Efeito da curcumina e três análogos ­ DMAD, DMAM E RI75 ­ sobre viabilidade, diferenciação e expressão gênica em pré-osteoblastos / Effect of curcumin and three of its analogues - DMAD, DMAM and RI75 - on pre-osteoblast cells' viability, differentiation, and gene expression

A curcumina, encontrada nos rizomas da cúrcuma (Curcuma longa L.), tem sido amplamente estudada devido aos seus potenciais benefícios à saúde que, entre outros, incluem propriedades anti-inflamatórias, antioxidantes e cicatrizantes. No entanto, devido à sua baixa biodisponibilidade e farmacocinética desfavorável, compostos análogos são desenvolvidos e estudados para obter melhores características biofarmacêuticas e aumentar os efeitos biológicos. Neste trabalho, avaliamos a atividade da curcumina e três de seus análogos sintéticos (DMAD, DMAM e RI75) sobre a viabilidade e diferenciação de uma linhagem celular pré-osteoblástica (MC3T3-E1). A expressão dos genes fator de crescimento endotelial vascular (vegf), do ligante 12 da quimiocina de motivo C-X-C (cxcl12) e do fator de transcrição runt- related 2 (runx2) também foi avaliada. As células foram estimuladas com curcumina e os três análogos usando concentrações de 10, 30 ou 50 µM. A curcumina e os análogos testados apresentaram nenhuma, ligeira ou moderada citotoxicidade celular em comparação com o controle negativo, após 24, 48 e 72 horas, nas concentrações testadas. Os resultados de atividade de mineralização para a curcumina e o análogo DMAD não apresentaram diferença estatística com o grupo controle, enquanto os análogos DMAM e RI75 apresentaram menor atividade de mineralização. Nenhuma das substâncias apresentou expressão gênica diferencial de cxcl12, um ativador de células endoteliais e inflamatórias. Por outro lado, em comparação com o controle, o análogo de curcumina RI75 mostrou regulação positiva de runx2 e vegf, ambos relacionados ao reparo tecidual. Os resultados sugerem que os análogos DMAD, DMAM e RI75 apresentam citotoxicidade semelhante ou inferior à curcumina natural e maior atividade biológica, com destaque para o análogo RI75, sendo substâncias com potencial promissor para biomodificações de materiais.

Curcumin, found in the rhizomes of turmeric (Curcuma longa L.), has been widely studied due to its potential health benefits which, among others, include anti inflammatory, antioxidant and healing properties. However, due to their low bioavailability and unfavorable pharmacokinetics, analogous compounds are developed and studied to obtain better biopharmaceutical characteristics and increase biological effects. In this work, we evaluated the activity of curcumin and three of its synthetic analogues (DMAD, DMAM and RI75) on the viability and differentiation of a pre-osteoblastic cell line (MC3T3-E1). The expression of the genes vascular endothelial growth factor (vegf), C-X-C motif chemokine ligand 12 (cxcl12) and runt related transcription factor 2 (runx2) was also evaluated. Cells were stimulated with curcumin and the three analogues using concentrations of 10, 30 or 50 µM. Curcumin and the analogues tested showed no, slight or moderate cellular cytotoxicity compared to the negative control, after 24, 48 and 72 hours, at the concentrations tested. The mineralization activity results for curcumin and the DMAD analogue showed no statistical difference with the control group, while the DMAM and RI75 analogues showed lower mineralization activity. None of the substances showed differential gene expression of cxcl12, an activator of endothelial and inflammatory cells. On the other hand, compared to the control, the curcumin analogue RI75 showed upregulation of runx2 and vegf, both related to tissue repair. The results suggest that the DMAD, DMAM and RI75 analogues present similar or lower cytotoxicity compared to natural curcumin and greater biological activity, especially the RI75 analogue, being, therefore, substances with promising potential for biomodification of materials.

Efeitos do laser de baixa intensidade em osteoblastos cultivados em arcabouços nanofibrosos de ácido polilático / Effects of low-intensity laser on osteoblasts cultured on polylactic acid nanofibrous scaffolds

A engenharia de tecidos ósseos é um ramo importante da medicina regenerativa e envolve o desenvolvimento de arcabouços com composição e arquitetura favoráveis à integração celular, além do estudo de fatores capazes de promover a adesão e proliferação celular, incluindo estímulos químicos e biofísicos. O objetivo do estudo foi avaliar a utilização do laser de baixa intensidade (LBI) como uma ferramenta para promover a bioestimulação in vitro de células osteoblásticas cultivadas em arcabouços nanofibrosos de ácido polilático (PLA). Os arcabouços foram produzidos pela técnica de eletrofiação e caracterizados quanto à molhabilidade, composição pela espectroscopia no infravermelho por transformada de Fourier (FTIR), morfologia da superfície por microscópica eletrônica de varredura (MEV), caracterização termogravimétrica (TGA), calorimetria diferencial exploratória (DSC) e cristalinidade por difração de raios-X (DRX). Os ensaios biológicos foram conduzidos com osteoblastos da linhagem OFCOL II cultivados na superfície dos arcabouços e submetidos ou não (grupo controle) a irradiação com laser diodo InGaAIP na potência de 30 mW, nas doses de 1, 4 e 6 J/cm² e nos comprimentos de onda de 660 nm (grupos V1, V4, V6, respectivo as doses) e 780 nm (grupos I1, I4 e I6, respectivo as doses). Os efeitos do LBI na proliferação dos osteoblastos foram avaliados através do método bioquímico Alamar Blue, nos intervalos de 24, 48 e 72h, enquanto a viabilidade e a morfologia celular foram analisadas no intervalo de 72h, através do ensaio Live/Dead e da microscopia eletrônica de varredura (MEV), respectivamente. Os dados do ensaio bioquímico de Alamar Blue mostraram uma maior proliferação celular nos grupos V6 em todos os intervalos analíticos em comparação ao grupo controle (p<0,05). Outras diferenças entre o grupo controle e irradiados foram encontradas apenas nos intervalos de 48h e 72h para V1, e para o grupo IV6 em 72h. O ensaio Live/Dead revelou um aumento na viabilidade celular nos grupos trados com LBI, sendo significativamente maior no grupo V1 quando comparado ao grupo controle. A análise por MEV mostrou adequada interação dos osteoblastos aos arcabouços, com o corpo celular se espalhando ao longo do eixo da nanofibra e a presença de contatos físicos mais evidentes, através da formação de ligação por meio de filopódios e lamelipódios, nos grupos V1, V6 e I6. Em conjunto, os dados do presente trabalho mostraram que o LBI promove a bioestimulação de osteoblastos cultivados sobre nanofibras de PLA, o que aponta para o seu uso potencial nas técnicas de engenharia tecidual óssea, sobretudo no que se refere ao uso do comprimento de onda de 660 nm, a qual apresentou grupos com mais resultados significativos (AU).

