Effect of ProRoot MTA® and Biodentine® on osteoclastic differentiation and activity of mouse bone marrow macrophages

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.

Toxicity of synthetic flavorings, nature identical and artificial, to hematopoietic tissue cells of rodents / Toxicidade de aromatizantes alimentares sintéticos idênticos aos naturais e artificiais a células de tecido hematopoiético de roedores

Abstract The goal of this study was to analyze cytotoxicity, genotoxicity and mutagenicity to bone marrow cells of mice of nature identical synthetic flavorings, passion fruit and strawberry, and artificial synthetic flavorings, vanilla, chocolate, tutti-frutti and cookie, at doses 0.5; 1.0; 2.0; 5.0 and 10.0 mL/kg. The additives were given to the animals by gavage in a single daily application for seven days. Data were subjected to analysis of variance (ANOVA) followed by post Tukey's post hoc test, p <0.05. Animals treated with 2.0; 5.0 and 10.0 mL/Kg of flavorings chocolate, strawberry and cookie, and 5.0 and 10.0 mL/Kg of flavorings vanilla and passion fruit died on the fifth and sixth day of the experiment, respectively. The doses 0.5 and 1.0 mL/Kg of the six additives significantly reduced erythropoiesis in the examined tissue. Also, treatments 0.5 and 1.0 mL/Kg of chocolate, and 1.0 mL/Kg of strawberry and biscuit induced the formation of micronuclei in the bone marrow erythrocytes, at a significant frequency. Therefore, under the study conditions, the six microingredients analyzed were cytotoxic and genotoxic, and additives strawberry, chocolate and cookie were also mutagenic in at least one of the evaluated doses.

Resumo Os aromatizantes são essenciais para a indústria na confecção de alimentos industrializados. Porém, pouco se sabe sobre o potencial tóxico desses microingredientes alimentares. Dessa forma, objetivou-se neste trabalho analisar, em células de medula óssea de camundongos, a citotoxicidade, genotoxicidade e mutagenicidade de aromatizantes alimentares sintéticos idênticos ao natural, de maracujá e morango, e artificiais, de baunilha, chocolate, tutti-frutti e biscoito, nas doses 0,5; 1,0; 2,0; 5,0 e 10,0 mL/Kg. Os aditivos foram administrados aos animais via gavagem em aplicação diária única durante sete dias. Os dados obtidos foram submetidos ao procedimento estatístico ANOVA com pós teste de Tukey, com p < 0.05. Os animais tratados com 2,0; 5,0 e 10,0 mL/Kg dos aromatizantes de chocolate, morango e biscoito, e 5,0 e 10,0 mL/Kg dos aromatizantes de baunilha e maracujá vieram a óbito no quinto e sexto dia de experimento, respectivamente. As doses 0,5 e 1,0 mL/Kg dos seis aditivos reduziram significativamente a eritropoiese do tecido analisado. Ainda, os tratamentos 0,5 e 1,0 mL/kg de chocolate, e 1,0 mL/Kg de morango e biscoito induziram a formação de micronúcleos aos eritrócitos de medula em frequência significante. Portanto, nas condições de estudo estabelecidas, os seis microingredientes analisados foram citotóxico e genotóxicos, e os aditivos de morango, chocolate e biscoito também foram mutagênicos em pelo menos uma das doses avaliadas.

Reduction of doxorubicin-induced genotoxicity by Handroanthus impetiginosus in mouse bone marrow revealed by micronucleus assay / Redução da genotoxicidade induzida pela doxorrubicina por Handroanthus impetiginosus na medula óssea de camundongos revelada pelo ensaio do micronúcleo

Abstract Handroanthus impetiginosus has long been used in traditional medicine and various studies have determined the presence of bioactive chemical compounds and potential phytotherapeutics. In this study, the genotoxicity of the lyophilized tincture of H. impetiginosus bark (THI) was evaluated in mouse bone marrow using micronucleus assays. The interaction between THI and genotoxic effects induced by the chemotherapeutic agent, doxorubicin (DXR), was also analyzed. Experimental groups were evaluated 24 to 48 h after treatment with N-nitroso-N-ethylurea (NEU; 50 mg/kg), DXR (5 mg/kg), sodium chloride (NaCl; 150 mM), and THI (0.5-2 g/kg). Antigenotoxic assays were carried out using THI (0.5 g/kg) in combination with NEU or DXR. Analysis of the micronucleated polychromatic erythrocytes (MNPCEs) indicated no significant differences between treatment doses of THI (0.5-2 g/kg) and NaCl. Polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) ratios did not indicate any statistical differences between DXR and THI or NaCl, but there were differences between THI and NaCl. A significant reduction in MNPCEs and PCE/NCE ratios was observed when THI was administered in combination with DXR. This study suggested the absence of THI genotoxicity that was dose-, time-, and gender-independent and the presence of moderate systemic toxicity that was dose-independent, but time- and gender-dependent. The combination of THI and DXR also suggested antigenotoxic effects, indicating that THI reduced genotoxic effects induced by chemotherapeutic agents.

