Association of blood heavy metal levels with osteocalcin abnormality and incidence of osteoporosis in Saudi subjects

Abstract Serum toxic metals have been implicated in development of many diseases. This study investigated the association between blood levels of lead and cadmium with abnormal bone mineral density (BMD) and incidence of osteoporosis. Sixty Saudi male adults age matching were assigned into two groups: A healthy control group (n = 30) and osteoporosis patients diagnosed according to T-score (n = 30). Serum calcium, vitamin D, osteocalcin, lead, cadmium were measured. Osteoporotic group showed a highly significant elevation of blood lead and cadmium levels compared to the control group (p 0.001). BMD was negatively correlated with serum osteocalcin level compared with control. There was a significant negative correlation between the cadmium and lead levels (r=-0.465 and p-value = 0.01) and calcium (p 0.004). Our findings suggested that high cadmium and lead were negative correlated to BMD and increased the risk factor for osteoporosis.

Resumo Os metais tóxicos do soro têm sido implicados no desenvolvimento de muitas doenças. Este estudo investigou a associação entre os níveis sanguíneos de chumbo e cádmio com densidade mineral óssea anormal (DMO) e incidência de osteoporose. Sessenta adultos sauditas do sexo masculino com idades iguais foram divididos em dois grupos: um grupo de controle saudável (n = 30) e pacientes com osteoporose diagnosticados de acordo com o T-score (n = 30). Cálcio sérico, vitamina D, osteocalcina, chumbo, cádmio foram medidos. O grupo osteoporótico apresentou elevação altamente significativa dos níveis de chumbo e cádmio no sangue em comparação ao grupo controle (p 0,001). A DMO foi negativamente correlacionada com o nível de osteocalcina sérica em comparação com o controle. Houve correlação negativa significativa entre os níveis de cádmio e chumbo (r = -0,465 ep = 0,01) e cálcio (p 0,004). Nossos achados sugeriram que cádmio e chumbo elevados foram correlacionados negativamente à DMO e aumentaram o fator de risco para osteoporose.

Association of blood heavy metal levels with osteocalcin abnormality and incidence of osteoporosis in Saudi subjects / Associação de anormalidades de osteocalcina nos níveis de metais pesados no sangue e incidência de osteoporose em indivíduos sauditas

Serum toxic metals have been implicated in development of many diseases. This study investigated the association between blood levels of lead and cadmium with abnormal bone mineral density (BMD) and incidence of osteoporosis. Sixty Saudi male adults age matching were assigned into two groups: A healthy control group (n = 30) and osteoporosis patients diagnosed according to T-score (n = 30). Serum calcium, vitamin D, osteocalcin, lead, cadmium were measured. Osteoporotic group showed a highly significant elevation of blood lead and cadmium levels compared to the control group (p <0.001). BMD was negatively correlated with serum osteocalcin level compared with control. There was a significant negative correlation between the cadmium and lead levels (r=-0.465 and p-value = 0.01) and calcium (p < 0.004). Our findings suggested that high cadmium and lead were negative correlated to BMD and increased the risk factor for osteoporosis.

Os metais tóxicos do soro têm sido implicados no desenvolvimento de muitas doenças. Este estudo investigou a associação entre os níveis sanguíneos de chumbo e cádmio com densidade mineral óssea anormal (DMO) e incidência de osteoporose. Sessenta adultos sauditas do sexo masculino com idades iguais foram divididos em dois grupos: um grupo de controle saudável (n = 30) e pacientes com osteoporose diagnosticados de acordo com o T-score (n = 30). Cálcio sérico, vitamina D, osteocalcina, chumbo, cádmio foram medidos. O grupo osteoporótico apresentou elevação altamente significativa dos níveis de chumbo e cádmio no sangue em comparação ao grupo controle (p < 0,001). A DMO foi negativamente correlacionada com o nível de osteocalcina sérica em comparação com o controle. Houve correlação negativa significativa entre os níveis de cádmio e chumbo (r = -0,465 ep = 0,01) e cálcio (p < 0,004). Nossos achados sugeriram que cádmio e chumbo elevados foram correlacionados negativamente à DMO e aumentaram o fator de risco para osteoporose.