Bone tissue engineering is a relevant branch of regenerative medicine and involves the development of scaffolds with composition and architecture favorable to cell integration, in addition to studying factors capable of promoting cell adhesion and proliferation, including chemical and biophysical stimuli. The study aimed to evaluate the use of low-level laser irradiation (LLLI) to promote in vitro biostimulation of osteoblastic cells cultured on polylactic acid (PLA) nanofibrous scaffolds. The scaffolds were produced by the electrospinning technique and characterized in terms of wettability, composition by Fourier transform infrared spectroscopy (FTIR), surface morphology by scanning electron microscopy (SEM), thermogravimetric characterization (TGA), differential scanning calorimetry (DSC) and crystallinity by Xray diffraction (XRD). The biological assays were conducted with osteoblasts of the OFCOL II lineage cultured on the surface of the scaffolds and submitted or not (control group) to irradiation with InGaAIP diode laser, power of 30 mW, with doses of 1, 4 and 6 J/cm² and wavelengths of 660 nm (groups V1, V4, V6, respectively doses) and 780 nm (groups I1, I4 and I6, respectively doses). The effects of LLLT from the perspective of osteoblasts were evaluated using the biochemical method Alamar Blue assay, at intervals of 24, 48 and 72h, while cell viability and morphology were observed at 72h, using the Live/Dead assay and electron microscopy. scan (SEM), respectively. The Alamar Blue assay data showed more significant cell proliferation in groups in the V6 groups at all analytical intervals compared to the control group (p<0.05). Other differences between the control and irradiated groups were found only at intervals of 48h and 72h for V1, and for group IV6 at 72h. The Live/Dead assay revealed an increase in cell viability in the groups treated with LLLT, being significantly higher in the V1 group when compared to the control group. SEM analysis showed adequate interaction between osteoblasts and scaffolds, with the cell body spreading along the nanofiber axis and the presence of more evident physical contacts, through the formation of bonds through filopodia and lamellipodia, in groups V1, V6 and I6. Together, the data from the present study observed that LLLT promotes the biostimulation of osteoblasts cultured on PLA nanofibers, which pointed to its potential use in bone tissue engineering techniques, especially with regard to the use of the wavelength of 660 nm, which presented groups with more significant results (AU).

Influence of titanium nanotubular surfaces, produced by anodization, on the behavior of osteogenic cells: in vitro evaluation / Influência da superfície nanotubular do titânio, produzido por anodização, no comportamento de células osteogênicas: avaliação in vitro

Objective: The objective of this study was to evaluate in vitro the influence of the anodized surface of Ti35Nb7Zr alloy on the behavior of osteogenic cells, for future application in biomedical implants. Material and Methods: For the development of this research, samples of commercially pure titanium (TiCp) and samples of Ti35Nb7Zr alloy were anodized, both were characterized by scanning electron microscopy (SEM) and were plated afterwards with human osteoblast-like cells (MG63 line) (2 x 104). Cell adhesion, cytotoxicity test, formation of mineralization nodules and a comet assay were also performed in different periods. The bottom of the plate was used as a control, without a sample. Results: SEM analysis showed that the topography of both samples presented surfaces covered by nanotubes. Cellular morphology exhibited spreading in both samples proposing an intimate cell- material liaison. After 3 days, the Ti35Nb7Zr group exhibited greater cell viability than the TiCp group (p<0.01). Regarding calcium content, there was no statistical difference between the anodized groups, but there was a difference between the experimental groups and the control group (p<0.01). In the comet assay, the percentage of DNA in the comet tail did not exhibit any significant difference (p>0.05) among the groups in the evaluated periods. Conclusion: It was concluded that this process of anodization was efficient to form nanotubes, as well as promote a positive influence on the behavior of osteogenic cells without promoting cell damage. (AU)

Objetivo: O objetivo deste estudo foi avaliar in vitro a influência da superfície anodizada da liga Ti35Nb7Zr no comportamento de células osteogênicas, para futura aplicação em implantes biomédicos. Material e Métodos: Para o desenvolvimento desta pesquisa, amostras de titânio comercialmente puro (TiCp) e amostras da liga Ti35Nb7Zr foram anodizadas, ambas foram caracterizadas por microscopia eletrônica de varredura (MEV) e posteriormente plaqueadas com células semelhantes a osteoblastos humanos (linha MG63) (2 x 104). Foram realizados em diferentes períodos a adesão celular, teste de citotoxicidade, formação de nódulos de mineralização e ensaio do cometa. O fundo da placa foi usado como controle, sem amostra. Resultados: A análise em MEV mostrou que a topografia de ambas as amostras apresentava superfícies cobertas por nanotubos. A morfologia celular exibiu espalhamento em ambas as amostras, propondo uma ligação íntima célula-material. Após 3 dias, o grupo Ti35Nb7Zr exibiu maior viabilidade celular do que o grupo TiCp (p<0.01). Em relação ao teor de cálcio, não houve diferença estatística entre os grupos anodizados, mas houve diferença entre os grupos experimentais e o grupo controle (p<0.01). No ensaio do cometa, a porcentagem de DNA na cauda do cometa não apresentou diferença significativa (p> 0.05) entre os grupos nos períodos avaliados. Conclusão:Concluiu-se que esse processo de anodização foi eficiente para formar nanotubos, além de promover uma influência positiva no comportamento das células osteogênicas sem promover dano celular. (AU)

Effects of salbutamol, montelukast and prednisone on orthodontic tooth movement in rats / Efeitos do salbutamol, montelucaste e prednisona no movimento dentário ortodôntico em ratos

Objective: To evaluate the effect of salbutamol, montelukast, and prednisone on orthodontic tooth movement in rats. Material and Methods: In vivo experimental preclinical study. The sample consisted of 48 rats randomly distributed in four study groups. Group A was given saline solution; to group B, salbutamol 4 mg/Kg; to group C, montelukast 2.5 mg/Kg and to group D, prednisone 2.5 mg/Kg. All were fitted with orthodontic devices and the medications were administered intraperitoneally every 12 hours for 5 days. The clinical evaluation (variation in the interincisal distance) was performed at one, three, five, and seven days and the histopathological analysis (cell count) at five and seven days. Results: In the clinical evaluation of the variation in the interincisal distance, a significant difference was found in all the evaluations (p <0.05). It was found that the salbutamol group presented higher variation values in the interincisal distance on all the days evaluated. In the histopathological analysis at five and seven days, it was found that the osteoblast and osteocyte count was significantly higher in the salbutamol group compared to the other groups (p <0.05). However, in the subgroup analysis, it was found that there was no significant difference in the osteoblast and osteocyte count between the prednisone, montelukast, and control group (p> 0.05). Conclusion: The administration of salbutamol increased the magnitude of orthodontic tooth movement; nonetheless, the administration of montelukast and prednisone did not modify the magnitude of orthodontic tooth movement in rats. (AU)