Resumo Handroanthus impetiginosus tem sido usada durante um longo período pela medicina tradicional e vários estudos têm demonstrados a presença de compostos químicos e potencial fitoterapêutico. Esta pesquisa avaliou a genotoxicidade da tintura da casca liofilizada de H. impetiginosus (THI) usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre THI e os efeitos genotóxicos induzidos pelo quimioterápico doxorrubicina (DXR) também foram analisados. Grupos experimentais foram analisados a 24-48 h após o tratamento com N-Nitroso-N-etiluréia (NEU; 50 mg/kg), DXR (5 mg/kg), NaCl (150 mM) e THI (0,5-2 g/kg). O ensaio antigenotóxico foi conduzido utilizando THI (0,5 g/kg) em combinação com NEU ou DXR. A análise de eritrócitos policromáticos micronucleados (EPCMNs) não mostrou diferenças significativas entre as doses de tratamento (0,5-2 g/kg) e NaCl. As proporções de eritrócitos policromáticos (EPC)/eritrócitos normocromáticos (ENC) não revelaram diferenças estatísticas entre DXR e THI ou NaCl, porém houve diferenças entre THI e NaCl. Uma redução significativa em EPCMNs e na razão EPC/ENC foi observada quando THI foi administrado em combinação com DXR. Essa pesquisa sugere ausência de genotoxicidade de THI, dose-, tempo- e sexo-independente, e moderada toxicidade sistêmica dose-independente, mas tempo- e sexo-dependente. A associação do THI e DXR também sugere efeitos antigenotóxicos. Por conseguinte, THI pode reduzir os efeitos genotóxicos induzidos pelo quimioterapêutico.

Antigenotoxicity protection of Carapa guianensis oil against mitomycin C and cyclophosphamide in mouse bone marrow

ABSTRACT The aim of this study was to evaluate the possible protective of C. guianensis oil against MMC and CP, which are direct- and indirect-acting chemical mutagens, using the micronucleus test. Three experiments were performed. First the C. guianensis oil was co-administered to mice at doses of 250, 500 and 1000 mg/kg bw with 4 mg/kg bw MMC or 50 mg/kg bw CP. Second, the mutagenic drug (CP) was administered ip 50 mg/kg bw and after 6 and 12 hours 250 and 500 mg/kg bw of C. guianensis oil were administered. In the last, C. guianensis oil was administrated (250 and 500 mg/kg bw) during five days and after it was administered ip 50 mg/kg bw CP. The results obtained showed that the C. guianensis oil is not cytotoxic neither genotoxic to mouse bone marrow. Regarding the antimutagenic effect, all doses of C. guianensis oil were significantly (p < 0.05) effective in reducing the frequency of micronucleated polychromatic erythrocytes, when compared with MMC or CP alone. Based on these results, our results suggest that the C. guianensis oil shows medicinal potential as an antimutagenic agent, modulating the mutagenicity caused by both direct- and indirect-acting chemical mutagens, in a mammalian model.

Efectos in vivo del ranelato de estroncio sobre células progenitoras de médula ósea de ratas diabéticas / In vivo effects of strontium ranelate on bone marrow progenitor cells of diabetic rats

La diabetes mellitus (DM) crónica se asocia con reducción en el contenido mineral óseo (osteopenia y osteoporosis). El objetivo de este trabajo fue evaluar la acción del ranelato de estroncio (RaSr) administrado por vía oral a animales control y diabéticos, sobre el potencial osteogénico de células progenitoras de médula ósea (CPMO). Dieciséis ratas Wistar macho jóvenes se dividieron en dos grupos: controles (C) y diabéticas (D) con destrucción parcial de células b-pancreáticas mediante inyecciones intraperitoneales consecutivas de nicotinamida y estreptozotocina. Siete días después de la inyección, cada grupo se subdividió: sin tratamiento, o tratadas oralmente con RaSr (625 mg/kg/día) durante seis semanas, luego de lo cual los animales fueron sacrificados. Las CPMO se obtuvieron de ratas de los cuatro grupos, por lavados del canal diafisario medular (húmero o fémur o ambos) y cultivo hasta confluencia en DMEM-10% FBS. La proliferación celular se evaluó mediante el ensayo de MTT. Luego las CPMO se replaquearon e incubaron en un medio osteogénico durante 14 días (fosfatasa alcalina [FAL] y colágeno tipo 1) o 21 días (mineralización). Las CPMO del grupo C+RaSr mostraron un aumento significativo versus control en la proliferación (133%) y en la diferenciación osteogénica (colágeno 143%, FAL 168%, mineralización 117%). La DM (grupo D) disminuyó significativamente la proliferación y diferenciación osteoblástica de las CPMO. El tratamiento con RaSr (grupo D+RaSr) previno completamente estos efectos antiosteogénicos de la DM. Así, en nuestro modelo experimental in vivo, la DM disminuye el potencial osteogénico de CPMO, efecto que puede ser prevenido por un tratamiento oral con RaSr. (AU)