Association of blood heavy metal levels with osteocalcin abnormality and incidence of osteoporosis in Saudi subjects / Associação de anormalidades de osteocalcina nos níveis de metais pesados no sangue e incidência de osteoporose em indivíduos sauditas

Serum toxic metals have been implicated in development of many diseases. This study investigated the association between blood levels of lead and cadmium with abnormal bone mineral density (BMD) and incidence of osteoporosis. Sixty Saudi male adults age matching were assigned into two groups: A healthy control group (n = 30) and osteoporosis patients diagnosed according to T-score (n = 30). Serum calcium, vitamin D, osteocalcin, lead, cadmium were measured. Osteoporotic group showed a highly significant elevation of blood lead and cadmium levels compared to the control group (p <0.001). BMD was negatively correlated with serum osteocalcin level compared with control. There was a significant negative correlation between the cadmium and lead levels (r=-0.465 and p-value = 0.01) and calcium (p < 0.004). Our findings suggested that high cadmium and lead were negative correlated to BMD and increased the risk factor for osteoporosis.(AU)

Os metais tóxicos do soro têm sido implicados no desenvolvimento de muitas doenças. Este estudo investigou a associação entre os níveis sanguíneos de chumbo e cádmio com densidade mineral óssea anormal (DMO) e incidência de osteoporose. Sessenta adultos sauditas do sexo masculino com idades iguais foram divididos em dois grupos: um grupo de controle saudável (n = 30) e pacientes com osteoporose diagnosticados de acordo com o T-score (n = 30). Cálcio sérico, vitamina D, osteocalcina, chumbo, cádmio foram medidos. O grupo osteoporótico apresentou elevação altamente significativa dos níveis de chumbo e cádmio no sangue em comparação ao grupo controle (p < 0,001). A DMO foi negativamente correlacionada com o nível de osteocalcina sérica em comparação com o controle. Houve correlação negativa significativa entre os níveis de cádmio e chumbo (r = -0,465 ep = 0,01) e cálcio (p < 0,004). Nossos achados sugeriram que cádmio e chumbo elevados foram correlacionados negativamente à DMO e aumentaram o fator de risco para osteoporose.(AU)

Association of blood heavy metal levels with osteocalcin abnormality and incidence of osteoporosis in Saudi subjects / Associação de anormalidades de osteocalcina nos níveis de metais pesados no sangue e incidência de osteoporose em indivíduos sauditas

Abstract Serum toxic metals have been implicated in development of many diseases. This study investigated the association between blood levels of lead and cadmium with abnormal bone mineral density (BMD) and incidence of osteoporosis. Sixty Saudi male adults age matching were assigned into two groups: A healthy control group (n = 30) and osteoporosis patients diagnosed according to T-score (n = 30). Serum calcium, vitamin D, osteocalcin, lead, cadmium were measured. Osteoporotic group showed a highly significant elevation of blood lead and cadmium levels compared to the control group (p <0.001). BMD was negatively correlated with serum osteocalcin level compared with control. There was a significant negative correlation between the cadmium and lead levels (r=-0.465 and p-value = 0.01) and calcium (p < 0.004). Our findings suggested that high cadmium and lead were negative correlated to BMD and increased the risk factor for osteoporosis.

Resumo Os metais tóxicos do soro têm sido implicados no desenvolvimento de muitas doenças. Este estudo investigou a associação entre os níveis sanguíneos de chumbo e cádmio com densidade mineral óssea anormal (DMO) e incidência de osteoporose. Sessenta adultos sauditas do sexo masculino com idades iguais foram divididos em dois grupos: um grupo de controle saudável (n = 30) e pacientes com osteoporose diagnosticados de acordo com o T-score (n = 30). Cálcio sérico, vitamina D, osteocalcina, chumbo, cádmio foram medidos. O grupo osteoporótico apresentou elevação altamente significativa dos níveis de chumbo e cádmio no sangue em comparação ao grupo controle (p < 0,001). A DMO foi negativamente correlacionada com o nível de osteocalcina sérica em comparação com o controle. Houve correlação negativa significativa entre os níveis de cádmio e chumbo (r = -0,465 ep = 0,01) e cálcio (p < 0,004). Nossos achados sugeriram que cádmio e chumbo elevados foram correlacionados negativamente à DMO e aumentaram o fator de risco para osteoporose.

