Titanium dioxide nanotubes applied to conventional glass ionomer cement influence the expression of immunoinflammatory markers: An in vitro study.

Objectives: To assess the impact of different concentrations TiO2-nt incorporated into a glass ionomer cement on the proliferation, mitochondrial metabolism, morphology, and pro- and anti-inflammatory cytokine production of cultured fibroblasts (NIH/3T3), whether or not stimulated by lipopolysaccharides (LPS-2 µg/mL, 24 h). Methods: TiO2-nt was added to KM (Ketac Molar EasyMix™, 3 %, 5 %, 7 % in weight); unblended KM was used as the control. The analyses included: Cell proliferation assay (n = 6; 24/48/72h); Mitochondrial metabolism assay (n = 6; 24/48/72h); Confocal laser microscopy (n = 3; 24/48/72h); Determination of biomarkers (IL-1ß/IL-6/IL-10/VEGF/TNF) by using both multiplex technology (n = 6; 12/18 h) and the quantitative real-time PCR assay (q-PCR) (n = 3, 24/72/120 h). The data underwent analysis using both the Shapiro-Wilk and Levene tests, and by generalized linear models (α = 0.05). Results: It demonstrated that cell proliferation increased over time, regardless of the presence of TiO2-nt or LPS, and displayed a significant increase at 72 h; mitochondrial metabolism increased (p < 0.05), irrespective of exposure to LPS (p = 0.937); no cell morphology changes were observed; TiO2-nt reverted the impact of KM on the secreted levels of the evaluated proteins and the gene expressions in the presence of LPS (p < 0.0001). Conclusions: TiO2-nt did not adversely affect the biological behavior of fibroblastic cells cultured on GIC discs.

Glycogen synthase kinase 3 inhibition enhances mineral nodule formation by cementoblasts in vitro.

This study aimed to investigate whether GSK-3 inhibition (CHIR99021) effectively promoted mineralization by cementoblasts (OCCM-30). OCCM-30 cells were used and treated with different concentrations of CHIR99021 (2.5, 5, and 10 mM). Experiments included proliferation and viability, cellular metabolic activity, gene expression, and mineral nodule formation by Xylene Orange at the experimental time points. In general, CHIR99021 did not significantly affect OCCM-30 viability and cell metabolism (MTT assay) (p > 0.05), but increased OCCM-30 proliferation at 2.5 mM on days 2 and 4 (p < 0.05). Data analysis further showed that inhibition of GSK-3 resulted in increased transcript levels of Axin2 in OCCM-30 cells starting as early as 4 h, and regulated the expression of key bone markers including alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx-2), osteocalcin (Ocn), and osterix (Osx). In addition, CHIR99021 led to an enhanced mineral nodule formation in vitro under both osteogenic and non-osteogenic conditions as early as 5 days after treatment. Altogether, the results of the current study suggest that inhibition of GSK-3 has the potential to promote cementoblast differentiation leading to increased mineral deposition in vitro.

In vitro cytotoxicity of resin cement and its influence on the expression of antioxidant genes.

AIM: This study evaluated cytotoxicity and antioxidant gene expression of resin cements on human gingival fibroblasts (hGF). MATERIALS AND METHOD: RelyX Ultimate™(RXU), Variolink™II(VLII), and RelyXU200™(RXU200) resin cements were incubated with culture medium for 24 h to obtain eluates. Then, the eluates were applied over hGF to assess cell viability at 24 h, 48 h, and 72 h and antioxidant gene expression at 24 h. hGF cultures non-exposed to the eluates were used as Control. Data were submitted to ANOVA and Bonferroni tests (α≤0.05). RESULTS: RXU and RXU200 reduced the number of viable cells in 24 h. Longer exposure to cement extracts caused cell death. Gene expression showed peroxiredoxin 1 (PRDX1) induction by all resin cement types, and superoxide dismutase 1 (SOD1) induction by RXU200 and VLII. Moreover, RXU200 induced not only PRDX1 and SOD1, but also glutathione peroxidase 1 (GPX1), catalase (CAT), and glutathione synthetase (GSS). CONCLUSIONS: All resin cements showed toxicity, and induced antioxidant genes in hGF. Antioxidant gene induction is at least partly associated with cytotoxicity of tested cements to oxidative stress experience.

