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This study aimed to evaluate the effect of maternal ethanol consumption during gestation and lactation on bone mass and osteogenic differentiation of mesenchymal stem cells of the bone marrow (BMMSCs) in rats. Thirteen adult Wistar rats were used. The rats were mated, and after confirmation of gestation, (day 0) they were distributed in two groups: the control group and the ethanol-treated group. From the ninth day of gestation, the rats of the ethanol and control groups were administered 40% alcoholic solution (4 g ethanol/kg) and distilled water, respectively, daily via gavage until the thirtieth day of lactation. The BMMSCs were extracted from the right femurs and tibiae and cultured using an osteogenic medium for 7, 14, and 21 days. The conversion of MTT to formazan crystals, alkaline phosphatase activity, and percentages of cells per field were analyzed. The number of mineralized nodules per field was examined, and quantification of the gene transcripts for osteopontin, osteocalcin, and BMP-2 was evaluated on day 21 by real-time RT-PCR. Morphometric evaluations of the percentage of trabecular bone and cortical thickness in the left femur and tibia were performed. The means were compared by the Student's 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 subjected to osteogenic differentiation in vitro, demonstrated higher conversion of MTT to formazan, higher alkaline phosphatase activity, a higher percentage of cells per field, higher expression of BMP-2, and higher synthesis of mineralized nodules when compared to those of control rat cells. However, there was no significant difference in the percentage of trabecular bone or cortical thickness between both groups. Hence, 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 BMMSCs showing higher osteogenic differentiation under in vitro conditions.

Osteoporosis is a metabolic disease that affects bone tissue and is highly associated with bone fractures. Due to the low bioavailability of conventional treatments for this disease, an alternative has been developed to project systems for the local release of therapeutic agents, such as bioactive glass (BG) containing therapeutic ions and molecules that are released during dissolution. In this study, submicron BG spheres derived from the sol–gel process containing Sr, a potential ion to prevent bone loss and increase bone formation, were evaluated. For the first time, the effect of these particles was investigated on the reduced osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) obtained from osteoporotic rats. The obtained submicron and spherical Sr-containing BG particles showed controlled ion release capacity, and osteogenic differentiation assays showed that these materials could stimulate the reduced differentiation potential of BMMSCs from osteoporotic rats due to Sr release.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 μl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 μl of extract/L containing 0.4 μg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 μg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 μg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