Polysaccharides from aloe vera target the Wnt/ß-catenin pathway to impact the tooth density of pulpitis rats

Purpose: To investigate the mechanism of polysaccharides from aloe vera (PAV), a main active ingredient of Aloe vera, treatment in pulpitis rats. Methods: Pulpitis were modeled by drilling the occlusal central fossa with Sprague Dawley rats. Next, the rats were treated with 20, 40, and 80 mg/kg PAV for three weeks, respectively. Computed tomography scanning assay, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining were used to detect the pathology change. Then, levels of tumor necrosis factor-α, interleukin-1ß, prostaglandin E2, and ciclooxigenase 2 were detected by enzyme-linked immunosorbent assay. The expressions of bone morphogenetic protein 2 human (BMP-2), osteocalcin, osterix, and runt-related transcription factor 2 (Runx2) were quantified by quantitative real-time polymerase chain reaction and Western blotting (WB). Finally, Wnt3a expression, p-GSK3ß/GSK3ß and p-ß-catenin/ß-catenin ratio were analyzed by WB. Results: PAV up regulated the bone mineral density, and reduced the breakage of the crown and cervical structures, and the necrosis of the crown and root pulp of pulpitis rats. In addition, results indicated that PAV could inhibit osteoblast formation. While osteoblasts' number was decreased, proteins of BMP-2, osteocalcin, osterix, and Runx2 were up-regulated by PAV. Furthermore, PAV increased the Wnt3a expression and the p-ß-catenin/ß-catenin ratio, and decreased p-GSK3ß/GSK3ß ratio. Interestingly, these effects were all in dose dependence. Conclusions: PAV could inhibit pulp inflammation and promote osteoblasts differentiation via suppressing the activation of the Wnt/ß-catenin signaling, enhancing the dental bone density.

Platelet-concentrated and platelet poor-plasma promote different pattern on immunohistochemical expression of TGF-β1, however they impairs the osteoneogensis in calvarial defects treated with autograft due suppression of osteocalcin

ABSTRACT Purpose Herein we evaluated the effects of platelet concentrate (PC) and platelet-poor plasma (PPP) on bone repair using noncritical defects in the calvaria of rabbits and compared them to the presence of TGF-β1 and osteocalcin on reparative sites. Methods Five noncritical defects of 8.7 mm in diameter were created on the calvaria of 15 animals. Each defect was treated differently, using autograft (ABG), ABG associated with PC (ABG + PC), ABG with PPP (ABG + PPP), isolated PPP, and blood clot (control). The animals were submitted to euthanasia on the second, fourth and sixth week post-surgery. Results The defects that received ABG+PC or PPP demonstrated lower bone formation when compared to specimens that received ABG in the same period. These results coincided to significant higher immunopositivity for TGF-β1 for specimens that received PC, and lower presence of cytokine in the group PPP. However, either higher or lower presence of TGF-β1 were also correlated to lower presence of osteocalcin. Likewise, these results were similar to findings in specimens treated only with PPP when compared to control. Conclusions PC and PPP were not effective when applied in association with ABG. Similarly, isolated use of PPP was not beneficial in optimizing the bone repair.

Effects of ergosteroside combined risedronate on fracture healing and BMP-2, BMP-7 and VEGF expression in rats

Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A

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)

Influence of low-level laser irradiation on osteocalcin protein and gene expression in bone tissue1

Purpose: To evaluate osteocalcin gene and protein expression in vitro and in an in vivo model of ostectomy. Methods: Twenty Wistar rats were assigned into two groups A (n=10, laser) and B (n=10, control). Ostectomy was performed in the femur diaphysis; the twenty fragments removed, composed in vitro groups named as in vivo (A and B) and cultivated in CO2 atmosphere for thirteen days. Low-level laser irradiation was performed in groups A (in vivo and in vitro) by an GaAlAs device (λ=808 nm, dose of 2J/cm2, power of 200mW, power density of 0.2W/cm2, total energy of 1.25J, spot diameter of 0.02mm) for 5 seconds, at one point, daily. It was performed immunocytochemistry assays in vivo and in vitro groups. In vitro groups were also submitted to RNA extraction, cDNA synthesis and gene expression by quantitative PCR. Statistical analysis was realized with p<0.05. Results: Immunocytochemistry scores showed no significant differences between control and laser groups either in vivo and in vitro. Gene expression also showed no statistical differences. Conclusion: Low-level laser irradiation did not alter osteocalcin protein and gene expression in vivo and in vitro in the studied period but it may have been expressed them in an earlier period.(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)