OBJETIVO: O objetivo deste estudo foi avaliar a toxicidade dos cimentos resinosos Rely X Ultimate 2, Rely X U200 e Variolink II, bem como sua influência na expressão de genes antioxidantes em fibroblastos gengivais humanos. Materiais e Método: Corpos de prova de cada cimento foram colocados em meio de cultura por 24 h e os extratos correspondentes foram aplicados aos fibroblastos. A viabilidade celular foi avaliada após 24, 48 e 72 h de exposição pelo ensaio de exclusão do azul de tripano e MTT. A expressão gênica foi avaliada por PCR quantitativo após 24 h de exposição aos extratos. Estes parâmetros foram comparados aos das células não expostas aos cimentos. Os dados foram submetidos ao teste ANOVA, seguido pelo pós-teste de Bonferroni (a≤0.05). RESULTADOS: Os resultados demonstraram que todos os cimentos promoveram redução do número de células viáveis e da atividade mitocondrial nos períodos de 48 e de 72 h (p< 0,01), sendo que o Variolink II apresentou o menor efeito e os cimentos Rely X Ultimate e Rely X U200 promoveram similarmente os maiores efeitos. A análise de expressão gênica evidenciou influência significativa em todos os cimentos avaliados sobre os níveis de transcritos de PRDX1, SOD1, GPX1 e GSS (p> 0,05), com um aumento considerável no Rely X U200. Conclusão: A indução de genes antioxidantes está, pelo menos em parte, associada à citotoxicidade dos cimentos testados para a experiência de estresse oxidativo.

Hybrid light applied with 37% carbamide peroxide bleaching agent with or without titanium dioxide potentializes color change effectiveness.

BACKGROUND: The effectiveness of dental color change was assessed by incorporating titanium dioxide (TiO2) into 37% carbamide peroxide bleaching agent associated with hybrid light. METHODOLOGY: Fifty bovine incisors were selected to receive the bleaching treatment, and separated into five groups (n = 10): 35% hydrogen peroxide (HP) (Whiteness HP, FGM/HP); 37% carbamide peroxide (CP) (Whiteness SuperEndo, FGM/CP); CP + hybrid light (HL) (CP HL); CP + 1% TiO2 (CP TiO2); CP TiO2 + hybrid light (CP TiO2 HL). The bleaching gels were applied to the dental surface for 30 min. Hybrid light (Whitening Plus, DMC/infrared laser diodes + blue LEDs +violet LEDs) was applied with 1 min of active light, alternating with 1 min of pause. A spectrophotometer (VITA Easyshade® Advance, Vita) was used to determine the color of the dental elements at baseline and time points after the 1st, 2nd and 3rd bleaching sessions. Color change effectiveness was evaluated using Vita Classical, CIEL*a*b*, WID and ΔEab, ΔE00 and ΔWID parameters. RESULTS: Generalized mixed linear models for repeated measures (α = 5%) showed significant decrease in Vita Classical scores and a* and b* values, as well as an increase in L* and ∆WID values for all the groups. Higher color change values for ΔEab were observed for CP HL and CP TiO2 HL, while those of ΔE00 and ΔWID were higher for CP TiO2 HL at the end of the bleaching treatment. CONCLUSION: Hybrid light applied with TiO2 incorporated into CP potentiated the effectiveness of the color change in the tooth structure.

In vitro cytotoxicity of resin cement and its influence on the expression of antioxidant genes / Avaliação in vitro da toxicidade de cimentos resinosos e sua influência na expressão de genes antioxidantes em fibroblastos humanos

ABSTRACT Aim: This study evaluated cytotoxicity and antioxidant gene expression of resin cements on human gingival fibroblasts (hGF). Materials and Method: RelyX Ultimate™(RXU), Variolink™II(VLII), and RelyXU200™(RXU200) resin cements were incubated with culture medium for 24 h to obtain eluates. Then, the eluates were applied over hGF to assess cell viability at 24 h, 48 h, and 72 h and antioxidant gene expression at 24 h. hGF cultures non-exposed to the eluates were used as Control. Data were submitted to ANOVA and Bonferroni tests (α≤0.05). Results: RXU and RXU200 reduced the number of viable cells in 24 h. Longer exposure to cement extracts caused cell death. Gene expression showed peroxiredoxin 1 (PRDX1) induction by all resin cement types, and superoxide dismutase 1 (SOD1) induction by RXU200 and VLII. Moreover, RXU200 induced not only PRDX1 and SOD1, but also glutathione peroxidase 1 (GPX1), catalase (CAT), and glutathione synthetase (GSS). Conclusions: All resin cements showed toxicity, and induced antioxidant genes in hGF. Antioxidant gene induction is at least partly associated with cytotoxicity of tested cements to oxidative stress experience.