The hypothesis of this experiment is that mesenchymal stem cells (MSCs) are involved in the genesis of the bone metaplasia caused by Solanum glaucophyllum intoxication. We determined using liquid chromatography that 1 mL of plant extract contained 3.8 μl of 1,25(OH)2D3. The ability of 100 μL, 1 mL and 5 mL of extract/L, containing 1 nM (0.4 μg/L), 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3, respectively, in inducing the osteogenic differentiation in bone marrow MSCs from rats was tested. At the concentrations of 1 and 5 mL of extract/L of culture medium without osteogenesis-inducing factors, the plant extract induced the osteogenic differentiation of the MSCs, as was evidenced by the greater synthesis of mineralized matrix. At the higher concentration (5 mL of extract/L), an increase in the relative expression of BMP-2 gene was observed. It was concluded that rat bone marrow MSC culture is a good model for studying the effects of the S. glaucophyllum extract on the osteogenic differentiation of undifferentiated cells. Also, S. glaucophyllum extracts containing 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3 induce the osteogenic differentiation of MSCs, suggesting that this is one of the mechanisms by which S. glaucophyllum causes bone metaplasia.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As plantas calcinogênicas têm importância na medicina veterinária mundial por conterem glicosídeos, que após a sua ingestão, liberam metabólitos da vitamina D, potencialmente nocivos. No Brasil, as espécies Solanum glaucophyllum (S. malacoxylon) e Nierembergia veitchii são responsáveis pela calcinose enzoótica, intoxicação de caráter endêmico que acomete ruminantes. A calcinose é caracterizada pela calcificação de tecidos moles e está frequentemente associada à metaplasias óssea e cartilaginosa. A hipótese do primeiro experimento é que as células tronco mesenquimais (CTM) estejam envolvidas na gênese do processo de metaplasia óssea causado pela Solanum glaucophyllum, o que ainda carece de comprovação científica. Além da metaplasia, a intoxicação pela Solanum glaucophyllum causa alterações ósseas, representadas por osteopetrose, osteonecrose e osteoporose. Por ser o osteoblasto uma célula que deriva da diferenciação osteogênica das CTM da medula óssea, a hipótese do segundo experimento é que a osteoporose induzida pela intoxicação por Solanum glaucophyllum seja decorrente não somente da ação da planta sobre os osteoblastos, como também do seu efeito sobre a diferenciação osteogênica das CTM. A concentração de 1,25(OH)2D3 obtida, por meio da técnica de cromatografia líquida, no extrato da planta foi de 3,8 µg/ml. Em ambos os experimentos, foram testados os efeitos de 100µl, 1ml e 5ml de extrato/L, contendo respectivamente, 1nM (0,4 µg/L), 10nM (4 µg/L) e 50nM (20 µg/L) de 1,25(OH)2D3. Foram realizados os seguintes ensaios: ensaio de MTT, atividade de fosfatase alcalina, determinação da porcentagem de células/campo, número de nódulos de mineralização em 50 campos e avaliação da expressão dos transcritos gênicos para osteopontina, sialoproteína óssea e BMP-2, por RT-PCR tempo real. No primeiro experimento,culturas de CTM da medula óssea foram utilizadas como modelo para estudo dos efeitos da Solanum glaucophyllum na indução da diferenciação osteogênica de células indiferenciadas. Na concentração de 5ml de extrato de Solanum glaucophyllum por litro de meio de cultura desprovido de fatores indutores da osteogênese, o extrato da planta induziu diferenciação osteogênica das CTM, comprovada pela maior síntese de matriz mineralizada e pelo aumento da expressão relativa dos transcritos gênicos para BMP-2. A diferenciação osteogênica induzida pelo extrato da planta foi estatisticamente semelhante à diferenciação induzida por meio de cultura contendo fatores que comprovadamente induzem osteogênese, representados pelo β-glicerofosfato, ácido ascórbico e pela dexametasona. Conclui-se que culturas de CTM da medula óssea de ratos são um bom modelo para estudar o efeito do extrato da Solanum glaucophyllum sobre a diferenciação osteogênica de células indiferenciadas e que os extratos de Solanum glaucophyllum contendo 10nM (4 µg/L) e 50nM (20 µg/L) de 1,25(OH)2D3 causam diferenciação osteogênica de CTM, sugerindo ser este um dos mecanismos pelo qual a Solanum glaucophyllum produz metaplasia óssea. No segundo experimento, a fim de estudar os efeitos da Solanum glaucophyllum em CTM submetidas à diferenciação em osteoblastos, diferentes concentrações do extrato da planta foram adicionadas ao meio de cultura, juntamente com fatores conhecidamente promotores de diferenciação osteogênica, representados pelo β-glicerofosfato, ácido ascórbico e pela dexametasona. Aos 21 dias, nas concentrações de 1ml e de 5ml de extrato/L, houve redução significativa do número e do tamanho dos nódulos de mineralização, respectivamente. A concentração de 5ml de extrato/L também reduziu significativamente a expressão do transcrito para osteopontina em comparação ao controle. Conclui-se que o extrato de Solanum glaucophyllum apresenta efeito negativo sobre culturas de células tronco mesenquimais comprometidas com a diferenciação osteogênica, o que sugere ser este um dos mecanismos pelo qual ocorre osteoporose nos animais intoxicados pela planta.

The effects of two concentrations of triiodothyronine (T3; 0.01 and 1,000 nM) on the osteogenic and chondrogenic differentiation abilities of equine adipose-derived mesenchymal stem cells (AD-MSCs) were evaluated. The osteogenic study evaluated the effect of T3 using alkaline phosphatase activity (ALP) assay; cell viability and density; and formation of mineralized nodules at Days 7, 14, and 21 in culture. The chondrogenic study tested the effect of T3 through ALP assay, mitochondrial metabolism, cell density, and periodic acid-Schiff-positive (PAS+) matrix percentage at Days 7 and 14. In both experiments, analysis of variance was used to compare averages through the Student-Newman-Keuls test. In the osteogenic study, no differences in any variable were detected between groups at Day 7. At Day 14, 0.01 nM T3 reduced cell density and the number of mineralized nodules despite the increase in ALP activity and mitochondrial metabolism (P.05). ALP activity increased at 1,000 nM T3 concentration (P.05). At Day 21, 0.01 nM T3 treatment increased ALP activity compared with control treatment (P.05). At 1,000 nM concentration, T3 reduced mitochondrial metabolism and cell density (P.05). In the chondrogenic study, the two T3 concentrations increased cell density compared with control treatment at Day 7. At Day 14, higher T3 concentration reduced mitochondrial metabolism, ALP activity, cell density, and PAS+ chondrogenic matrix percentage compared with control treatment (P.05). Thus, T3 addition to equine AD-MSC cultures has no enhancement effect on osteogenic or chondrogenic differentiation and may, in fact, negatively affect cell density and matrix synthesis depending on hormone concentration and culture time.