Objetivo: Avaliar o efeito do salbutamol, montelucaste e prednisona no movimento dentário ortodôntico em ratos. Material e métodos: Estudo pré-clínico experimental in vivo. A amostra foi composta por 48 ratos distribuídos aleatoriamente em quatro grupos de estudo. O grupo A recebeu solução salina; para o grupo B, salbutamol 4 mg/kg; ao grupo C, montelucaste 2,5 mg/kg e ao grupo D, prednisona 2,5 mg/kg. Todos foram equipados com dispositivos ortodônticos e os medicamentos foram administrados por via intraperitoneal a cada 12 horas por 5 dias. A avaliação clínica (variação da distância interincisal) foi realizada em um, três, cinco e sete dias e a análise histopatológica (contagem de células) em cinco e sete dias. Resultados: Na avaliação clínica da variação da distância interincisal, houve diferença significativa em todas as avaliações (p <0,05). Verificou-se que o grupo salbutamol apresentou maiores valores de variação na distância interincisal em todos os dias avaliados. Na análise histopatológica aos cinco e sete dias, verificou-se que a contagem de osteoblastos e osteócitos foi significativamente maior no grupo salbutamol em comparação aos demais grupos (p<0,05). No entanto, na análise de subgrupos, verificou-se que não houve diferença significativa na contagem de osteoblastos e osteócitos entre os grupos prednisona, montelucaste e controle (p>0,05). Conclusão: A administração de salbutamol aumentou a magnitude do movimento dentário ortodôntico; no entanto, a administração de montelucaste e prednisona não modificou a magnitude do movimento dos dentes ortodônticos em ratos. (AU)

Avaliação dos efeitos de novas hidroxiapatitas e suas modificações superficiais utilizadas para regeneração óssea / Evaluation effects of new hydroxyapatites and their surface modifications used for bone regeneration

O objetivo deste estudo foi sintetizar um substituto ósseo a base de Hidroxiapatita (HAp), modificá-lo superficialmente com hexametafosfato (HMP) e colágeno tipo I (COL) e analisar o comportamento in vitro e in vivo. A síntese de HAp foi realizada pelo método de coprecipitação controlada a partir de H3PO4, CaCl2 e NH4OH. Após processamento foram realizadas as modificações superficiais em soluções de HMP e COL. As partículas de hidroxiapatita e suas modificações foram caracterizadas através das técnicas de potencial-zeta (ζ), tamanho de partícula, espectroscopia de infravermelho com transformada Fourrier (FTIR) e difração de raios X (DRX), as quais evidenciaram alta semelhança química com a HAp biológica. A morfologia foi avaliada através da técnica de microscopia eletrônica de varredura (MEV), a qual mostrou que as nanopartículas de HAp obtidas possuíam aproximadamente 130 nm, pode ser visualizada uma película recobrindo as superfícies modificadas com HMP e COL. Foi realizada cultura de células MC3T3, com análises de MTT, ALP e nódulos de mineralização. Nas análises in vivo, foram realizados defeitos críticos em calvaria de 150 ratos, divididos em 5 grupos (GC:autógeno; G1:HAp; G2:HMP; G3:COL; G4:BioOss) e submetidos a eutanásia após 7,14,30,60 dias. Os espécimes foram avaliados em cortes calcificados MicroCt e confocal, apresentando fechamento do defeito e formação óssea significante em G1,G3 e G4. Portanto conclui-se que G1 e G3 apresentaram comportamento favorável e viável na neoformação óssea comparado ao G4 substituto ósseo comercialmente disponível, tornando-se uma futura alternativa para regeneração óssea(AU)

The aim of this study was to synthesize a bone substitute based on Hydroxyapatite (HAp), superficially modify it with hexametaphosphate (HMP) and collagen type I (COL) and analyze its behavior in vitro and in vivo. The synthesis of HAp was carried out by the controlled co-precipitation method from H3PO4, CaCl2 and NH4OH. After processing, surface modifications were performed in HMP and COL solutions. HAp particles and their modifications were characterized using the techniques of zeta-potential (ζ), particle size, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), which showed high chemical similarity with the Biological HAp. The morphology was evaluated using the technique of scanning electron microscopy (SEM), which showed that the HAp nanoparticles obtained had approximately 130 nm, a film covering the modified surfaces with HMP and COL can be visualized. Culture of MC3T3 cells was performed with analysis of MTT, ALP and mineralization nodules. In the in vivo analysis, critical calvarian defects were performed in 150 rats, divided into 5 groups (GC:autogenous bone; G1:HAp; G2:HMP; G3:COL; G4:BioOss) and euthanized after 7,14,30,60 days. The specimens were evaluated in calcified MicroCt and confocal sections, showing defect closure and significant bone formation in G1,G3 and G4. Therefore, it is concluded that G1 and G3 presented favorable and viable behavior in bone neoformation compared to the commercially available G4 bone substitute, becoming a future alternative for bone regeneration(AU)

Avaliação da ozonioterapia no tratamento da osteonecrose induzida por zoledronato em ratas senis / Evaluation of ozone therapy in the treatment of zoledronate-induced osteonecrosis in senile rats

A ozonioterapia vem se demonstrando uma ferramenta promissora na prevenção de infecções e no auxílio da reparação tecidual, conciliando com os desafios no tratamento da osteonecrose dos maxilares induzida por medicamentos (ONM-M), este projeto objetiva analisar os efeitos da ozonioterapia, em 55 ratas senis (18 meses), entre 300-350g, induzidas a osteonecrose via medicamentosa (Zoledronato 100µg/kg), após exodontia do primeiro molar inferior. Os animais foram divididos em 4 grupos equitativos (10 ratas por grupo), o primeiro grupo SAL, recebeu aplicações de soro fisiológico por 7 semanas, grupo SAL + OZ recebeu aplicações de soro fisiológico por 7 semanas e o tratamento com a ozonioterapia (0,7mg/kg) a cada 2 dias por 28 dias, o grupo ZOL recebeu aplicações de zoledronato (100µg/kg) por 7 semanas e por último o grupo ZOL + OZ recebeu também aplicações de zoledronato no mesmo protocolo e foi tratado com a ozonioterapia (0,7mg/kg) a cada 2 dias por 28 dias. Todos as ratas receberam a antibioticoterapia (Cristacilina® 0,1ml/kg por dia) iniciando 3 dias antes do procedimento de extração, se estendendo até 4 dias de pós-operatório, passaram pela extração do molar na terceira semana de experimento e foram submetidas a eutanásia na sétima semana de experimento. Após a eutanásia as mandíbulas foram ressecadas, reduzidas e preparadas para as análises microtomográficas (caracterização óssea do osso senil (MCT0) e após terapia com zoledronato (MCT1ZOL) contra seu par controle (MCT1SAL), parâmetros volumétricos (Bv,Bv.Tv,Tb.Th,Tb.N,Tb.Sp,Po.Tot) dos grupos experimentais), histométricas (porcentagem de osso neoformado e porcentagem de osso não vital) e imunoistoquímicas (expressão de TNFa, IL-1b, VEGF, OCN e TRAP). Os resultados da caracterização óssea não apresentaram diferença quando comparado os grupos experimentais (p> 0,05), possivelmente devido ao pouco tempo decorrido na terapia com zoledronato. Os demais resultados comparando os grupos experimentais mostrou com diferenças estatisticamente significativas (p< 0,05) uma característica de osso vítreo, denso, sem vitalidade, pobre em vascularização, com elevados valores para marcadores de inflamação, traduzindo isso em osteonecrose dos maxilares relacionada com a medicação, destoando principalmente do grupo controle SAL, que apresentou melhora na reparação alveolar e características de osso vital e vascularizado. A ozonioterapia (ZOL+OZ, SAL+OZ) apresentou valores significantes estatisticamente quando comparado ao grupo sem tratamento, traduzindo em melhora na vascularização do tecido ósseo, em melhora reparacional do alvéolo, modulação da inflamação local e o aparecimento/manutenção de células osteoblásticas ativas (p< 0,05). Mostrando-se uma terapia viável no controle/tratamento da osteonecrose dos maxilares relacionado com medicamentos(AU)