RESUMO Objetivo: O objetivo deste estudo foi avaliar a toxicidade dos cimentos resinosos Rely X Ultimate 2, Rely X U200 e Variolink II, bem como sua influência na expressão de genes antioxidantes em fibroblastos gengivais humanos. Materiais e Método: Corpos de prova de cada cimento foram colocados em meio de cultura por 24 h e os extratos correspondentes foram aplicados aos fibroblastos. A viabilidade celular foi avaliada após 24, 48 e 72 h de exposição pelo ensaio de exclusão do azul de tripano e MTT. A expressão gênica foi avaliada por PCR quantitativo após 24 h de exposição aos extratos. Estes parâmetros foram comparados aos das células não expostas aos cimentos. Os dados foram submetidos ao teste ANOVA, seguido pelo pós-teste de Bonferroni (a≤0.05). Resultados: Os resultados demonstraram que todos os cimentos promoveram redução do número de células viáveis e da atividade mitocondrial nos períodos de 48 e de 72 h (p < 0,01), sendo que o Variolink II apresentou o menor efeito e os cimentos Rely X Ultimate e Rely X U200 promoveram similarmente os maiores efeitos. A análise de expressão gênica evidenciou influência significativa em todos os cimentos avaliados sobre os níveis de transcritos de PRDX1, SOD1, GPX1 e GSS (p> 0,05), com um aumento considerável no Rely X U200. Conclusão: A indução de genes antioxidantes está, pelo menos em parte, associada à citotoxicidade dos cimentos testados para a experiência de estresse oxidativo.

TiO2 nanotube-based nanotechnology applied to high-viscosity conventional glass-ionomer cement: ultrastructural analyses and physicochemical characterization.

This study characterized TiO2 nanotube (TiO2-nt) ultrastructure and morphology, and the physicochemical impact on high-viscosity conventional glass-ionomer cement (GIC). TiO2-nt was synthesized by the alkaline method (n = 3), assessed by scanning (SEM) and transmission electron microscope (TEM), and was added (3%, 5%, 7%-in weight) to KM (Ketac Molar EasyMix™). Analyses included: SEM; Energy-dispersive spectroscopy (EDS); Raman spectroscopy (RAMAN); Setting time with Gillmore needles (ST); Color (Co); Radiopacity (XR); Water sorption (WS); and solubility (SO). Quantitative data were submitted to ANOVA and Tukey's tests (chr = 0.05). External and internal TiO2-nt diameters were 11 ± 2 nm and 6 ± 0 nm, respectively. Data analyses showed: (i) TiO2-nt present into KM matrix, with a concentration-dependent increase of Ti levels into KM, (ii) physical interaction between KM and TiO2-nt, (iii) longer initial ST for the 7% group compared to KM and 3% groups (p ≤ 0.01), (iv) decreased luminosity and yellowness for the 5% and 7% groups, (v) 36% greater radiopacity for the 5% group compared to enamel, dentin, and KM, and (vi) lower SO values for the 5% group, with no significant differences on WS across the groups. TiO2-nt displayed physical interaction with KM matrix, and also modified SO, XR and Co, without affecting ST. This study provides information on the potential impact of TiO2-nt on GIC performance. TiO2-nt may be proposed to boost confidence among dental surgeons in terms of GIC's handling characteristics, success rate and differential diagnostic.

Titanium dioxide nanotubes in a hydrogen peroxide-based bleaching agent: physicochemical properties and effectiveness of dental bleaching under the influence of a poliwave led light activation.

OBJECTIVES: The effects of different concentrations of titanium dioxide (TiO2) into 40% hydrogen peroxide (HP) were evaluated as regards the effectiveness of dental color change either associated with activation by polywave LED light or not. MATERIALS AND METHODS: TiO2 (0, 1, 5, or 10%) was incorporated into HP to be applied during in-office bleaching (3 sessions/40 min each). Polywave LED light (Valo Corded/Ultradent) was applied or not in activation cycles of 15 s (total time of 2 min). The color of 80 third molars separated into groups according to TiO2 concentration and light activation (n = 10) was evaluated at baseline and at time intervals after the 1st, 2nd, and 3rd bleaching sessions. RESULTS: WID value was significantly higher when using HP with 5% TiO2 in the 2nd session than the values in the other groups (p < 0.05). After the 2nd and 3rd sessions, the ΔEab value was significantly higher when activated with light (p < 0.05) for all agents containing TiO2 or not. Zeta potential and pH of the agents were not modified by incorporating TiO2 at the different concentrations. CONCLUSIONS: The 5% TiO2 in the bleaching agent could enhance tooth bleaching, even without light application. Association with polywave LED light potentiated the color change, irrespective of the presence of TiO2 in the bleaching gel. CLINICAL SIGNIFICANCE: HP with 5% TiO2 could lead to a greater tooth bleaching response in the 2nd clinical session, as well as the polywave light can enhance color change.

Physicochemical evaluation of hydrogen peroxide bleaching gels containing titanium dioxide catalytic agent, and their influence on dental color change associated with violet LED.