Chronic diabetes mellitus (DM) is associated with a reduction in bone mineral content (osteopenia and osteoporosis). The object of this study was to evaluate the in vivo effect of he anti-osteoporotic drug strontium ranelate (SrRa) administered orally to control and diabetic animals, on the osteogenic potential of bone marrow progenitor cells (BMPC). Sixteen young male Wistar rats were divided into two groups: control (C) and diabetic with partial beta-cell destruction via consecutive intra-peritoneal injections of nicotinamide and streptozotocin (D). Seven days postinjection, each group was sub-divided: without treatment, or oral treatment with SrRa (625 mg/kg/day) for six weeks, after which the animals were euthanised (groups C, C+SrRa, D, D+SrRa). BMPC were obtained from rats of all four groups by flushing of the diaphysary canal (humerus and/or femur). Adherent cells were then cultured until confluence in DMEM10% FBS. Cell proliferation was evaluated with the MTT mitogenic bioassay. BMPC were replated and incubated in an osteogenic medium for 14 days (determination of alkaline phosphatase [ALP] and type-1 collagen) or 21 days (evaluation of mineralisation). BMPC from C+SrRa rats showed a significant increase versus control in proliferation (133%) and in osteogenic differentiation (collagen 143%, ALP 168%, mineralisation 117%). Induction of diabetes (group D) significantly decreased the proliferation and osteoblastic differentiation of BMPC. Treatment of diabetic animals with SrRa (group D+SrRa) completely prevented these anti-osteogenic effects of Diabetes. Thus, in our experimental in vivo model, Diabetes decreases the osteogenic potential of BMPC, an effect that can be prevented by oral treatment with strontium ranelate. (AU)

Evaluation of the genotoxicity and mutagenicity of Ca3SiO5-based cement

Abstract Ca3SiO5 is new cement based on the composition of Portland that has been developed to have superior physicochemical and biological properties. In a clinical evaluation, the cement did not appear to have cytotoxic properties and allowed for the proliferation of pulp cells and gingival fibroblasts. However, no previous studies have evaluated the genotoxicity or the mutagenicity of Ca3SiO5in vivo. Therefore, the goal of this study is to evaluate the genotoxic and mutagenic potential of Ca3SiO5-based cement in vivo. Twenty-four male Wistar rats were divided into 3 groups (n = 8). Group A rats received subcutaneous implantation of Ca3SiO5 in the dorsum. Group B rats received a single dose of cyclophosphamide (positive control). Group C rats received subcutaneous implantation of empty tubes in the dorsum (negative control). After 24 hours, all animals were euthanized and the bone marrow of the femurs was collected for use in the comet assay and the micronucleus test. The comet assay revealed that the Ca3SiO5 group had a tail intensity of 23.57 ± 7.70%, the cyclophosphamide group had a tail intensity of 27.43 ± 7.40%, and the negative control group had a tail intensity of 24.75 ± 5.55%. The average number of micronuclei was 6.25 (standard deviation, SD = 3.53) in the Ca3SiO5 group, 9.75 (SD = 2.49) in the cyclophosphamide group, and 0.75 (SD = 1.03) in the negative control group. There was an increase in the micronuclei frequency in the Ca3SiO5 group compared to that of the negative control group (p < 0.05). Our data showed that exposure to the Ca3SiO5-based cement resulted in an increase in the frequency of micronuclei, but no genotoxicity was detected according to the comet assay.

Inducible protective processes in animal systems XIV: Cytogenetic adaptive response induced by EMS or MMS in bone marrow cells of diabetic Mouse

Background: Adaptive response has been well studied by employing physical and chemical agents in normal test systems, whereas in diseased conditions very little data are available

Aim of the study: To know the presence or absence of adaptive response in diseased condition, alkylating agents such as EMS or MMS have been employed in diabetic mouse

Material and methods: To induce diabetes, mice were injected with 180 mg/kg body weight of Stz. Diabetic mice were treated with conditioning [100 mg/kg body weight of EMS or 40 mg/kg body weight of MMS], challenging [300 mg/kg body weight of EMS or 160 mg/kg body weight of MMS] and combined doses of EMS or MMS with 8 h time lag. Parallelly controls were maintained. Mice were sacrificed at 24 or 48 or 72 h RTs. Bone marrow was extracted and slides were prepared by a routine air dry technique by Evans et al. [1964] to analyze the chromosomal aberrations

Results: The results show that both the alkylating agents induced exclusively chromatid type of aberrations in both diabetic and non diabetic mice, but it is to be underlined that MMS is a more potent inducer of aberrations than EMS. Eventhough, combined treatment of EMS or MMS induced significantly less chromosomal breaks compared to challenging treatment [p< 0.05] in diabetic mice, EMS induced 40% reduction of breaks, compared to 51.74% by MMS at 24 h RT. This is true to other tested RTs

Conclusion:[1] Methylating agents are a more effective inducer of adaptive response than ethylating agents in diabetic mouse. [2] Further, it is interesting to note that the percentage reduction of chromosomal breaks in diabetics is comparatively much less than in non diabetic mouse, inferring that there is variation in adaptive response between diseased and non diseased condition