Impacto da Osteocalcina na Esteroidogenese Ovariana

A osteocalcina é conhecida como uma proteína óssea contendo ácido gama-carboxi glutâmico (BGLAP), é um pequeno (49 aminoácidos) hormônio proteico não-colágeno secretado por osteoblastos, geralmente relatado como um marcador de formação óssea. Mais recentemente, outros papéis da osteocalcina na secreção e sensibilidade à insulina foram sugeridos, embora seu impacto na secreção de esteróides ovarianos permaneça obscuro. Portanto, o presente estudo tem como objetivo investigar a presença do receptor de osteocalcina (GPCR6A) nas células da granulosa e da teca do ovário bovino e o possível efeito da osteocalcina sobre enzimas esteroidogênicas. Nossos resultados indicam, pela primeira vez, a presença do receptor osteocalcina GPRC6A nas células e testes da teca e sua ausência ou muito baixa expressão nas células da granulosa. Após o tratamento com osteocalcina não carboxilada (100ng / ml), um tamanho de efeito significativo nas principais enzimas esteroidogênicas (CYP17A1, CYP11A1) e proteína (STAR) foi observado na primeira hora (mas não 24 horas) apenas na presença de co-tratamento com hormônio luteinizante (LH). Doses mais baixas de osteocalcina não carboxilada (1 ng / ml) não foram eficazes para influenciar essas enzimas. Embora limitados, nossos resultados demonstram que parece haver alguma ação da osteocalcina na primeira hora após o tratamento, aumento a expressão de mRNA em enzimas esteroidogenicas chaves como a STAR, CYP11A1 e CYP17A1 de forma sinérgica com o uso de LH (100 ng/ml).

Osteocalcin, known as bone gamma-carboxy glutamic acid-containing protein (BGLAP), is a small (49-amino-acid) noncollagenous protein hormone secreted by osteoblasts usually reported as a marker of bone formation. More recently, other roles for osteocalcin in insulin secretion and sensibility have been suggested, although its impact on the ovarian steroid secretion remains obscure. Therefore, the present study aims to investigate the presence of osteocalcin receptor (GPCR6A) in the granulosa and theca cells of the bovine ovary and the possible effect of osteocalcin on steroidogenic enzymes. Our results indicate, for the first time, the presence of the osteocalcin receptor GPRC6A in theca cells and testis and its absence or very low expression in the granulosa cells. After treatment with non-carboxylated osteocalcin (100ng/ml), a significant effect size on the key steroidogenic enzymes (CYP17A1, CYP11A1) and protein (STAR) was observed in the first hour (but not 24-hour) only in the presence of cotreatment with luteinizing hormone (LH). Lower doses of non-carboxylated osteocalcin (1ng/ml) were not effective to influence these enzymes. Although limited, our results demonstrate that there appears to be some action of osteocalcin in the first hour after treatment, increasing mRNA expression in key steroidogenic enzymes such as STAR, CYP11A1, and CYP17A1 synergistically with the use of LH (100 ng/ml)

Uso do chá mate (Ilex paraguariensis) descafeinado na prevenção do dano oxidativo e das alterações ósseas em ratos Wistar obesos

A obesidade é uma doença crônica e um dos principais problemas de saúde no mundo, sendo o consumo de alimentos como o refrigerante a base de cola (RBC) associado à obesidade e aos danos oxidativos e ósseos em humanos. A ingestão de chá mate (CM) pode minimizar os efeitos indesejáveis do excesso de gordura, porém é contraindicado aos sensíveis à cafeína. Neste trabalho verificamos se o CM descafeinado (CMD) mantém as propriedades antioxidantes, prevenção da obesidade e lesões ósseas da forma in natura. Foi realizado um estudo randomizado com mascaramento, em que 60 ratos foram distribuídos aleatoriamente em seis grupos (n = 10): controle (C); RBC, ratos tratados com RBC; CM, ratos tratados com CM; CMD, ratos tratados com CMD; RBC + CM e RBC + CMD. O grupo controle foi comparado aos demais quanto a sua constituição corporal (peso e Índice de Lee) e perfil bioquímico plasmático [triglicerídeos, colesterol total, fosfatases alcalina (FA), cálcio total (Ca), leptina, adiponectina, osteocalcina (OCN), osteoprotegerina (OPG) e fosfatase ácida resistente ao tartarato (TRAP)]. Para quantificar o estresse oxidativo (EO) e a lesão tecidual foram mensuradas a densidade mineral óssea areal (DMOa), as substâncias reativas ao ácido tiobarbitúrico (TBARS), as atividades de superóxido dismutase (SOD) e glutationa peroxidase (GPx). O CMD mantém propriedades benéficas da forma in natura quanto ao controle da obesidade e EO ósseo, sendo uma alternativa promissora dietética e fitoterápica para indivíduos sensíveis à cafeína.