Ozone therapy has been shown to be a promising tool in the prevention of infections and in the aid of tissue repair, reconciling with the challenges in the treatment of medication-induced jaw osteonecrosis (ONM-M), this project aims to analyze the effects of ozone therapy in 55 rats senile (18 months), between 300-350g, induced to osteonecrosis via medication (Zoledronate 100µg / kg), after extraction of the lower first molar. The animals were divided into 4 equitable groups (ten rats per group), the first SAL group, received saline applications for 7 weeks, SAL + OZ group received saline applications for 7 weeks and ozone therapy (0, 7mg / kg) every 2 days for 28 days, the ZOL group received applications of zoledronate (100µg / kg) for 7 weeks and lastly the ZOL + OZ group also received applications of zoledronate in the same protocol and was treated with ozone therapy (0.7mg / kg) every 2 days for 28 days. All rats received antibiotic therapy (Cristacilina® 0.1ml / kg per day) starting 3 days before the extraction procedure, extending up to 4 days after the operation, underwent molar extraction in the third week of the experiment and were submitted to euthanasia in the seventh week of experiment. After euthanasia, the mandibles were resected, reduced and prepared for microtomographic analysis (bone characterization of senile bone (MCT0) and after therapy with zoledronate (MCT1ZOL) against its control pair (MCT1SAL), volumetric parameters (Bv, Bv.Tv, Tb .Th, Tb.N, Tb.Sp, Po.Tot) of the experimental groups), histometric (percentage of newly formed bone and percentage of non-vital bone) and immunohistochemistry (expression of TNFa, IL-1b, VEGF, OCN and TRAP) . The results of bone characterization did not show any difference when comparing the experimental groups (P> 0.05), possibly due to the short time elapsed in zoledronate therapy. The other results comparing the experimental groups showed with statistically significant differences (P < 0.05) a characteristic of vitreous bone, dense, without vitality, poor in vascularization, with high values for inflammation markers, translating this into a related jaw osteonecrosis with medication, disagreeing mainly with the SAL control group, which showed improvement in alveolar repair and characteristics of a vital and vascularized bone. Ozone therapy (ZOL + OZ, SAL + OZ) showed statistically significant values when compared to the untreated group, translating into an improvement in bone tissue vascularization, a reparational improvement of the alveolus, modulation of local inflammation and the appearance/maintenance of cells active osteoblasts (P < 0.05). Showing to be a viable therapy in the control/treatment of osteonecrosis of the jaws related to drugs(AU)

Citotoxicidade e efeito indutor de mineralização de flavonoides sobre células osteoblásticas / Cytotoxicity and inducing effect of mineralization of flavonoids on osteoblastic cells

A perda óssea dentária e a formação de lesões periapicais surgem como uma consequênc ia do desequilíbrio da homeostase óssea. Os osteoblastos, juntamente com os osteoclastos e osteócitos, atuam na formação e na reabsorção óssea. Vários marcadores de formação óssea são produzidos por osteoblastos ativos e refletem diferentes aspectos da dif erenciação osteoblástica e da remodelação óssea. Com isso, muitos autores têm explorado o uso de fitoterápicos, visando obter novos compostos que apresentem propriedades terapêuticas, como os flavonoides, e que estimulem a neoformação óssea e o reparo da r egião periapical. O objetivo deste estudo foi avaliar in vitro a citotoxicidade e efeito indutor de mineralização de flavonoides sobre células osteoblásticas humanas. Para isso, células osteoblásticas da linhagem Saos expostas aos seguintes flavono2 foram ides: quercetina, miricetina e seus derivados taxifolina, isoquercitrina, rutina, ampelopsina e EGCG, além de pinocembrina, crisina e canferol, de forma isolada e combinada. Foi avaliado o efeito citotóxico, a atividade de fosfatase alcalina e indução de n mé todo de Shapiroódulos de mineralização. Os resultados foram analisados p elo Wilk, e as variáveis foram submetidas à análise de ANOVA seguida pelo teste de Tukey para comparar entre os grupos e/ou concentrações ou teste de Dunnett para comparar entre cada grupo e o controle, com nível de significância de 5%. A viabilidade da cultura de osteoblastos não teve uma redução estatisticamente significativa na presença da maioria dos compostos, exceto crisina a 100µM. Taxifolina, isoquercitrina, rutina, ampelopsina e EGCG foram os compostos que estimularam significativamente a atividade da fosfatase alcalina, juntamente com as combinações taxifolina+isoquercitrina, taxifolina+ampelopsina e taxifolina+rutina a 25/25 µM. Quanto a formação de nódulos de mine ralização, ampelopsina, isoquercitrina, rutina, pinocembrina e miricetina isolados e taxifolina+isoquercitrina, taxifolina+ampelopsina e taxifolina+rutina combinados obtiveram os melhores resultados, variando de acordo com as concentrações. Concluise que a taxifolina, isoquercitrina, rutina e ampelopsina e combinações de taxifolina com esses flavonoides são citocompatíveis e apresentam efeito indutor de mineralização em osteoblastos Saos-2(AU)

Dental bone loss and the formation of periapical lesions arise as a consequence of imbalance of bone homeostasis. Osteoblasts, together with osteoclasts and osteocytes, act in bone formation and resorption. Several markers of bone formation are produced by active osteoblasts and reflect different aspects of osteoblastic differentiation and bone remodeling. Thus, many authors have explored the use of phytotherapics in order to obtain new compounds with therapeutic properties, such as flavonoids, and also stimulate bone neoformation and periapical region repair. The objective of this study was to evaluate in vitro the cytotoxicity and inducing effect of flavonoid mineralization on human osteoblastic cells. For this, osteoblastic cells of the Saos-2 lineage were exposed to the following flavonoids: quercetin, myricetin and its derivatives taxifoline, isoquercitrin, rutin, ampelopsin and EGCG, in addition to pinocembrin, chrysin and kaempferol, in an isolated and combined manner. The cytotoxic effect, the activity of alkaline phosphatase and the induction of mineralization nodules were evaluated. The results were analyzed using the Shapiro-Wilk method, and the variables were submitted to ANOVA analysis followed by the Tukey test to compare between groups and/or concentrations or Dunnett's test to compare between each group and the control, with a level of 5% significance. The viability of the osteoblast culture did not have a statistically significant reduction in the presence of most compounds, except 100 µM chrysin. Taxifoline, isoquercitrin, rutin, ampelopsin and EGCG were the compounds that significantly stimulated the activity of alkaline phosphatase, together with the combinations taxifoline+isoquercitrin, taxifoline+ampelopsin and taxifoline+rutin at 25/25 µM. As for the formation of mineralization nodules, ampelopsin, isoquercitrin, rutin, pinocembrin and myricetin alone and taxifoline+isoquercitrin, taxifoline+ampelopsin and taxifoline+rutin combined obtained the best results, varying according to the concentrations. It is concluded that taxifoline, isoquercitrin, rutin and ampelopsin and combinations of taxifolin with these flavonoids are cytocompatible and have a mineralization-inducing effect on Saos-2 osteoblasts(AU)