BACKGROUND: The purpose of this study was: 1) to analyze the physical-chemical properties of hydrogen peroxide (HP) agents at 7.5% (HP7) and 35% (HP35), and the association with or without TiO2 nanotubes; 2) to evaluate dental bleaching effectiveness by using HP7 and HP35 together with or without TiO2 nanotubes, and applied with or without violet LED (VL). METHODOLOGY: 80 bovine incisors were treated according to groups (n = 10): HP35; HP35 + VL; HP35T (HP35 + TiO2); HP35T + VL; HP7; HP7 + VL; HP7T (HP7 + TiO2); HP7T + VL. Bleaching effectiveness was measured at 4 time points according to the Vita Classical, CIEL*a*b*, CIEDE2000, and WID parameters. HP35, HP35T, HP7, and HP7T were evaluated for mass change, pH, mean particle size (P), polydispersity (PDI), and zeta potential (ZP), over 6 months of storage. RESULTS: The pH of HP35 thickener was higher when associated to TiO2. At baseline, both of the bleaching gels containing TiO2 had lower P, PDI, and PZ (p < 0.05). All groups showed a significant decrease in Vita Classical color scores (p = 0.0037). There was a higher L* value, and lower b* values for HP7 when associated to VL after the 3rd session. (p < 0.05). HP35T showed higher color change (ΔEab, ΔE00), and lower a* value in the presence of VL (p < 0.05). ΔWID presented lower values for both gels, when TiO2 was incorporated (p ≤ 0.05). CONCLUSION: The incorporation of TiO2 to the bleaching gel showed good stability with minimal variations in physical-chemical properties. The color change in HP35 was more effective than in HP7, but the VL boosted the bleaching effectiveness of HP7, whereas TiO2 did not increase bleaching effectiveness.

Glycogen synthase kinase 3 inhibition enhances mineral nodule formation by cementoblasts in vitro

Abstract This study aimed to investigate whether GSK-3 inhibition (CHIR99021) effectively promoted mineralization by cementoblasts (OCCM-30). OCCM-30 cells were used and treated with different concentrations of CHIR99021 (2.5, 5, and 10 mM). Experiments included proliferation and viability, cellular metabolic activity, gene expression, and mineral nodule formation by Xylene Orange at the experimental time points. In general, CHIR99021 did not significantly affect OCCM-30 viability and cell metabolism (MTT assay) (p > 0.05), but increased OCCM-30 proliferation at 2.5 mM on days 2 and 4 (p < 0.05). Data analysis further showed that inhibition of GSK-3 resulted in increased transcript levels of Axin2 in OCCM-30 cells starting as early as 4 h, and regulated the expression of key bone markers including alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx-2), osteocalcin (Ocn), and osterix (Osx). In addition, CHIR99021 led to an enhanced mineral nodule formation in vitro under both osteogenic and non-osteogenic conditions as early as 5 days after treatment. Altogether, the results of the current study suggest that inhibition of GSK-3 has the potential to promote cementoblast differentiation leading to increased mineral deposition in vitro.

Effect of thermocycling on internal microhardness of high and low viscosity bulk fill composite resins in class I cavities / Efeito da termociclagem na microdureza interna de resinas compostas bulk fill de alta e baixa viscosidade aplicadas em cavidades classe I

ABSTRACT Objective: To evaluate the effect of thermocycling on the Knoop internal microhardness of high- and low viscosity bulk fill resins applied in Class I cavities. Methods: Thirty third molars with Class I cavity preparations were randomly divided into 3 groups according to the restorative system: nanoparticulated composite resin (Filtek™ Z350 XT 3M ESPE) (oblique increments of 2mm); low viscosity bulk fill resin (Filtek™ Bulk fill Flow, 3M ESPE) (3mm increments covered with a 1-mm layer of nanoparticulated resin); high viscosity bulk fill resin (Filtek™ Bulk fill, 3M ESPE) (single 4-mm increment). After 24h, half of samples was submitted to thermocycling (1,000 cycles). All samples (n=5) were sectioned in half to measure the internal microhardness at bottom and top of restoration. Results: Analysis of variance indicated that, for nanoparticulated composite resin, without thermocycling, the microhardness at the top was statistically lower than at the bottom. After thermocycling, there was no difference in microhardness between the top and the bottom. For low viscosity bulk fill resin, without thermocycling, there was no significant difference in microhardness means between the top and the bottom. After thermocycling, significantly higher microhardness was found at the top than at the bottom (p <0.05). For high viscosity bulk fill resin there was no significant difference between the microhardness values at the top and bottom, regardless of thermocycling (p> 0.05). In all composite resins, an increase in microhardness was observed after thermocycling (p <0.05). Conclusion: Thermocycling increased the internal microhardness of resin restorations, and, for the low viscosity bulk fill resin, the microhardness at the top was higher than at the bottom after thermocycling.