Obesity is a chronic disease and one of the main health problems in the world, with the consumption of foods such as cola soft drinks (CSD) associated with obesity and oxidative and bone damage in humans. Intake of mate tea (MT) can minimize the undesirable effects of excess fat, however, this tea is contraindicated in humans and animals sensitive to caffeine. Accordingly, we investigated the hypothesis that the decaffeinated mate tea (DMT) maintaining the antioxidant properties and prevention of obesity in natura of the bone lesions. A systematic review was carried out based on the suggested hypothesis. Furthermore, there was randomized and blind study, where 60 rats were randomly assigned to six groups (n = 10): control (C); CSD, rats treated with CSD; MT, rats treated with DMT; CSD + MT and CSD + DMT. The control group was compared to the others regarding its body constitution (weight and Lee index) and plasma biochemical profile (triglycerides, total cholesterol, alkaline phosphatases (FA), total calcium (Ca), leptin, adiponectin, osteocalcin (OCN), osteoprotegerin (OPG) and tartrateresistant acid phosphatase (TRAP)). To quantify the oxidative stress OS and tissue lesion, the bone mineral density areal (BMDa), the reactive substances to thiobarbituric acid (TBARS), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured. The DMT maintains beneficial properties of the in natura form regarding the control of obesity and bone OS, being a promising dietary and herbal alternative for individuals sensitive to caffeine.

CALCIUM BALANCE, BONE DENSITY, AND PERFORMANCE OF LATE-LACTATION DAIRY COWS WITH SUPRAPHYSIOLOGICAL CONCENTRATION OF BLOOD VITAMIN D

Em bovinos a análise de densitometria óptica radiográfica tem sido muito pouco utilizada. 14 Com a padronização da técnica e facilidade de utilização da ferramenta e obtenção do 15 dado, esta técnica pode ser utilizada para avaliar o metabolismo ósseo da vaca leiteira. 16 Com isto, objetivou-se padronizar a técnica de densitometria óptica radiográfica e valores 17 da densidade mineral em bovinos leiteiros e correlacionar os valores com idade, peso e 18 marcadores séricos (Ca total, Ca ionizado, P, Mg, osteocalcina, fosfatase alcalina do osso 19 e telopeptídeo carboxiterminal do colágeno tipo I (CTX-1)). Foram avaliadas trinta vacas 20 holandesas (230 ± 137 DEL, 8 primíparas) produção de leite 31,3 ± 6,3 kg/d, ingestão de 21 matéria seca 23,0 ± 3,8 kg/d e peso 666 ± 83 kg. Foi avaliado no primeiro dia (d 0) e após 22 84 dias (d 84), a densidade mineral óssea, peso corporal, idade e concentrações séricas de 23 Ca ionizado, Ca total, P, Mg. Os parâmetros sanguíneos osteocalcina, fosfatase alcalina 24 óssea e CTX-1 foram avaliados somente no dia 84, com intuito de verificar a correlação 25 entre os parâmetros. A densidade mineral óssea (DMO) é maior com o avançar da idade 26 das vacas, apresentando resultado significativos para três regiões de avaliação. Animais 27 com maior idade aumentaram peso (681 vs 623 Kg) e reduziram fósforo (6,10 vs 6,66 28 mg/dL) e telopeptídeo carboxiterminal do colágeno tipo I (CTX-1) (0,32 vs 0,56 ng/mL), 29 e tenderam a reduzir fosfatase óssea (25,31 vs 32,21 ng/mL) comparado com animais 30 mais jovens. Com avançar do dia de lactação (DEL) a DMO das vacas leiteiras foi maior, 31 excesso para a variável do carpo acessório. Com o avançar do DEL, o peso (670 vs 633 32 Kg) e as concentrações sanguíneos de cálcio total (9,38 vs 8,95 mg/dL) e cálcio ionizado 33 (5,11 vs 4,87 mg/dL) aumentaram. Os valores médios da DMO de vacas em lactação do 33 34 carpo acessório, e metacarpo na região de avaliação lateral, medial e medular, 35 apresentaram valores médios de 14,91; 27,45; 25,25 e 16,06 mmAl respectivamente. A 36 DMO em vacas leiteiras correlacionou positivamente com cálcio total e cálcio ionizado 37 das regiões do carpo acessório, e metacarpo córtex lateral, medial e medular. Pesos 38 corporais das vacas apresentaram boa correlação com a DMO das radiografias do córtex 39 lateral, medial e medular do metacarpo. A fosfatase alcalina do osso correlacionou 40 negativamente com a DMO do metacarpo lateral. E o marcador de reabsorção óssea CTX41 1 tendeu a correlacionar negativamente com todas as medidas das radiografias do córtex 42 do metacarpo lateral e medial. Conclui-se que a DMO determinada por densitometria 43 óptica em imagem radiográfica é uma metodologia de fácil execução, reprodutibilidade e 44 baixo custo que pode ser empregado na avaliação da DMO em vacas leiteiras.