Osteoporosis affects functional activity and gene expression of osteoblastic cells derived from rat alveolar bone

Abstract Recent studies suggest that osteoporosis, in addition to the damage caused in long bones, may cause deterioration in the jaws, especially in alveolar bone sites, with effects in the progress of periodontal disease as well as in bone healing. The aim of this study was to evaluate the effect of osteoporosis in the metabolism of rat alveolar bone osteoblasts. There were used 10 female rats divided in two experimental groups (Sham and OVX), which were ovariectomized and after 8 weeks euthanized to collect mandibular bone samples in order to isolate osteoblastic cells. The cells were cultured in 24-well plates to perform the in vitro experiments. After 7, 10 and 14 days, there were evaluated cell proliferation by MTT assay, in situ detection of alkaline phosphatase (ALP) as well as mineralized nodules and expression of genes associated to bone remodeling. Results showed that at 7, 10 and 14 days cell proliferation was lower for OVX group. In situ detection of ALP was higher at 7 days and lower at 10 and 14 days in OVX group. At 17 and 21 days, OVX group had a significative decrease of mineralization nodules. There was a downregulation in the expression of Alp, Bglap and Runx2 genes and an upregulation of Opg in OVX group, whereas Opn and Rankl modulation was similar between the evaluated groups. Our results suggest that osteoporosis has a deleterious effect on alveolar bone cells from ovariectomized rats, which might affect the treatment of diseases associated to the jaw bones.

Resumo Estudos recentes sugerem que a osteoporose, além dos danos provocados em ossos longos, pode causar deterioração dos ossos maxilares, especialmente na região do osso alveolar, com efeitos na progressão da doença periodontal assim como no reparo ósseo. O objetivo deste estudo foi avaliar o efeito da osteoporose no metabolismo de osteoblastos do osso alveolar mandibular de ratos. Foram utilizadas 10 ratas fêmeas divididas em dois grupos experimentais (Sham e OVX), que foram ovariectomizadas e após 8 semanas, eutanasiadas para coletar amostras do osso mandibular e isolar as células osteoblásticas. As células foram cultivadas em placas de cultura de 24 poços para serem realizados os experimentos in vitro. Após 7, 10 e 14 dias foram avaliados a proliferação celular pelo ensaio de MTT, detecção in situ de fosfatase alcalina (ALP) assim como de nódulos mineralizados e expressão quantitativa de genes associados à remodelação óssea. Os resultados mostraram que aos 7, 10 e 14 dias a proliferação celular foi menor para o grupo OVX. A detecção in situ de ALP foi maior aos 7 dias e menor aos 10 e 14 dias no grupo OVX. Aos 17 e 21 dias o grupo OVX apresentou uma diminuição dos nódulos mineralizados. Houve uma repressão na expressão dos genes Alp, Bglap e Runx2 e uma indução do gene Opg no grupo OVX, enquanto que a modulação dos genes Opn e Rankl foi similar entre os grupos experimentais. Nossos resultados sugerem que a osteoporose tem um efeito deletério no metabolismo de células do osso alveolar em ratas ovariectomizadas, o que pode afetar o tratamento de doenças associadas aos ossos maxilares

Influence of bisphosphonates on the behavior of osteoblasts seeded onto titanium discs

Abstract Among other factors, types of bisphosphonates and treatment regimens seem to be strongly associated with the success or failure of installation of osseointegrated implants. This study investigated the influence of two bisphosphonates, sodium alendronate (SA) and zoledronic acid (ZA), on the metabolism of osteoblasts. Human osteoblasts (Saos-2) were seeded onto machined or acid-treated titanium discs previously placed on 24-well plates in complete culture medium. After 24 h, cells were exposed to bisphosphonates at 0.5, 1 or 5 µM for 24 h, 48 h or 7 days. The effects of SA and ZA on osteoblasts were assessed based on the adhesion of these cells to the titanium surfaces by direct fluorescence, cell viability, total protein and collagen synthesis. Alkaline phosphatase activity and mineral nodule deposition by these cells were also evaluated. Data were evaluated by ANOVA and Tukey tests (α=0.05). Decreased adhesion of cells to the titanium discs was observed when exposed to both bisphosphonates; however, this lack of cell adhesion was more evident for ZA-treated cells. In addition, the exposure of osteoblasts to ZA decreased the viability, ALP activity and mineral nodule deposition, which may be related to poor osseointegration after implant installation.

Resumo Entre outros fatores, os tipos de bisfosfonatos bem como os regimes de tratamento parecem estar diretamente associados com o sucesso ou falhas na instalação de implantes osseointegrados. Este estudo avaliou a influência de dois bisfosfonatos, o alendronato de sódio (AS) e o ácido zoledrônico (AZ), no metabolismo de osteoblastos. Osteoblastos humanos (Saos-2) foram cultivados sobre discos de titânio polidos ou submetidos a tratamento ácido superficial, previamente alocados em placas de 24 compartimentos, utilizando meio de cultura completo. Após 24 horas, as células foram expostas aos bisfosfonatos, nas concentrações de 0,5, 1 ou 5 µM, por 24 h, 48 h, ou 7 dias. Os efeitos do AZ e AZ sobre os osteoblastos foram determinados considerando a adesão destas células às superfícies de titânio, por meio de fluorescência direta, a viabilidade celular, produção de proteína total e síntese de colágeno. A atividade de fosfatase alcalina e a deposição de nódulos mineralizados também foram avaliadas. Os dados foram analisados por meio do teste ANOVA complementado por Tukey (α = 0.05). Menor adesão dos osteoblastos foi observada quando estas células foram expostas a ambos os bisfosfonatos, porém, esta falha na adesão foi mais evidente para as células tratadas com AZ. Além disso, a exposição dos osteoblastos ao AZ também resultou em diminuição da viabilidade, atividade de ALP e deposição de nódulos mineralizados, o que pode estar relacionado a uma pobre osseointegração após a instalação do implante.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Transcriptome profile of highly osteoblastic/cementoblastic periodontal ligament cell clones

Abstract Heterogeneous cell populations of osteo/cementoblastic (O/C) or fibroblastic phenotypes constitute the periodontal dental ligament (PDL). A better understanding of these PDL cell subpopulations is essential to propose regenerative approaches based on a sound biological rationale. Objective Our study aimed to clarify the differential transcriptome profile of PDL cells poised to differentiate into the O/C cell lineage. Methodology To characterize periodontal-derived cells with distinct differentiation capacities, single-cell-derived clones were isolated from adult human PDL progenitor cells and their potential to differentiate into osteo/cementoblastic (O/C) phenotype (C-O clones) or fibroblastic phenotype (C-F clones) was assessed in vitro. The transcriptome profile of the clonal cell lines in standard medium cultivation was evaluated using next-generation sequencing technology (RNA-seq). Over 230 differentially expressed genes (DEG) were identified, in which C-O clones showed a higher number of upregulated genes (193) and 42 downregulated genes. Results The upregulated genes were associated with the Cadherin and Wnt signaling pathways as well as annotated biological processes, including "anatomical structure development" and "cell adhesion." Both transcriptome and RT-qPCR showed up-regulation of WNT2, WNT16, and WIF1 in C-O clones. Conclusions This comprehensive transcriptomic assessment of human PDL progenitor cells revealed that expression of transcripts related to the biological process "anatomical structure development," Cadherin signaling, and Wnt signaling can identify PDL cells with a higher potential to commit to the O/C phenotype. A better understanding of these pathways and their function in O/C differentiation will help to improve protocols for periodontal regenerative therapies.

