ligadoras dezinco, assim como relacioná-las com suas respectivas funções diante da esfera reprodutiva.(AU)

In the past few years, the interesting in dogs has provided an increase of this commercial activity. Thus,there is an interest in the increase of reproductive performance using biotechnology such as semencryopreservation and artificial insemination. The identification of proteins present in seminal plasma canineamazed initiates further discussion on the use of protein additives that improve sperm viability and it can used ascontraception in female dogs. Thus, this study aimed to describe the canine seminal plasma proteins, describedin the scientific literature, such as lactoferrin, arginine esterase, the heparin-binding proteins, osteopontin andzinc binding proteins, as well as relate them to their respective functions on the reproductive sphere.(AU)

Proteínas do plasma seminal canino: revisão / Protein patterns of canine seminal plasma: review

Nas últimas décadas, o crescente interesse pela cinofilia motivou o crescimento de uma atividadecomercial lucrativa. Assim, há um interesse em aumentar a performance reprodutiva por meio de biotécnicas dareprodução, como a criopreservação de gametas e a inseminação artificial. A identificação de proteínas presentesno plasma seminal canino dá início a novas discussões sobre o uso de proteínas em aditivos que melhorem aviabilidade espermática e possam ser usados como método contraceptivo em cadelas. Dessa forma, este trabalhoobjetivou descrever as proteínas do plasma seminal canino verificadas na literatura científica, como alactoferrina, a arginina esterase, as proteínas ligadoras de heparina, a osteopontina e as proteínas ligadoras dezinco, assim como relacioná-las com suas respectivas funções diante da esfera reprodutiva.

In the past few years, the interesting in dogs has provided an increase of this commercial activity. Thus,there is an interest in the increase of reproductive performance using biotechnology such as semencryopreservation and artificial insemination. The identification of proteins present in seminal plasma canineamazed initiates further discussion on the use of protein additives that improve sperm viability and it can used ascontraception in female dogs. Thus, this study aimed to describe the canine seminal plasma proteins, describedin the scientific literature, such as lactoferrin, arginine esterase, the heparin-binding proteins, osteopontin andzinc binding proteins, as well as relate them to their respective functions on the reproductive sphere.

OSTEOSSARCOMA EM CÃES: CARACTERIZAÇÃO CLÍNICO-PATOLÓGICA E AVALIAÇÃO DE FATORES PROGNÓSTICOS HISTOPATOLÓGICOS E DE IMUNO-HISTOQUÍMICA

Neoplasias em animais domésticos são afecções crescentes em medicina veterinária, especialmente devido ao aumento da longevidade destes animais e aos melhores métodos de diagnóstico. Em cães, os tumores ósseos são relativamente comuns e o osteossarcoma, representa 85% das neoplasias ósseas diagnosticadas nesta espécie. Este trabalho teve como objetivo descrever os aspectos epidemiológicos, clínicos e patológicos do osteossarcoma canino de 2006 a 2018 e avaliar fatores prognósticos associados ao neoplasma. Os materiais e métodos aplicados e resultados obtidos foram apresentados em dois artigos científicos. O primeiro artigo descreve os aspectos epidemiológicos e patológicos de 36 casos de osteossarcoma extraesquelético em cães. No período analisado, 2006 a 2016, o osteossarcoma acometeu os tecidos moles em 16,7% dos casos. As fêmeas, com média de 10,4 anos de idade e peso médio de 19,5 kg foram mais acometidas. Não houve predisposição racial. Os principais locais foram: glândula mamária (80,6%), tecido subcutâneo (5,6%) e fígado (5,6%). Na histologia, os osteossarcomas osteoblásticos (61,1%) de grau II e III foram os mais frequentes. Metástases nodais ocorreram em 21,4% dos casos na mama. Metástases distantes ocorreram nos pulmões (57,1%), fígado (14,3%), baço (14,3%) e em múltiplos sítios (14,3%). Metástases pulmonares foram mais frequentes em cadelas com osteossarcoma de glândula mamária. O segundo artigo descreve os aspectos clínico-patológicos de 153 casos de ostessarcoma apendicular em cães, dos quais os dados de sobrevida de 22 caninos submetidos a intervenções cirúrgicas e à quimioterapia foram confrontados estatisticamente com os dados clínicos, histopatológicos e imunohistoquímicos para correlação prognóstica. No período analisado, 2008 a 2018, o osteossarcoma acometeu o tecido ósseo em 83% dos casos e deste, 82% envolviam o esqueleto apendicular. A média de idade foi de nove anos e o peso médio de 33,4 kg. Não houve predileção sexual. A raça pura mais acometida foi o Rottweiler (42%). Os principais locais acometidos foram: úmero proximal (95%), fêmur distal (70%), rádio distal (96%) e tíbia proximal (56%). Na histologia, o subtipo osteoblástico foi o mais frequente (57%). Metástases nodais ocorreram em 14% dos casos. Metástases distantes ocorreram em 75% dos casos, nos quais o pulmão (43%) foi o principal órgão acometido. Quanto à análise de fatores prognósticos, o peso, idade, sexo, membro e osso afetado, classificação histológica, contagem mitótica e grau histológico não influenciaram na sobrevida (p > 0,05). Neoplasmas na extremidade proximal dos membros tiverem uma tendência a um pior prognóstico (p=0,06). Para estes casos, a taxa de sobrevida em um ano foi de 14,3%, enquanto que na extremidade distal a taxa foi de 46,7%. Não houve diferença significativa em relação à sobrevida quando utilizadas as técnicas de amputação ou preservação do membro, desde que associadas à quimioterapia (p=0,20). A sobrevida para estes casos variou de 73 a 1185 dias e a média foi de 394 dias. Todos os casos apresentaram marcação citoplasmática para osteopontina, porém a intensidade e o percentual de células neoplásicas marcadas não influenciaram na sobrevida (p > 0,05). Em 80% dos casos com metástases deste estudo houve marcação acentuada para osteopontina.