Vitamin D can affect Ca metabolism, bone remodeling, and lactation performance of dairy cows. The 15 objective of this experiment was to evaluate lactation performance, body Ca retention, and bone 16 density of late lactation dairy cows subjected to supraphysiological concentration of blood 25(OH)D 17 for 12 weeks. We hypothesized that the increase in blood 25(OH)D would improve Ca retention, bone 18 mineralization, and lactation performance. Thirty Holsteins cows (230 ± 137 days in lactation, 8 19 primiparous) were individually fed a standard total mixed ration for 14 days and a treatment for 84 20 days. Treatments were Phy (placebo) or Supra (40,000 IU/d of calcidiol) vitamin D level or placebo. 21 The basal diet contained 1,033 UI/kg of dry matter (DM) of cholecalciferol and 0.77% Ca in DM. The 22 intake of vitamin D3 (cholecalciferol) from the diet was 23,800 IU/d. Data was analyzed with Proc 23 Mixed of as a covariated adjusted randomized block design with repeated measures over time. 24 Significance was declared at P 0.01 and trends at P 0.10. The average plasma concentration of 25 25(OH)D of Supra was twice the concentration of Phy (117 vs 53 ng/mL). The increase in the 26 concentration of plasma 25(OH)D in response to calcidiol supplementation was of larger magnitude 27 on day 84 (+ 84.4 ng/mL) than on day 28 (+ 45.3 ng/mL). There was not a deficiency of vitamin D in 28 this group of cows. The concentrations of both forms of total Ca and ionized Ca were increased by 51 29 vitamin D on days 28 and 56, but there was no treatment difference on day 84 (9.4 mg/dL). Cows on 30 Supra excreted more Ca in urine (g/d) in week 12 than cows on Phy and there was no difference in the 31 flow of urinary Ca on week 4. The total tract apparent digestibility of Ca was not affected by vitamin 32 D level. The markers of bone deposition osteocalcin (OC) and bone alkaline phosphatase (BAP) were 33 reduced in Supra on day 84 and did not differ in day 28. The marker of bone resorption CTX-1 did not 34 differ. The maintenance of a constant blood Ca concentration with high vitamin D supply in day 84, 35 in spite of the greater loss of urinary Ca and similar Ca absorption from the digestive tract, was 36 explained by a reduction in Ca deposition in bones. The estimated retention of Ca in the body did not 37 differ. There were trends for reductions in the optical density of accessory carpal and medullar region 38 of metacarpal bones with Supra, suggestive of osteoporosis. The Supra level of vitamin D was 39 associated to increased milk yield (31.6 vs 30.9 kg/d) and had no effect on DMI (23.0 kg/d). The 40 energy-corrected milk, feed efficiencies, body condition score, body weight gain, and the total tract 41 apparent digestibility of dry matter and organic matter did not differ. Supra was associated with a 42 reduction in milk somatic cell count (SCC) from 105,000 to 83,500 cells/mL, suggestive of a positive 43 action of vitamin D on mammary gland immunity. Dairy cows exposed for 12 weeks to twice as much 44 concentration of blood 25(OH)D than cows with 50 ng/mL produced more milk at similar intake, had 45 lower SCC in milk, had higher concentration of blood Ca and excretion of Ca in urine, and had reduced 46 bone mineral deposition markers and bone optical density.