Efeito da administração local e sistêmica da atorvastatina no reparo ósseo de defeitos críticos em calvária de ratos / Effect of local or systemic administration of atorvastatin on bone repair of critical defects in rats' calvaria

Objetivo: O presente estudo teve como objetivo comparar o efeito da Atorvastatina, aplicada de forma local e sistêmica, em defeitos críticos de calotas de ratos. Material e médodos: 36 (trinta e seis) ratos (Rattus norvegicus, albinus, Wistar), adultos foram divididos aleatoriamente em 3 grupos, o grupo aplicação de membrana de colágeno com água destilada (GAD) com defeitos de tamanho crítico contendo água destilada; o grupo aplicação Sistêmica de Atorvastatina (GAS) foram realizado defeitos de tamanho crítico e os animais foram tratados com atorvastatina (3,6mg/kg/dia) por gavagem; e o grupo de aplicação local de Atorvastatina (GAL) com defeitos de tamanho crítico contendo Atorvastatina. Cada grupo foi avaliado através da histometria, mensuração do defeito residual, área de osso néo formado (AON), área de membrana e tecido mole, contagem de células (osteócitos, osteoblastos, células inflamatórias) e imunoistoquímica (OSTEOCALCINA e CD31), nos períodos de 14 e 28 dias. Resultados: Os dados mostraram diminuição do defeito residual para GAS quando comparada ao GAL (p=0,024) e ao GAD (p=0,033), o GAS revelou diminuição de número de osteócitos em comparação ao GAD (p=0,026), e em comparação com GAL (p=0,020). Os osteoblastos não apresentaram diferença entre os grupos (p>0,05), a quantidade de fibroblastos mostrou maiores somente para o GAL de 14 para 28 dias (p=0,019). Aos 28 dias, tanto para GAL quanto GAS, a quantidade de células inflamatórias foram maiores comparadas ao GAD (p< 0,05). A marcação de imunoistoquimica para CD31 não apresentou alteração, e OCN nos osteoblastos mostrou maior imunomarcação aos 14 dias em comparação aos 28 dias somente para o GAS (p=0,026; Holm-Sidak), OCN na marcação da matriz extra celular (MEC) mostrou aumento da imunomarcação aos 14 quando comparado aos 28 dias no GAL e GAS (p=0,041; Holm-Sidak). Conclusão: A Atorvastatina promoveu efeito positivo na osteogênese e sugere-se que ela não exerça função anti-inflamatória(AU)

Objective: This study aimed to compare the effect of Atorvastatin, applied locally and systemically, on critical defects of rat caps. Material and medicaments: 36 (thirty-six) rats (Rattus norvegicus, albinus, Wistar), adults were randomly divided into 3 groups, the collagen membrane application group with distilled water (GAD) with critical size defects containing distilled water; the systemic application group of Atorvastatin (GAS) was performed with critical size defects and the animals were treated with atorvastatin (3.6mg/kg/day) per gavage; and the local application group of Atorvastatin (GAL) with critical size defects containing Atorvastatin. Each group was evaluated by histometry, measurement of the residual defect, area of neo formed bone (AON), area of membrane and soft tissue, cell count (osteocytes, osteoblasts, inflammatory cells) and immunohistochemistry (OSTEOCALCINE and CD31), in periods of 14 and 28 days. Results: The data showed a decrease of residual defect for GAS when compared to GAL (p=0.024) and GAD (p=0.033), GAS showed a decrease of osteocytes in comparison to GAD (p=0.026), and in comparison to GAL (p=0.020). The osteoblasts did not present difference between the groups (p>0.05), the amount of fibroblasts showed higher only for the GAL from 14 to 28 days (p=0.019). At 28 days, both for GAL and GAS, the amount of inflammatory cells were higher compared to GAD (p<0.05). The immunohistochemistry marking for CD31 showed no change, and CSF in osteoblasts showed higher immunomarkings at 14 days compared to 28 days only for GAS (p=0.026; Holm-Sidak), CSF in extra cellular matrix marking (SCM) showed increased immunomarkings at 14 days compared to 28 days in GAL and GAS (p=0.041; Holm-Sidak). Conclusion: Atorvastatin promoted a positive effect on osteogenesis and it is suggested that it does not have an anti-inflammatory function(AU)

Updating the role of matrix metalloproteinases in mineralized tissue and related diseases

Abstract Bone development and healing processes involve a complex cascade of biological events requiring well-orchestrated synergism with bone cells, growth factors, and other trophic signaling molecules and cellular structures. Beyond health processes, MMPs play several key roles in the installation of heart and blood vessel related diseases and cancer, ranging from accelerating metastatic cells to ectopic vascular mineralization by smooth muscle cells in complementary manner. The tissue inhibitors of MMPs (TIMPs) have an important role in controlling proteolysis. Paired with the post-transcriptional efficiency of specific miRNAs, they modulate MMP performance. If druggable, these molecules are suggested to be a platform for development of "smart" medications and further clinical trials. Thus, considering the pleiotropic effect of MMPs on mammals, the purpose of this review is to update the role of those multifaceted proteases in mineralized tissues in health, such as bone, and pathophysiological disorders, such as ectopic vascular calcification and cancer.

Terapia anticoagulante oral: complicações hemorrágicas em indivíduos submeticos à cirurgia oral menor e avaliação in vitro do efeito sobre células ósseas / Oral anticoagulant therapy: Bleeding complications in individuals submitted to dental surgery and in vitro effects on bone cells