Re-emergence of Chorioptes bovis (Acari: Psoroptidae) in cattle in the state of Rio Grande do Sul, Brazil / Reemergência de Chorioptes bovis (Acari: Psoroptidae) em bovinos no Estado do Rio Grande do Sul, Brasil

Here we describe an outbreak of chorioptic mange in cattle, 56 years after its first identification in Brazil. Between the months of June and July 2011, dermatitis characterized by alopecia and crusted and thickened skin at the insertion of the tail and in the ischiorectal fossa was recognized in 40 (35.7%) out of 112 Holstein cows on a farm in the northeastern mesoregion of the state of Rio Grande do Sul, Brazil. After diagnosing mange caused by Chorioptes bovis, the cows were weighed and treated with 0.5% ivermectin, as a pour-on single dose, and were separated into two groups: cows in early lactation and those in late lactation. The survival rate of C. bovis and the healing rate in the two groups of infested cows were monitored every seven days through skin scrapings. After 28 days of evaluation, the cure rate through treatment was greater among cows in early lactation (p <0.0001). The survival rate of C. bovis was higher in cows in late lactation.

O objetivo deste estudo foi descrever um surto de sarna corióptica em bovinos, 56 anos após a sua primeira identificação no Brasil. Entre os meses de junho a julho de 2011, a dermatite caracterizada por alopecia, com crosta e espessamento da pele na inserção da cauda e na fossa isquiorretal, foi observada em 40 (35,7%) de 112 vacas holandesas de uma propriedade rural pertencente à Mesorregião do Nordeste do Estado do Rio Grande do Sul, Brasil. Após o diagnóstico da sarna causada por Chorioptes bovis, as vacas foram pesadas, tratadas com 0,5% de ivermectina pour on em dose única e separadas em dois grupos: vacas no início da lactação e no final da lactação. A taxa de sobrevivência de C. bovis e a taxa de cura dos dois grupos de vacas infestadas foram monitoradas a cada sete dias por meio de raspas de pele. Após 28 dias do estudo, a taxa de cura com o tratamento foi maior em vacas no início da lactação (p <0,0001). A taxa de sobrevivência de C. bovis foi maior em vacas no final da lactação.

Potential chemoprotective effects of green propolis, L-lysine and celecoxib on bone marrow cells and peripheral blood lymphocytes of Wistar rats subjected to bladder chemical carcinogenesis

PURPOSE: To evaluate the genotoxicity of propolis and L-lysine, as well as their effects on the possible cellular damage in erythroblasts (bone marrow) and leukocytes (peripheral blood) caused by the carcinogen BBN (n - butyl - n {4 - hydroxybutyl} nitrosamine) in rats subjected to bladder carcinogenesis and treated with green propolis and L-lysine. METHODS: One hundred and twenty five rats were distributed into the following groups: I, IIA, IIB, III, K, L M N, X, XI, XII and XIII. Groups I to X received BBN in drinking water for 14 weeks (wks). Group I was treated with intragastric (ig) propolis at 150 mg/kg body weight, for 44 wks, beginning 30 days before start of BBN. Groups IIA and III were treated with propolis (150 mg/kg), for 40 wks, subcutaneous (sc) and ig, respectively, beginning simultaneously with BBN. On the 32nd wk, the animals of groups L, M and N were treated ig with L-lysine (300 mg/kg), celecoxib (30 mg/kg) and propolis (300 mg/kg), respectively, up to the 40th wk. The groups that received only BBN (IIB and K) were treated with water, sc and orally, respectively, for 40 wks. Groups XI, XII and XIII received respectively propolis (150 mg/kg), L-lysine (150 mg/kg) and water ig for 40 wks. After 40 wks, the surviving animals were anesthetized and subjected to femoral bone marrow aspiration and blood collection from the aorta, for CA and MNT, respectively, for investigation of genotoxicity. RESULTS: Groups IIB and K, which received only BBN and water, showed the greatest DNA damage in peripheral leukocytes (CA) and largest number of micronuclei in bone marrow erythrocytes (MNT) in relation to all other groups that received BBN and lysine and/or propolis (p<0.001). CONCLUSIONS: Both propolis and L-lysine are effective in protecting against genotoxicity, as well not being genotoxic themselves toward the cells evaluated, at the doses and times administered and according to the two tests utilized. .