Estudo comparativo da diferenciação osteogênica das células tronco mesenquimais da medula óssea e do tecido adiposo de cães adultos / Comparative study of the osteogenic differentiation of mesenchymal stem cells from bone marrow and adipose tissue of adult dogs

O objetivo deste estudo foi comparar o potencial osteogênico das células tronco mesenquimais extraídas da medula óssea (CTM-MO) com as do tecido adiposo (CTM-AD) de cães adultos. As células foram caracterizadas fenotipicamente quanto à expressão de CD29, CD90, CD34 e CD45 e submetidas à diferenciação adipogênica e condrogênica por 21 dias e osteogênica por 7, 14 e 21 dias. Foram constituídos quatro grupos: 1) CTM-MO em meio osteogênico, 2) CTM-MO em meio basal, 3) CTM-AD em meio osteogênico e 4) CTM-AD em meio basal. Aos 7, 14 e 21 dias de diferenciação osteogênica as culturas foram submetidas às avaliações da conversão de MTT em formazan, da atividade da fosfatase alcalina (FA), da síntese de colágeno e de matriz mineralizada, avaliação do número de células por campo e foram quantificados os transcritos gênicos para osterix, sialoproteina óssea (BSP), osteonectina (ON) e osteocalcina (OC). Tanto as células extraídas da medula óssea quanto do tecido adiposo mostraram elevada expressão de marcadores para células tronco e baixa expressão de marcadores de células hematopoiéticas (menor que 2%). Além disso, foram capazes de se diferenciar em osteoblastos, condrócitos e adipócitos. As CTM-AD submetidas à diferenciação osteogênica mostraram maior conversão do MTT em formazan que as CTM-MO, sob mesmas condições aos 7 e 21 dias. O número de células por campo, a atividade da FA, a síntese de colágeno e de matriz mineralizada foram superior nas CTM-AD em diferenciação, em relação às CTM-MO sob as mesmas condições, em todos os tempos estudados. As expressões de osterix, BSP e OC foram predominantemente superiores nas CTM-MO diferenciadas, mas a expressão de ON foi superior nas CTM-AD diferenciadas aos 7, 14 e 21 dias. Conclui-se que as CTM-AD apresentam maior potencial osteogênico que as CTM-MO quando extraídas de cães adultos.(AU)

The aim of this study was to compare the osteogenic potential of mesenchymal stem cells obtained from bone marrow (BM-MSC) with those extracted from adipose tissue (AT-MSC) of adult dogs. The cells were phenotypically categorized according to the expression of CD29, CD90, CD34 and CD45, and submitted to adipogenic and chondrogenic differentiation for 21 days and osteogenic differentiation for 7, 14 and 21 days. Four groups were formed: BM-MSC in osteogenic medium (1), BM-MSC in basal medium (2), AT-MSC in osteogenic medium (3) and ATMSC in basal medium (4). On days 7, 14 and 21 of osteogenic differentiation, the cultures were submitted to evaluations of MTT conversion in formazan, of alkaline phosphatase activity (AP), of collagen and mineralized matrix synthesis, evaluation of the number of cells per field and there was quantification of the gene transcripts for osterix, bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). Both the cells obtained from bone marrow and those from adipose tissue showed high expression of stem cells markers and low expression of hematopoietic cells markers (lower than 2%). Besides, they were able to differentiate into osteoblasts, chondrocytes and adipocytes. AT-MSC submitted to osteogenic differentiation showed higher MTT conversion in formazan than BM-MSC, under the same conditions on days 7 and 21. The number of cells per field, the AP activity, the collagen and mineralized matrix synthesis were higher in AT-MSC en differentiation, in relation to BM-MSC under the same conditions in all evaluated times. Expressions of osterix, BSP and OC were predominantly higher in differentiated BMMSC, however the expression of ON was higher AT-MSC differentiated on days 7, 14 and 21. In conclusion, AT-MSC present higher osteogenic potential than BM-MSC when extracted from adult dogs.(AU)