Os objetivos deste trabalho foram 1) avaliar o impacto da terapia anticoagulante oral no sangramento associado à exodontias durante os períodos intraoperatório e pósoperatório; 2) investigar os efeitos do etexilato de dabigatrana, um inibidor direto da trombina, sobre as células ósseas. Para atender o objetivo 1, foram recrutados indivíduos em uso de anticoagulantes orais do tipo antagonista de vitamina K (AVK) e alvo-específico (DOAC, do inglês direct oral anticoagulant) e indivíduos sem terapia anticoagulante com indicação de exodontia. As exodontias foram realizadas sem a suspensão da terapia anticoagulante e parâmetros associados a desfechos hemorrágicos foram avaliados. A avaliação quantitativa do sangramento intraoperatório foi realizada por meio da mensuração do volume e análise dos fluidos aspirados durante o procedimento e normalizada por um escore. Obtivemos como resultados que as complicações hemorrágicas pós-operatórias bem como o escore de sangramento intraoperatório foi similar entre os grupos, sendo que nenhum evento hemorrágico foi observado no grupo DOAC. A história prévia de complicações hemorrágicas em procedimentos odontológicos (p=0,001) e uso de medidas hemostáticas locais (p=0,017) foram estatisticamente maiores no grupo AVK. Para atender o objetivo 2, experimentos foram conduzidos a partir de modelo in vitro, no qual o efeito da terapia anticoagulante foi avaliado diretamente sobre as células ósseas e em modelo animal ex-vivo. Neste modelo ex-vivo, células de animais previamente tratados com etexilato de dabigatrana foram diferenciadas em osteoclastos. Culturas primárias de células-tronco de camundongos e ratos foram diferenciadas em osteoclastos e osteoblastos e tratadas com o fármaco disponível comercialmente, etexilato de dabigatrana (Pradaxa® 1-6 µg/mL) bem como seu princípio ativo, dabigatrana (0,1, 0,3, 3 e 6 µg/mL). Células não expostas aos medicamentos foram utilizadas como controle. A diferenciação de osteoclastos foi inibida pelo tratamento em ambos os modelos, in vitro e ex-vivo. Paralelamente, observou-se a redução da expressão gênica e proteica do marcador Catepsina K e da atividade reabsortiva destas células. Nas culturas de osteoblastos, o tratamento inibiu a expressão gênica dos marcadores fosfatase alcalina (ALP) e osteocalcina, reduziu a atividade in situ de ALP e a deposição de matriz extracelular, indicando um efeito negativo na diferenciação dos osteoblastos. Concluiu-se que o uso de anticoagulantes orais não aumentou a ocorrência de desfechos hemorrágicos na população estudada, o que reforça a manutenção da terapia para a realização de exodontias. O tratamento sobre culturas celulares utilizando etexilato de dabigatrana impactou negativamente a diferenciação e atividade de osteoclastos e osteoblastos.(AU)

The objectives of this study were 1) to evaluate the impact of oral anticoagulant therapy on the pattern of intraoperative and postoperative bleeding in dental surgery; 2) to investigate the effects of dabigatran etexilate, a direct thrombin inhibitor, on bone cells. To fulfill objective 1, individuals undergoing oral anticoagulant therapy with vitamin K antagonists (VKA) or direct oral anticoagulants (DOAC) and individuals without anticoagulant therapy, who had indication of dental extraction were included. Dental surgery procedures were performed without interruption of anticoagulant therapy and parameters associated with hemorrhagic outcomes were evaluated. Intraoperative bleeding was evaluated by means of the measurement of the total amount of blood collected during the procedure corrected by absorbance reading and normalized by score. The results showed that the occurrence of bleeding events and the intraoperative blood loss were similar among groups and hemorrhagic episodes were not observed amongst the individuals taking DOACs. The previous history of complications in dental procedures (p=0.001) and the use of additional hemostatic measures (p=0.017) were significantly higher in the VKA group. To fulfill objective 2, experiments were conducted by means of an in vitro model in which the direct effect of anticoagulant therapy on bone cells was evaluated. An ex-vivo animal model in which cells of animals previously treated with dabigatran etexilate were differentiated was also carried out into osteoclasts. Primary cultures of mice and rats cells were differentiated into osteoclasts and osteoblasts and treated with dabigatran etexilate solution (Pradaxa® 1-6 µg/mL) and its active principle dabigatran (0.1, 0.3, 3 and 6 µg/mL). Untreated cells were used as controls and the effects of the treatment on cell viability and differentiation were evaluated. Both dabigatran etexilate and its active principle, dabigatran inhibited osteoclast differentiation and activity in vitro and in the ex-vivo model, as demonstrated by the reduction of resorption pits and cathepsin K gene and protein expression. In osteoblast cultures, dabigatran etexilate reduced the in situ alkaline phosphatase (ALP) activity, matrix mineralization and gene expression of ALP and osteocalcin. These findings indicated osteoblast inhibition. In conclusion, oral anticoagulant therapy did not result in increased bleeding outcomes in this sample, which strengthen the advocacy of the maintenance of the therapy during dental surgery. Dabigatran etexilate treatment impaired the activity and differentiation of osteoclasts and osteoblasts.(AU)

Addition of zirconium oxide to Biodentine increases radiopacity and does not alter its physicochemical and biological properties

Abstract Objectives: To evaluate the radiopacity of Biodentine (BD) and BD associated with 15% calcium tungstate (BDCaWO4) or zirconium oxide (BDZrO2), by using conventional and digital radiography systems, and their physicochemical and biological properties. Materials and Methods: Radiopacity was evaluated by taking radiographs of cement specimens (n=8) using occlusal film, photostimulable phosphor plates or digital sensors. Solubility, setting time, pH, cytocompatibility and osteogenic potential were also evaluated. Data were analyzed using one-way ANOVA and Tukey post-test or two-way ANOVA and Bonferroni post-test (α=0.05). Results: BD radiopacity was lower than 3 mm Al, while BD ZrO2 and BD CaWO4 radiopacity was higher than 3 mm Al in all radiography systems. The cements showed low solubility, except for BDCaWO4. All cements showed alkaline pH and setting time lower than 34 minutes. MTT and NR assays revealed that cements had greater or similar cytocompatibility in comparison with control. The ALP activity in all groups was similar or greater than the control. All cements induced greater production of mineralized nodules than control. Conclusions: Addition of 15% ZrO2 or CaWO4 was sufficient to increase the radiopacity of BD to values higher than 3 mm Al. BD associated with radiopacifiers showed suitable properties of setting time, pH and solubility, except for BDCaWO4, which showed the highest solubility. All cements had cytocompatibility and potential to induce mineralization in Saos-2 cells. The results showed that adding 15% ZrO2 increases the radiopacity of BD, allowing its radiography detection without altering its physicochemical and biological properties.

Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Papel dos receptores ß1 e ß2 adrenérgicos sobre a diferenciação osteoblástica de células mesenquimais estromais da medula óssea de ratos espontaneamente hipertensos / Role of ß1 and ß2 adrenergic receptor on bone marrow mesenquimal stem cells osteoblastic differentiation from hypertensive rats