Triiodotironina não aumenta a diferenciação osteogênica reduzida pela idade de células-tronco mesenquimais da medula óssea de ratas / Triiodothyronine does not increase osteogenic differentiation reduced by age in bone marrow mesenchymal stem cells of female rats

OBJETIVO: Avaliar se a adição de T3 aumenta o potencial osteogênico das células-tronco mesenquimais da medula óssea (CTM-MO) de ratas adultas normais comparado ao de ratas jovens. MATERIAIS E MÉTODOS: CTM-MO foram cultivadas em meio osteogênico e separadas em seis grupos: 1) CTM-MO de ratas jovens; 2) CTM-MO de ratas adultas; 3, 4, 5 e 6) CTM-MO de ratas adultas com T3 nas concentrações de 0,01; 1; 100 e 1000 nM, respectivamente. Foram avaliados: atividade da fosfatase alcalina, conversão do dimetiltiazol (MTT) e síntese de colágeno aos sete, 14 e 21 dias e celularidade e número de nódulos de mineralização aos 21 dias de diferenciação. RESULTADOS: T3 reduziu significativamente a conversão do MTT, a atividade da fosfatase alcalina, a síntese de colágeno e a formação dos nódulos de mineralização em pelo menos uma das doses e dos períodos estudados (p < 0,05). Os valores foram menores quando comparados aos das CTM-MO de ratas jovens e adultas sem T3 (p < 0,05). CONCLUSÃO: T3 apresenta efeitos negativos sobre os fatores envolvidos na diferenciação osteogênica das CTM-MO de ratas adultas.

OBJECTIVE: To examine if triiodothyronine (T3) increases osteogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs) of adult rats compared with young rats. MATERIALS AND METHODS: BMMSCs were cultured in osteogenic medium and distributed into six groups: 1) BMMSCs of young rats; 2) BMMSCs of adult rats; 3, 4, 5 and 6) BMMSCs of adult rats with T3 (0.01, 1, 100 to 1000 nM). We analyzed alkaline phosphatase activity, dimethylthiazol (MTT) conversion, and collagen synthesis at 7, 14, and 21 days, and percentage of cells per field and number of mineralized nodules at 21 days of differentiation. RESULTS: T3 reduced MTT conversion, alkaline phosphatase activity, collagen synthesis, and the synthesis of mineralizalized nodules in at least one of the doses and periods studied (p < 0.05). Values were lower when compared with young and adult rats BMMSCs (p < 0.05) without T3. CONCLUSION: T3 has a negative effect on the factors involved in osteogenic differentiation of BMMSC from adult rats.

Inhibitory effects of osteoprotegerin on osteoclast formation and function under serum-free conditions

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kappaB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor kappaB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.

Efeito da triiodotironina na expressão das proteínas ósseas durante a diferenciação osteogênica de células-tronco mesenquimais / Effect of triiodothyronine on the bone proteins expression during osteogenic differentiation of mesenchymal stem cells

OBJETIVO: O objetivo deste estudo foi avaliar o efeito da T3 na expressão da osteocalcina, osteo­pontina e colágeno I durante a diferenciação osteogênica das células-tronco mesenquimais (CTM). MATERIAIS E MÉTODOS: As células da medula óssea de ratas Wistar jovens foram extraídas, cultivadas e separadas em cinco grupos: controle (indiferenciado), diferenciado (estímulo osteogênico) e diferenciado com T3 (10-3 nM, 10-2 nM e 100 nM). Para cada grupo, foram cultivadas quatro amostras que foram analisadas por RT-PCR tempo real aos 7, 14 e 21 dias, para quantificação dos transcritos gênicos para osteocalcina, osteopontina e colágeno I. RESULTADOS: Todos os grupos diferenciados sem T3 ou com T3 independentemente da concentração apresentaram expressão de colágeno I significativamente menor e expressão de osteocalcina e osteopontina significativamente maior em comparação a das CTM indiferenciadas. Mas o grupo T3 100 nM apresentou concentração de osteocalcina mais elevada e semelhante à da cultura de osteoblastos. CONCLUSÃO: Conclui-se que a triiodotironina não altera a expressão de osteopontina e de colágeno pelas CTM, mas aumenta a expressão da osteocalcina durante a diferenciação osteogênica in vitro, sendo esse efeito dose-dependente.

OBJECTIVE: The aim of this study was to evaluate the effect of T3 on the expression of osteocalcin, osteopontin and collagen I during osteogenic differentiation of mesenchymal stem cells (MSC). MATERIALS AND METHODS: The bone marrow cells of Wistar rats with 30 days of age were extracted, cultured and separated into five groups: control (undifferentiated), differentiated (osteogenic stimulus) and differentiated with T3 (10-3 nM, 10-2 nM and 100 nM). For each group, four samples were cultured and were analyzed by real time RT-PCR at 7, 14 and 21 days for quantification of gene transcripts for osteocalcin, osteopontin and collagen I. RESULTS: All the different groups without T3 or with T3 regardless of the concentration, showed the collagen I expression significantly lower expression, and osteocalcin and osteopontin expression significantly greater than that of undifferentiated MSC. Nevertheless, the group T3 100 nM showed higher expression of osteocalcin and a similar expression of the osteoblast culture. CONCLUSION: In conclusion, the triiodothyronine does not affect the expression of osteopontin and collagen I, but increases ostecalcin expression during osteogenic differentiation in vitro of the MSC, and this effect is dose-dependent.

Cadmium chloride toxicity suppresses osteogenic potential of rat bone marrow mesenchymal stem cells through reducing cell viability and bone matrix mineralization.