RESUMO Objetivo: Avaliar o efeito da termociclagem na microdureza Knoop interna de resinas bulk fill de alta e baixa viscosidade aplicadas em cavidades classe I. Métodos: Trinta terceiros molares com cavidades Classe I foram divididos aleatoriamente em três grupos de acordo com o sistema restaurador: Resina composta nanoparticulada (Filtek™ Z350 XT 3M ESPE) (incrementos oblíquos de 2mm); resina bulk fill de baixa viscosidade (Filtek™ Bulk fill Flow, 3M ESPE) (incremento de 3mm mais 1mm de resina nanoparticulada); resina bulk fill de alta viscosidade (Filtek ™ Bulk fill, 3M ESPE) (único incremento de 4mm). Após 24h, metade das amostras foi submetida a termociclagem (1.000 ciclos). Todas as amostras (n=5) foram seccionadas ao meio para mensuração da microdureza interna na base e topo da restauração. Resultados: Análise de variância indicou que, para a resina composta nanoparticulada e antes da termociclagem, a microdureza no topo foi estatisticamente inferior do que na base (p<0,05). Após a termociclagem, não houve diferença entre topo e base. Já para a resina bulk fill de baixa viscosidade, antes da termociclagem, não se constatou diferença significativa nos valores de microdureza entre topo e base. Após termociclagem, valores significativamente mais elevados de microdureza foram encontrados no topo do que na base (p<0,05). Para resina bulk fill de alta viscosidade, não houve diferença significativa na microdureza entre topo e base, realizando-se ou não termociclagem (p>0,05). Em todas as resinas compostas foi verificado aumento de microdureza após termociclagem (p<0,05). Conclusão: A termociclagem aumentou a microdureza para todas as resinas compostas, sendo que para a resina bulk fill de baixa viscosidade a microdureza no topo foi maior do que na base após termociclagem.

Influence of melatonin associated with the Bio-Gide® membrane on osteoblast activity: an in vitro Study.

Melatonin (MLT) is a hormone responsible for regulating several physiological processes. It has been shown that MLT can be an important mediator in bone formation and stimulation, promoting osteoblast differentiation. In clinical practice, in tissue regeneration procedures, it is necessary to use membranes or barriers, associated with biomaterials, or not. The aim of this in vitro study was to assess the effect of melatonin on the activity of osteoblastic cells, associated, or not, with a resorbable collagen membrane (Bio-Gideä). For this, mice-derived pre-osteoblastic cells MC3T3 obtained from the ATCC (American Type Culture Collection) were used. Cultured cells were subject to the following treatments: MLT with a concentration of 1mM, a Bio-Gideä membrane and a membrane associated with MLT (Bio-Gideä + MLT). Proliferation and cell viability assays and protein lysate (ELISA test) quantification for the BMP-2 protein were carried out, in periods of 72 hours, 7 days and 10 days. After analyzing the data (one-way ANOVA, alpha=5%) it was observed that when MLT was used in isolation, there was an increase in cell proliferation and viability in osteoblastic cells (p<0.05). But, when MLT was associated with resorbable membranes, there was an inverse behavior, both in terms of proliferation and viability (p<0.05). In the case of the ELISA test, no secretion of BMP-2 was detected in any of the analyzed groups. It is concluded that MLT has a stimulatory effect on osteoblasts, but, when associated with Bio-Gideä resorbable membranes, it does not show any viable action in osteoblastic cell stimulation.

A melatonina (MLT) é um hormônio responsável pela regulação de diversos processos fisiológicos no nosso organismo. Tem sido demonstrado que a melatonina possa ser um importante mediador na formação e estimulação óssea, promovendo a diferenciação dos osteoblastos. Clinicamente, para o procedimento de regeneração tecidual, faz-se necessário a utilização de membranas ou barreiras, associadas ou não a biomateriais. Assim, o objetivo deste estudo in vitro foi avaliar o efeito da melatonina na atividade de células osteoblásticas, associada ou não a uma membrana de colágeno reabsorvível (Bio-Gide®). Para isto foram utilizadas células pré-osteoblásticas MC3T3 do ATCC (American Type Culture Collection), de camundongos. As células em cultura foram submetidas aos seguintes tratamentos: MLT na concentração de 1mM, membrana Bio Gide® e membrana associada à MLT (Bio-Gide® + MLT). Foram realizados os ensaios de proliferação e viabilidade celular e quantificação do lisado proteico (teste ELISA), para a proteína BMP-2, nos períodos de 72 horas, 7 e 10 dias. Após a análise dos dados (ANOVA um critério, alfa=5%) pode-se observar que a MLT quando utilizada sozinha, resultou em um aumento na proliferação e viabilidade celular nas células osteoblásticas (p<0,05). Entretanto, quando a MLT foi associada à membrana reabsorvível foi observado um comportamento inverso, tanto na proliferação quanto na viabilidade (p<0,05). Para o teste ELISA realizado, não houve secreção detectável de BMP-2 para nenhum grupo analisado. Conclui-se que a melatonina possui uma ação estimuladora nos osteoblastos, mas quando associada à membrana reabsorvível Bio-Gide®, não demonstra uma ação viável na estimulação de células osteoblásticas.