Neoplasms in domestic animals are increasing in veterinary medicine, especially due to the increased longevity of these animals and the best diagnostic methods. In dogs, bone tumors are relatively common and osteosarcoma accounts for 85% of the bone neoplasms diagnosed in this species. The objective of this study was to describe the epidemiological, clinical and pathological aspects of canine osteosarcoma from 2006 to 2018 and to evaluate prognostic factors associated with neoplasm. The materials and methods applied and results obtained were presented in two scientific papers. The first article describes the epidemiological and pathological aspects of 36 cases of extra-skeletal osteosarcoma in dogs. In the analyzed period, from 2006 to 2016, osteosarcoma affected the soft tissues in 16.7% of cases. Females, with a mean age of 10.4 years, mean weight of 19.5 kg were the most affected. There was no racial predisposition. The main sites were: mammary gland (80.6%), followed by subcutaneous tissue (5.6%) and liver (5.6%). In histology, osteoblastic osteosarcomas (61.1%) of grade II and III were the most frequent. Nodal metastases were present in 21.4% of the cases that occurred in the breast. Distant metastases occurred in the lungs (57.1%), liver (14.3%), spleen (14.3%) and multiple sites (14.3%). Pulmonary metastases were more frequent in bitches with osteosarcoma of the mammary gland. The second article describes the clinical-pathological aspects of 153 cases of appendicular ostessarcoma in dogs, of which the survival data of 22 canine submitted to surgery and chemotherapy were statistically compared with the clinical, histopathological and immunohistochemical data for prognostic correlation. In the analyzed period, from 2008 to 2018, the osteosarcoma affected the bone tissue in 83% of the cases and of this, 82% involved the appendicular skeleton. The mean age was 9 years and the mean weight was 33.4 kg. There was no sexual predilection. The pure breed most affected was the Rottweiler (42%). The main sites were: proximal humerus (95%), distal femur (70%), distal radius (96%) and proximal tibia (56%). In histology, osteoblastic osteosarcomas were more frequent (57%). Nodal metastases occurred in 14% of cases. Distant metastases occurred in 75% of cases, in which the lung (43%) was the main organ affected. Regarding the analysis of prognostic factors, weight, age, sex, affected limb and bone, histological classification, mitotic count and histological grade did not influence survival (p> 0.05). Neoplasms located at the proximal extremities of the limbs tend to have a poor prognosis (p = 0.06). For these cases, the survival rate at one year was 14.3%, while at the distal end the rate was 46.7%. There was no significant difference in survival when using amputation or limb preservation techniques, since they were associated with chemotherapy (p = 0.20). The survival of these dogs ranged from 73 to 1185 days, with a mean of 394 days. All cases presented cytoplasmic marking for osteopontin, but the intensity and percentage of marked neoplastic cell did not influence the survival (p> 0.05). In 80% of the cases with metastases from this study there was marked marking for osteopontin.