Background and Objectives: Cadmium an environmental pollutant, exert several risks to human health. In this study we investigated the effect of cadmium chloride (CdCl 2 ) on Viability, morphology and bone Matrix Miniralization of Rat Bone Marrow Mesenchymal Stem Cells (rMSCs). Materials and Methods: rMSCs were cultured in DMEM containing 15% FBS and pen-strep. After 21 days of treatment with the selected doses of 750 and 2000 nM of CdCl 2 viability, colony forming unit, population doubling number, DAN breakage and the morphology of the cells were studied. Also to study the effects of CdCl2 on differentiation property, the morphology and bone matrix mineralization via estimation of intracellular calcium concentration and quantitative alizarin red were also evaluated in the cells using Hoechst, Acridine orange and Alizarin red staining. Data was analyzed using one-way ANOVA and Tukey ' s test and the means difference was considered significant at P<0.05. Results: The mean viability, colony forming unit, population doubling number and also the mean bone matrix mineralization of the rMSCs treated with CdCl 2 significantly reduced in a dose dependent manner. Nuclear fragmentation and cytoplasm shrinkage was also seen in the treated cells. Conclusion: CdCl 2 can reduce the viability and bone matrix mineralization of rMSCs even at low doses.

Evaluation of vascular endothelial growth factor (VEGF) in interradicular bone marrow in olpadronate treated animals

Vascular endothelial growth factor (VEGF) is a protein that increases vascular permeability and induces the proliferation, migration and survival of endothelial cells. Bisphosphonates (BPs) are antiresorptive drugs that are widely used in the treatment of bone metabolism diseases and bone metastases. Since 2003, cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) have been reported. Few papers explain the mechanisms that induce BRONJ; some authors mention alterations in bone remodelling and a certain antiangiogenic effect of BPs. The aim of this study is to evaluate the expression of VEGF in bone marrow cells and the number of blood vessels and area occupied by them in animals treated with the BP sodium olpadronate (OPD). We used 16 Wistar rats, 60 days old, divided into two groups, experimental (OPD) and control. The OPD group received 0.3 mg/kg/week intraperitoneal OPD for 5 weeks. The control group received an equivalent intraperitoneal volume of physiological saline solution. After euthanasia, hemimandibles were processed and mesio-distal histological sections of the first molar were prepared. Sections were stained with hematoxylin-eosin (HE), immunohistochemical detection of VEGF was performed (sc- 7269) and the following histomorphometric parameters were evaluated: In HE-stained sections - number of blood vessels per sq. mm. and percentage (%) of area occupied by blood vessels in relation to total area evaluated; in sections with immunohistochemical detection of VEGF – number of VEGF+ bone marrow cells per sq. mm. Data underwent statistical analysis. Number of blood vessels/mm2 was significantly lower in the OPD group (OPD: 92 ± 16; Control: 140 ± 31; p<0.05) and % vascular area/ total area evaluated showed no significant difference (OPD: 15.6 ± 6.1; Control: 10.2 ± 4.2). Number of VEGF+ cells/mm2 was lower in the OPD group than in the control group, with statistically significant differences (OPD: 7804.8 ± 597; Control: 13187.6 ± 894; p<0.001). The results of this study suggest that monosodium olpadronate has an antiangiogenic effect. Further studies are needed to reveal its potential as an antitumor agent and its connection with the onset of BRONJ.

El factor de crecimiento vascular (VEGF) es una proteina que incrementa la permeabilidad vascular, induce la proliferacion, migracion y supervivencia de las celulas endoteliales. Los bifosfonatos (BFs) son drogas antirresortivas ampliamente utilizadas en el tratamiento de enfermedades que alteran el metabolismo oseo y de metastasis oseas. Desde el 2003 se han reportado casos de osteonecrosis de maxilar asociada al uso de BFs (ONAB). Escasas publicaciones explican los mecanismos que inducen la ONAB, algunos autores mencionan las alteraciones en la remodelacion osea y un cierto efecto antiangiogenico de los BFs. El objetivo del presente trabajo es evaluar la expresion de VEGF en celulas de la medula osea y el numero y el area ocupada por vasos sanguineos en animales tratados con el BF olpadronato monosodico (OPD). Se utilizaron 16 ratas Wistar de 60 dias divididas en dos grupos, experimental (OPD) y control. El grupo OPD, recibio 0,3 mg/kg/sem de OPD via IP, durante 5 semanas. El grupo control, recibio un volumen equivalente via IP de solucion fisiologica. Luego de practicada la eutanasia se obtuvieron las hemimandibulas y se procesaron para obtener cortes histologicos mesio-distales del primer molar. Se realizo la coloracion hematoxilina-eosina (HE) y la deteccion inmunohistoquimica de VEGF (sc-7269) y se evaluaron los siguientes parametros histomorfometricos: En cortes con H&E: Numero de vasos sanguineos por mm2 y porcentaje (%) de area ocupada por los vasos sanguineos en relacion al area total evaluada; en cortes con la deteccion inmunohistoquimica de VEGF: Numero de celulas medulares VEGF+ por mm2. Los datos fueron estadisticamente analizados. El N° vasos sanguineos/mm2 fue significativamente menor en el grupo OPD (OPD: 92 ± 16; control: 140 ± 31; p<0,05) y el % area vascular/area total evaluada no mostro diferencias significativas (OPD: 15,6 ± 6.1; Control: 10.2 ± 4.2). El N° de celulas VEGF+/mm2 en el grupo OPD fue menor que en el grupo control con diferencias estadisticamente significativas (OPD: 7804,8 ± 597; Control: 13187,6 ± 894; p<0,001). Los resultados de este estudio sugieren que el olpadronato monosodico tiene un efecto antiangiogenico. Futuros estudios revelaran su potencial como agente antitumoral asi como tambien su relacion con la aparicion de ONAB.