Estudo comparativo da diferenciação osteogênica das células tronco mesenquimais da medula óssea e do tecido adiposo de cães adultos / Comparative study of the osteogenic differentiation of mesenchymal stem cells from bone marrow and adipose tissue of adult dogs

O objetivo deste estudo foi comparar o potencial osteogênico das células tronco mesenquimais extraídas da medula óssea (CTM-MO) com as do tecido adiposo (CTM-AD) de cães adultos. As células foram caracterizadas fenotipicamente quanto à expressão de CD29, CD90, CD34 e CD45 e submetidas à diferenciação adipogênica e condrogênica por 21 dias e osteogênica por 7, 14 e 21 dias. Foram constituídos quatro grupos: 1) CTM-MO em meio osteogênico, 2) CTM-MO em meio basal, 3) CTM-AD em meio osteogênico e 4) CTM-AD em meio basal. Aos 7, 14 e 21 dias de diferenciação osteogênica as culturas foram submetidas às avaliações da conversão de MTT em formazan, da atividade da fosfatase alcalina (FA), da síntese de colágeno e de matriz mineralizada, avaliação do número de células por campo e foram quantificados os transcritos gênicos para osterix, sialoproteina óssea (BSP), osteonectina (ON) e osteocalcina (OC). Tanto as células extraídas da medula óssea quanto do tecido adiposo mostraram elevada expressão de marcadores para células tronco e baixa expressão de marcadores de células hematopoiéticas (menor que 2%). Além disso, foram capazes de se diferenciar em osteoblastos, condrócitos e adipócitos. As CTM-AD submetidas à diferenciação osteogênica mostraram maior conversão do MTT em formazan que as CTM-MO, sob mesmas condições aos 7 e 21 dias. O número de células por campo, a atividade da FA, a síntese de colágeno e de matriz mineralizada foram superior nas CTM-AD em diferenciação, em relação às CTM-MO sob as mesmas condições, em todos os tempos estudados. As expressões de osterix, BSP e OC foram predominantemente superiores nas CTM-MO diferenciadas, mas a expressão de ON foi superior nas CTM-AD diferenciadas aos 7, 14 e 21 dias. Conclui-se que as CTM-AD apresentam maior potencial osteogênico que as CTM-MO quando extraídas de cães adultos.(AU)

The aim of this study was to compare the osteogenic potential of mesenchymal stem cells obtained from bone marrow (BM-MSC) with those extracted from adipose tissue (AT-MSC) of adult dogs. The cells were phenotypically categorized according to the expression of CD29, CD90, CD34 and CD45, and submitted to adipogenic and chondrogenic differentiation for 21 days and osteogenic differentiation for 7, 14 and 21 days. Four groups were formed: BM-MSC in osteogenic medium (1), BM-MSC in basal medium (2), AT-MSC in osteogenic medium (3) and ATMSC in basal medium (4). On days 7, 14 and 21 of osteogenic differentiation, the cultures were submitted to evaluations of MTT conversion in formazan, of alkaline phosphatase activity (AP), of collagen and mineralized matrix synthesis, evaluation of the number of cells per field and there was quantification of the gene transcripts for osterix, bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). Both the cells obtained from bone marrow and those from adipose tissue showed high expression of stem cells markers and low expression of hematopoietic cells markers (lower than 2%). Besides, they were able to differentiate into osteoblasts, chondrocytes and adipocytes. AT-MSC submitted to osteogenic differentiation showed higher MTT conversion in formazan than BM-MSC, under the same conditions on days 7 and 21. The number of cells per field, the AP activity, the collagen and mineralized matrix synthesis were higher in AT-MSC en differentiation, in relation to BM-MSC under the same conditions in all evaluated times. Expressions of osterix, BSP and OC were predominantly higher in differentiated BMMSC, however the expression of ON was higher AT-MSC differentiated on days 7, 14 and 21. In conclusion, AT-MSC present higher osteogenic potential than BM-MSC when extracted from adult dogs.(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

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.

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)