Influence of melatonin associated with the Bio-Gide® membrane on osteoblast activity: an in vitro Study / Influência da melatonina associada à membrana bio gide® na atividade de osteoblastos: um estudo in vitro

ABSTRACT Melatonin (MLT) is a hormone responsible for regulating several physiological processes. It has been shown that MLT can be an important mediator in bone formation and stimulation, promoting osteoblast differentiation. In clinical practice, in tissue regeneration procedures, it is necessary to use membranes or barriers, associated with biomaterials, or not. The aim of this in vitro study was to assess the effect of melatonin on the activity of osteoblastic cells, associated, or not, with a resorbable collagen membrane (Bio-Gideä). For this, mice-derived pre-osteoblastic cells MC3T3 obtained from the ATCC (American Type Culture Collection) were used. Cultured cells were subject to the following treatments: MLT with a concentration of 1mM, a Bio-Gideä membrane and a membrane associated with MLT (Bio-Gideä + MLT). Proliferation and cell viability assays and protein lysate (ELISA test) quantification for the BMP-2 protein were carried out, in periods of 72 hours, 7 days and 10 days. After analyzing the data (one-way ANOVA, alpha=5%) it was observed that when MLT was used in isolation, there was an increase in cell proliferation and viability in osteoblastic cells (p<0.05). But, when MLT was associated with resorbable membranes, there was an inverse behavior, both in terms of proliferation and viability (p<0.05). In the case of the ELISA test, no secretion of BMP-2 was detected in any of the analyzed groups. It is concluded that MLT has a stimulatory effect on osteoblasts, but, when associated with Bio-Gideä resorbable membranes, it does not show any viable action in osteoblastic cell stimulation.

RESUMO A melatonina (MLT) é um hormônio responsável pela regulação de diversos processos fisiológicos no nosso organismo. Tem sido demonstrado que a melatonina possa ser um importante mediador na formação e estimulação óssea, promovendo a diferenciação dos osteoblastos. Clinicamente, para o procedimento de regeneração tecidual, faz-se necessário a utilização de membranas ou barreiras, associadas ou não a biomateriais. Assim, o objetivo deste estudo in vitro foi avaliar o efeito da melatonina na atividade de células osteoblásticas, associada ou não a uma membrana de colágeno reabsorvível (Bio-Gide®). Para isto foram utilizadas células pré-osteoblásticas MC3T3 do ATCC (American Type Culture Collection), de camundongos. As células em cultura foram submetidas aos seguintes tratamentos: MLT na concentração de 1mM, membrana Bio Gide® e membrana associada à MLT (Bio-Gide® + MLT). Foram realizados os ensaios de proliferação e viabilidade celular e quantificação do lisado proteico (teste ELISA), para a proteína BMP-2, nos períodos de 72 horas, 7 e 10 dias. Após a análise dos dados (ANOVA um critério, alfa=5%) pode-se observar que a MLT quando utilizada sozinha, resultou em um aumento na proliferação e viabilidade celular nas células osteoblásticas (p<0,05). Entretanto, quando a MLT foi associada à membrana reabsorvível foi observado um comportamento inverso, tanto na proliferação quanto na viabilidade (p<0,05). Para o teste ELISA realizado, não houve secreção detectável de BMP-2 para nenhum grupo analisado. Conclui-se que a melatonina possui uma ação estimuladora nos osteoblastos, mas quando associada à membrana reabsorvível Bio-Gide®, não demonstra uma ação viável na estimulação de células osteoblásticas.

Polycaprolactone scaffolds as a biomaterial for cementoblast delivery: An in vitro study.

OBJECTIVE: To define the potential of polycaprolactone (PCL) scaffold for cementoblast delivery. BACKGROUND: Dental cementum is critical for tooth attachment and position, and its regenerative capabilities remain unpredictable. METHODS: PCL scaffolds were manufactured by the electrospinning technique at 10% and 20% (w/v) and seeded with cementoblasts (OCCM-30). Scaffolds were characterized for their morphology and biological performance by scanning electron microscopy (SEM), confocal and conventional histology, cytocompatibility (PrestoBlue assay), gene expression (type I collagen - Col1; bone sialoprotein - Bsp; runt-related transcription factor 2 - Runx-2; alkaline phosphatase - Alpl; osteopontin - Opn; osteocalcin - Ocn, osterix - Osx), and the potential to induce extracellular matrix deposition and mineralization in vitro. RESULTS: Overall, data analysis showed that PCL scaffolds allowed cell adhesion and proliferation, modulated the expression of key markers of cementoblasts, and led to enhanced extracellular matrix deposition and calcium deposition as compared to the control group. CONCLUSION: Altogether, our findings allow concluding that PCL scaffolds are a viable tool to culture OCCM-30 cells, leading to an increased potential to promote mineralization in vitro. Further studies should be designed in order to define the clinical relevance of cementoblast-loaded PCL scaffolds to promote new cementum formation.