Assessment of mutagenicity and cytotoxicity of Solanum paniculatum L. extracts using in vivo micronucleus test in mice / Avaliação da mutagenicidade e citotoxicidade de extratos de Solanum paniculatum L. usando o teste do micronúcleo in vivo em camundongos

Solanum paniculatum L. is a plant species widespread throughout tropical America, especially in the Brazilian Savanna region. It is used in Brazil for culinary purposes and in folk medicine to treat liver and gastric dysfunctions, as well as hangovers. Because of the wide use of this plant as a therapeutic resource and food, the present study aimed at evaluating the mutagenic and cytotoxic effects of S. paniculatum ethanolic leaf and fruit extracts using the mouse bone marrow micronucleus test. Our results indicate that neither S. paniculatum ethanolic leaf extract nor its ethanolic fruit extract exhibited mutagenic effect in mice bone marrow; however, at higher doses, both extracts presented cytotoxic activity.

Solanum paniculatum L., popularmente conhecida como jurubeba, ocorre em toda a América tropical, especialmente no Cerrado. No Brasil, é utilizada para fins culinários e na medicina popular para o tratamento de distúrbios gástricos e hepáticos, além de ressacas. Devido à grande utilização desta planta pela população como recurso terapêutico e alimentício, o presente estudo teve como objetivo avaliar as atividades mutagênica e citotóxica dos extratos etanólico das folhas e frutos de S. paniculatum utilizando o teste do micronúcleo em medula óssea de camundongos. Os resultados indicam que os extratos etanólicos tanto das folhas quanto dos frutos de S. paniculatum não apresentaram ação mutagênica em medula óssea de camundongos, porém, em doses mais elevadas, ambos os extratos exibiram atividade citotóxica.

Osteoblast response to commercially available demineralized bone matrices - An in-vitro study.

Objective: Reconstruction of lost attachment apparatus is a major goal of periodontal therapy. Although various osteoinductive bone replacement grafts (BRGs) have been used with apparent clinical success, unequivocal evidence of osteoinductivity may be obtained only through the demonstration of increased osteoblastic/osteoclastic differentiation following exposure to these materials. Materials and Methods: Bone marrow stem cells (BMSCs) obtained from rat femur were cultured in Dulbecco's Modified Eagles Medium (DMEM) and 10% fetal bovine serum (FBS). They were then exposed to two demineralized bone matrices (DBM's) - Grafton and Osseograft, and divided into three groups, comprising of a negative control (BMSC + DMEM + 10% FBS), Grafton, Osseograft. An osteogenic medium (OM) (10 hm dexamethasone, 10 hm b-glycerophosphate, and 50 μg/ml ascorbic acid) was added to create three subgroups comprising of a positive control (OM), Grafton with OM, Osseograft with OM. Results: After an initial phase (up to day 5), both Grafton and Osseograft induced an increased proliferative activity in the BMSCs, which reached a plateau after day 10. These grafts also induced increased alkaline phosphatase activity when compared to the control groups and to BMSCs with an OM. Conclusion: Both Osseograft and Grafton are capable of inducing osteoblastic proliferation and differentiation.

Bone resorptive activity of osteoclast-like cells generated in vitro by PEG-induced macrophage fusion

Normal bone remodeling is maintained by a balance between osteoclast and osteoblast activity, whereas defects in osteoclast activity affecting such balance result in metabolic bone disease. Macrophage-macrophage fusion leading to multinucleated osteoclasts being formed is still not well understood. Here we present PEG-induced fusion of macrophages from both U937/A and J774 cell lines and the induced differentiation and activation of osteoclast-like cells according to the expression of osteoclast markers such as tartrate resistant acid phosphatase (TRAP) and bone resorptive activity. PEG-induced macrophage fusion, during the non-confuent stage, signifcantly increased the osteoclastogenic activity of macrophages from cell lines compared to that of spontaneous cell fusion in the absence of PEG (polyethylene glycol). The results shown in this work provide evidence that cell fusion per se induces osteoclast-like activity. PEG-fused macrophage differential response to pretreatment with osteoclastogenic factors was also examined in terms of its ability to form TRAP positive multinucleated cells (TPMNC) and its resorptive activity on bovine cortical bone slices. Our work has also led to a relatively simple method regarding those previously reported involving cell co-cultures. Multinucleated osteoclast-like cells obtained by PEG-induced fusion of macrophages from cell lines could represent a suitable system for conducting biochemical studies related to basic macrophage fusion mechanisms, bone-resorption activity and the experimental search for bone disease therapeutic alternatives.