Introdução: O remodelamento ósseo é um processo complexo que depende do balanço entre formação e reabsorção óssea, mecanismo regulado pelas células ósseas e fatores sistêmicos, como o Sistema Nervoso Simpático (SNS). Os mediadores deste sistema são capazes de regular o metabolismo ósseo através dos receptores adrenérgicos expressos na superfície dos osteoblastos. Entretanto, o papel dos receptores ß-adrenérgicos ainda não está totalmente elucidado no processo de diferenciação osteogênica. Objetivos: avaliar o papel dos receptores ß-adrenérgicos na diferenciação osteoblástica de células tronco mesenquimais da medula óssea de ratos normotensos e espontaneamente hipertensos (SHR). Métodos: Ratos machos Wistar e SHR (10 semanas) foram utilizados para a coleta da medula óssea a partir do fêmur, as quais foram plaqueadas em garrafas de cultivo celular e depois em placas de 24 poços, onde receberam o meio osteogênico (MO: MEM, mais 50 µg/mL de ácido ascórbico, 10 mM de ß-glicerofosfato e 10-8 M de dexametasona), e o tratamento com Isoprenalina (0,01 µM), Carvedilol (1 µM), antagonista adrenérgico não seletivo, ou Nebivolol (0,1 µM), antagonista ß1-adrenérgico. O ensaio de proliferação celular (MTT) e a atividade de fosfatase alcalina (Fal) foram realizados nos dias 7, 10 e 14. A mineralização foi avaliada no dia 14 através do vermelho de Alizarina. A expressão gênica dos marcadores osteogênicos e dos receptores ß1 e ß2 adrenérgicos foi avaliada no dia 7 por RT-PCR em tempo real. A atividade proteolítica da metaloproteinase de matriz (MMP-2) foi avaliada no mesmo período utilizando zimografia. As vias da MAPK também foram avaliadas ao final de 7 dias. Resultados: A Isoprenalina reduz a fosfatase alcalina na linhagem de células Wistar nos dois primeiros períodos, e ao final de 14 dias apresenta um aumento significativo. A adição dos -bloqueadores reverte tal resposta. Em SHR a Isoprenalina proporciona aumento da atividade de fosfatase alcalina no período intermediário. O Nebivolol inibe essa resposta no mesmo período e, em 7 dias, é capaz de reverter a redução causada pelo agonista. A Isoprenalina aumentou a expressão de todos os fatores de transcrição e o bloqueio dos receptores reverteu essa condição A Isoprenalina aumenta a expressão de Opn, Ocn e BSP nas células de animais Wistar, e em SHR aumenta apenas Ocn e o tratamento com Carvedilol corrige. A atividade de MMP-2 também foi reduzida pelo Nebivolol apenas no grupo Wistar. Além disso, o Nebivolol reduziu a expressão gênica do receptor ß1-adrenérgico. O ensaio de mineralização mostrou menor deposição mineral em Wistar. O Nebivolol também mediou a redução da fosforilação das vias da MAPK neste mesmo grupo de células. Conclusão: Nossos dados sugerem que o receptor ß1-adrenérgico pode estar envolvido na diferenciação osteogênica de células de ratos Wistar mas não em células de ratos SHR(AU)

Introduction: Bone remodeling is a complex process that depends on the balance between formation and resorption bone, a process which is regulated by bone cells and systemic factors, like the Sympathetic Nervous system (SNS). The mediators of these system are able to regulate bone metabolism through adrenergic receptors on the surface os the osteoblastos. However, the role of ß-adrenergic receptors is not clear in he osteogenic differentiation process. Thus, in this study we aimed to evaluate the role of B-adrenergic receptor on osteoblastic differentiation of bone marrow mesenchymal stem cells from normotensive and Spontaneously Hypertensive Rats (SHR). Methods: 70-days-old male Wistar and SHR rats were used for bone marrow collection from femurs, which was placed in cell culture flasks and after in to 24-well plates, where they received osteogenic medium (OM: MEN, plus 50 µg/mL ascorbic acid, 10 mM ß glycerophosphate, and 10-8 M dexamethasone) and the treatment with Isoprenaline (0.01 µM), Carvedilol (1µM), non-selective adrenergic receptor antagonist, or Nebivolol (0,1 µM), ß1-adrenergic receptor antagonist. Cell proliferation (MTT assay) and alkaline phosphatase specific activity (Alp) were analyzed at day 7, 10 and 14. Mineralization was evaluated at day 14, by Alizarin Red S. Gene expression of osteogenic markers and B1 and B2-adrenergic receptor were evaluated at day 7, by real time-RT-PCR. The proteolytic activity of matrix metalloproteinase 2 (MMP-2) were evaluated at day 7 using gelatin zymography. The MAPK pathway was evaluated at the same period. Results: Isoprenaline provides increased alkaline phosphatase activity in the intermediate period. The addition of Nebivolol includes this response over this same period and, within 7 days, was able to reverse the reduced agonist reduction. Isoprenaline increased expression of all transcription factors and receptor blockade reversed this condition Isoprenaline increases expression of Opn, Ocn and BSP in Wistar animal cells, and in SHR only increases Ocn and Carvedilol-corrected treatment. MMP-2 was reduced by Nebivolol treatment just at Wistar cells. Besides that, Nebivolol reduced Adrb1 gene expression at day 7 in Wistar group. Mineralization showed that Nebivolol reduced mineral deposition in Wistar. Nebivolol reduced MAPK proteins phosphorylation. Conclusion: Our results suggest that ß1 adrenergic receptor seems to be involved in the osteogenic differentiation of cells from Wistar rats but not in SHR cells(AU)

Avaliação topográfica e in vitro de superfícies de titânio revestidas com vidro bioativo / Topographic and in vitro evaluation of titanium surfaces coated with bioative glass

Objetivo: Avaliar e comparar a rugosidade superficial e a atividade dos osteoblastos em contato com uma nova superfície bioativa e nanoestruturada de titânio grau 4 revestida com vidro bioativo contendo fosfato de cálcio, sintetizada pelo método sol-gel. Material e método: Sessenta e três discos de titânio, medindo 4 mm de diâmetro por 2 mm de altura, foram preparados e divididos em três grupos: microtexturizado (Ticp - controle); revestido com vidro bioativo e seco a vácuo a 37 °C por 10 dias (BGTi37), e revestido com vidro bioativo e aquecido a 600 °C por cinco horas (BGTi600). Três espécimes de cada grupo foram utilizados para avaliação da topografia superficial e 18 espécimes, para cultura celular. Resultado: O revestimento de vidro bioativo diminuiu a rugosidade média quando comparado ao titânio microtexturizado. A proporção de células viáveis, a produção de fosfatase alcalina e o grau de mineralização da matriz óssea em contato com os espécimes de titânio do grupo BGTi600 foram significativamente menores em relação aos grupos controle e do titânio microtexturizado. Conclusão: Apesar de sua marcante menor rugosidade, a superfície BGTi37 apresentou comportamento biológico semelhante a uma superfície de titânio microtexturizada e moderadamente rugosa. A outra superfície experimental (BGTi600), a de menor rugosidade entre todas as testadas, apresentou os piores resultados de ativação dos osteoblastos.

Objective: To evaluate and compare the surface roughness and the activity of the osteoblasts in contact with a new bioactive and nanostructured surface of grade 4 titanium coated with bioactive glass containing calcium phosphate synthesized by the sol-gel method. Material and method: Sixty-three titanium disks, measuring 4 × 2 mm, were prepared and divided into three groups: rough surface, obtained by sandblasted, large-grit, acid-etched (SLA) treatment (Ticp); SLA surface coated with bioglass and dried in a vacuum at 37 °C for 10 days (BGTi37) and SLA surface coated with bioglass and dried in air at 600 °C for 5 hours (BGTi600). Three specimens of each group were used for evaluation of surface topography and 18 for cell cultures. Result: The bioactive glass coating decreased the average roughness when compared to rough titanium surface. The proportion of viable cells, the production of alkaline phosphatase and the degree of mineralization of the bone matrix in contact with the titanium specimens of the BGTi600 group was significantly lower in relation to the control and rough titanium surface groups. Conclusion: Despite its marked lower roughness, BGTi37 surface presented a similar biological behavior to a titanium rough surface obtained by SLA treatment. The other experimental surface (BGTi600), the one with the least roughness among all tested, presented the worst results of osteoblast activation.

A SERM increasing the expression of the osteoblastogenesis and mineralization-related proteins and improving quality of bone tissue in an experimental model of osteoporosis

Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.