TiO2 nanotube-containing glass ionomer cements display reduced aluminum release rates.

Titanium dioxide nanotubes (TiO2-nts) were incorporated into a glass ionomer cement (GIC) with improved mechanical properties and antibacterial activity. The aims of the present in vitro study were to define the elemental characterization, aluminum (Al) release rate, and initial working time for GIC reinforced with TiO2-nts, in an experimental caries model. TiO2-nts were incorporated into GIC powder components at 5% by weight, and compared with unblended GIC. Experimental approaches used energy-dispersive spectrometry (EDS), atomic absorption spectrophotometry (AAS), and brightness loss to define surface element properties, Al release rates, and initial working time, respectively. Statistical analysis was performed by 2-way ANOVA, Tukey's test, generalized linear models, and Student's t test (a = 0.05). EDS data analysis revealed that TiO2-nts incorporated into GIC had no significant impact on the typical elemental composition of GICs in an in vitro caries model. Regarding the demineralizing solution, GIC with TiO2-nt significantly decreased the Al release rate, compared with the control group (p < 0.0001). Moreover, TiO2-nt incorporated into GIC did not alter the initial working time of the material (p > 0.05). These findings add information to our scientific body of knowledge concerning the potential impact of TiO2-nt on the performance of conventional GICs.

Childhood Hypophosphatasia Associated with a Novel Biallelic ALPL Variant at the TNSALP Dimer Interface.

The goal of this study was to perform a clinical and molecular investigation in an eight-year-old female child diagnosed with hypophosphatasia (HPP). The proband and her family were evaluated by medical and dental histories, biochemical analyses, radiographic imaging, and genetic analysis of the tissue-nonspecific alkaline phosphatase (ALPL) gene. A bioinformatic analysis was performed to predict the structural and functional impact of the point mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) molecule and to define their potential contribution to the phenotype. We identified a novel combination of heterozygous ALPL missense variants in the proband, p.Ala33Val and p.Asn47His, compatible with an autosomal recessive mode of inheritance and resulting in skeletal and dental phenotypes. Computational modeling showed that the affected Asn47 residue is located in the coil structure close to the N-terminal α-helix, whereas the affected Ala33 residue is localized in the N-terminal α-helix. Both affected residues are located close to the homodimer interface, suggesting they may impair TNSALP dimer formation and stability. Clinical and biochemical follow-up revealed improvements after six years of ERT. Reporting this novel combination of ALPL variants in childhood HPP provides new insights into genotype-phenotype associations for HPP and specific sites within the TNSALP molecule potentially related to a childhood-onset HPP and skeletal and dental manifestations. Beneficial effects of ERT are implicated in skeletal and dental tissues.

TiO2 nanotube-containing glass ionomer cements display reduced aluminum release rates

Abstract Titanium dioxide nanotubes (TiO2-nts) were incorporated into a glass ionomer cement (GIC) with improved mechanical properties and antibacterial activity. The aims of the present in vitro study were to define the elemental characterization, aluminum (Al) release rate, and initial working time for GIC reinforced with TiO2-nts, in an experimental caries model. TiO2-nts were incorporated into GIC powder components at 5% by weight, and compared with unblended GIC. Experimental approaches used energy-dispersive spectrometry (EDS), atomic absorption spectrophotometry (AAS), and brightness loss to define surface element properties, Al release rates, and initial working time, respectively. Statistical analysis was performed by 2-way ANOVA, Tukey's test, generalized linear models, and Student's t test (a = 0.05). EDS data analysis revealed that TiO2-nts incorporated into GIC had no significant impact on the typical elemental composition of GICs in an in vitro caries model. Regarding the demineralizing solution, GIC with TiO2-nt significantly decreased the Al release rate, compared with the control group (p < 0.0001). Moreover, TiO2-nt incorporated into GIC did not alter the initial working time of the material (p > 0.05). These findings add information to our scientific body of knowledge concerning the potential impact of TiO2-nt on the performance of conventional GICs.

Orthodontic tooth movement alters cementocyte ultrastructure and cellular cementum proteome signature.