[Effects of nitric oxide production inhibitor named, N[G]-nitro-L-arginine methyl ester [L-NAME], on rat mesenchymal stem cells differentiation]

Recently, the findings of some studies have shown that, nitric oxide [NO] probably has an important role in differentiation of mesenchymal stem cells to osteoblasts. The aim of the present investigation was to study the effects of nitric oxide production inhibitor named, N[G]-nitro-L-arginine methyl ester [L-NAME], on rat mesenchymal stem cells differentiation to osteoblasts in vitro. This was an experimental study conducted at Hamedan University of Medical Sciences in 2009, in which rat bone marrow stem cells were isolated in an aseptic condition and cultured in vitro. After third passage, the cells were cultured in osteogenic differentiation medium. To study the effects of L-NAME on osteogenic differentiation, the L-NAME was added to the culture medium at a concentration of 125, 250, and 500 micro M in some culture plates. During the culture procedure, the media were replaced with fresh ones, with a three days interval. After 28 days of culturing; the mineralized matrix was stained using Alizarian red staining method. The gathered data were analyzed by SPSS software version 12 using one way ANOVA. The findings of this study showed that in the presence of L-NAME, differentiation of bone marrow mesenchymal stem cells to osteoblasts was disordered and matrix mineralization significantly decreased in a dose dependent manner. This study revealed that, inhibition of nitric oxide production using L-NAME can prevent the differentiation of rat bone marrow mesenchymal stem cells to osteoblast. The results imply that NO is an important constituent in differentiation of mesenchymal stem cell to osteoblasts

Petroleum ether extract of Cissus quadrangularis (Linn. ) enhances bone marrow mesenchymal stem cell proliferation and facilitates osteoblastogenesis

OBJECTIVE: To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts (osteoblastogenesis) and extracellular matrix calcification. This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification. METHODS: MSCs were cultured in media with or without Cissus quadrangularis for 4 weeks and were then stained for alkaline phosphatase. Extracellular matrix calcification was confirmed by Von Kossa staining. marrow mesenchymal stem cells cultures in control media and osteogenic media supplemented with Cissus quadrangularis extract (100, 200, 300 µg/mL) were also subjected to a cell proliferation assay (MTT). RESULTS: Treatment with 100, 200 or 300 µg/mL petroleum ether extract of Cissus quadrangularis enhanced the differentiation of marrow mesenchymal stem cells into ALP-positive osteoblasts and increased extracellular matrix calcification. Treatment with 300 µg/mL petroleum ether extract of Cissus quadrangularis also enhanced the proliferation rate of the marrow mesenchymal stem cells. Cells grown in osteogenic media containing Cissus quadrangularis exhibited higher proliferation, differentiation and calcification rates than did control cells. CONCLUSION: The results suggest that Cissus quadrangularis stimulates osteoblastogenesis and can be used as preventive/alternative natural medicine for bone diseases such as osteoporosis.

Antimutagenic activity of egyptian propolis and bee pollen water extracts against cisplatin-induced chromosomal abnormalities in bone marrow cells of mice

Bee-collected pollen and propolis are apicultural products which are recognized as a well balanced food. These beehive products are composed of nutritionally valuable substances and contain considerable amounts of polyphenol substances which may have several useful pharmacological properties. The protective activity of Bee-collected pollen [BPE] and water-soluble derivative of propolis [WSDPE] aqueous extracts was studied on cisplatin[CDDP] induced genotoxicity in male albino mice [Mus mascullus]. The treatment of mice with Bee-collected pollen and propolis extracts at doses 140 and 8.4 mg/kg body weight/day, respectively for 14 days synergistically with the intraperitoneal administration of cisplatin at dose of 2.8 mg/kg b.wt exhibited significant chemoprotective activity. Genotoxicity and cytotoxicity was evaluated by the bone marrow chromosomal aberration assay and mitotic index, respectively. WSDPE and BPE, alone did not significantly induce chromosomal aberrations confirming their non-mutagenic effects. While, the animals in groups five and six [G5 and G6], that were injected i.p. with CDDP alone for one week and then for the next 14 days these animals were given WSDPE and BPE in synergistic with i.p. injection of CDDP, exhibited a significant decrease in cytogenetic damages induced by CDDP in bone marrow cells. The anti-cytotoxicity effects of WSDPE and BPE were also evident, as observed by significant increase in mitotic index, when compared to positive control group [G2]. Thus, results of the present investigation revealed that WSDP and BPE have chemoprotective potential against CDDP induced genotoxicity in bone marrow cells of male albino mice. Also, the present investigation indicated that the chemoprotective frequency of BPE was much greater than WSDPE