Cementum is a mineralized tissue that covers tooth roots and functions in the periodontal attachment complex. Cementocytes, resident cells of cellular cementum, share many characteristics with osteocytes, are mechanoresponsive cells that direct bone remodeling based on changes in loading. We hypothesized that cementocytes play a key role during orthodontic tooth movement (OTM). To test this hypothesis, we used 8-week-old male Wistar rats in a model of OTM for 2, 7, or 14 days (0.5 N), whereas unloaded contralateral teeth served as controls. Tissue and cell responses were analyzed by high-resolution micro-computed tomography, histology, tartrate-resistant acid phosphatase staining for odontoclasts/osteoclasts, and transmission electron microscopy. In addition, laser capture microdissection was used to collect cellular cementum, and extracted proteins were identified by liquid chromatography coupled to tandem mass spectrometry. The OTM model successfully moved first molars mesially more than 250 µm by 14 days introducing apoptosis in a small number of cementocytes and areas of root resorption on mesial and distal aspects. Cementocytes showed increased nuclear size and proportion of euchromatin suggesting cellular activity. Proteomic analysis identified 168 proteins in cellular cementum with 21 proteins found only in OTM sites and 54 proteins only present in control samples. OTM-down-regulated several extracellular matrix proteins, including decorin, biglycan, asporin, and periostin, localized to cementum and PDL by immunostaining. Furthermore, type IV collagen (COL14A1) was the protein most down-regulated (-45-fold) by OTM and immunolocalized to cells at the cementum-dentin junction. Eleven keratins were significantly increased by OTM, and a pan-keratin antibody indicated keratin localization primarily in epithelial remnants of Hertwig's epithelial root sheath. These experiments provide new insights into biological responses of cementocytes and cellular cementum to OTM.

Titanium dioxide nanotubes added to glass ionomer cements affect S. mutans viability and mechanisms of virulence.

This in vitro study evaluated the impact of TiO2 nanotubes (n-TiO2) incorporated into glass ionomer cement (GIC) on Streptococcus mutans (S. mutans) characteristics at cellular and molecular levels. n-TiO2, synthesized by the alkaline method (20 nm in size), was added to Ketac Molar EasyMix® at 0%, 3%, 5%, and 7% by weight. S. mutans strains were cultured on GIC disks with addition or not of n-TiO2 for 1, 3, and 7 days and the following parameters were assessed: inhibition halo (mm) (n=3/group); cell viability (live/dead) (n=5/group); cell morphology (SEM) (n=3/group); and gene expression by real-time PCR (vicR, covR, gtfB, gtfC, and gtfD) (n=6/group). The data were analyzed by the Kruskal-Wallis test, repeated-measures ANOVA or two-way ANOVA, and Tukey's and Dunn's post-hoc tests (α=0.05). The agar diffusion test showed a higher antibacterial property for 5% n-TiO2 compared with 3% and 7% (p<0.05) with no effect of time (1, 3, and 7 days). The cell number was significantly affected by all n-TiO2 groups, while viability was mostly affected by 3% and 5% n-TiO2, which also affected cell morphology and organization. Real-time PCR demonstrated that n-TiO2 reduced the expression of covR when compared with GIC with no n-TiO2 (p<0.05), with no effect of time, except for 3% n-TiO2 on vicR expression. Within-group and between-group analyses revealed n-TiO2 did not affect mRNA levels of gtfB, gtfC, and gtfD (p>0.05). Incorporation of n-TiO2 at 3% and 5% potentially affected S. mutans viability and the expression of key genes for bacterial survival and growth, improving the anticariogenic properties of GIC.

Cementocyte alterations associated with experimentally induced cellular cementum apposition in Hyp mice.

BACKGROUND: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology. METHODS: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp). RESULTS: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43. CONCLUSION: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation.

Fluoride release and remineralizing potential of varnishes in early caries lesions in primary teeth.

This study evaluated the fluoride (F) release and remineralizing potential of varnishes containing sodium fluoride (5% NaF), 5% NaF with CPP-ACP and 5% NaF with TCP in early caries lesions in primary teeth. To determine the F release at 1, 4, 6, 24, 72, and 168 hr, strips were covered with the varnishes and immersed in purified water (n = 7). The varnishes and purified water (negative control) were applied on enamel blocks with early caries lesions (n = 16). Enamel blocks were stored in artificial saliva and submitted to a pH-cycling. The area of enamel hardness loss (ΔS) was analyzed by microhardness, lesion depth by polarized light microscopy (PLM) and the chemical analysis by Energy-dispersive X-ray spectroscopy. Data were submitted to Shapiro-Wilk, two-way and one-way ANOVA, Tukey and paired t-tests (α = 5%). All varnishes released F, but 5% NaF with CPP-ACP had the highest release at 4, 6, 24, and 72 hr (p < .05) followed by 5% NaF with TCP and 5% NaF. No significant difference in ΔS was observed among varnishes (5% NaF = 4,098.4 ± 1,407.9; 5% NaF with CPP-ACP = 4,164.0 ± 1,019.3; 5% NaF with TCP = 4,183.2 ± 1,527.2; p = .999), but all of them differed from the negative control group (6,757.8 ± 2,274.7; p < .001). Lesion depth was lower in varnishes groups compared to negative control (% reduction: 5% NaF = 41.8%, 5% NaF with CPP-ACP = 38.8%, and 5% NaF with TCP = 36.3%; p < .001). Similar Ca, P, and Ca/P ratio percentages among groups and F was not detected after the treatments. All fluoride varnishes showed potential to enhance remineralization of early caries lesions in primary teeth.