Assessment of the regenerative potential of macro-porous chitosan-calcium simvastatin scaffolds on bone cells

Abstract This study evaluated the bioactive potential of a macro-porous chitosan scaffold incorporated with calcium hydroxide (CH-Ca) and functionalized with bioactive doses of simvastatin (SV) for bone tissue regeneration. Initially, the bioactive dose of SV in osteoblastic cells (SAOS-2) was determined. For the direct contact experiment, SAOS-2 cells were plated on scaffolds to assess cell viability and osteogenic differentiation. The second assay was performed at a distance using extracts from scaffolds incubated in culture medium to assess the effect of conditioned medium on viability and osteogenic differentiation. The initial screening showed that 1 μM SV presented the best biostimulating effects, and this dose was selected for incorporation into the CH-Ca and pure chitosan (CH) scaffolds. The cells remained viable throughout the direct contact experiment, with the greatest cell density in the CH-Ca and CH-Ca-SV scaffolds because of their higher porosity. The CH-Ca-SV scaffold showed the most intense bio-stimulating effect in assays in the presence and absence of osteogenic medium, leading to an increased deposition of mineralized matrix. There was an increase in the viability of cells exposed to the extracts for CH-Ca, CH-SV, and CH-Ca-SV during the one-day period. There was an increase in ALP activity in the CH-Ca and CH-Ca-SV; however, the CH-Ca-SV scaffold resulted in an intense increase in the deposition of mineralized nodules, approximately 56.4% at 7 days and 117% at 14 days, compared with CH (control). In conclusion, functionalization of the CH-Ca scaffold with SV promoted an increase in bioactivity, presenting a promising option for bone tissue regeneration.

Management of Tooth Discoloration after Orthognathic Surgery Complications: Report of Two Cases

ABSTRACT: Orthognathic surgery is performed for the correction of craniofacial discrepancies. However, complications, such as tooth discoloration are possible. This case report presents two patients who underwent bilateral sagittal split ramus osteotomy associated with segmental Le Fort I osteotomy and genioplasty. During surgeries, the apical region of anterior teeth was accidentally injured in both cases. After three-week surgery follow-up, the injured teeth showed a change in color to dark pink. In both teeth, the root canal treatment was performed followed by the non vital tooth bleaching. Three sessions were necessary to achieve a significant color change of the teeth. The two-year follow-up showed that both teeth preserved an acceptable color. It was concluded that tooth discoloration after orthognathic surgery is a possible complication, which could be overcome following a conservative approach. Additionally, patients should be informed preoperatively.

RESUMEN: La cirugía ortognática es comúnmente realizada para corregir las discrepancias cráneo-faciales. Sin embargo, se pueden producir complicaciones tales como la pigmentación dentaria. Este reporte de casos presenta a dos pacientes que fueron sometidos osteotomía sagital bilateral de la rama mandibular asociada a osteotomía segmentaria Le Fort I y genioplastía. Durante la fase quirúrgica, la región apical de dientes anteriores fueron accidentalmente dañados en ambos casos. Después de tres semanas de seguimiento de la cirugía, los dientes afectados mostraron un cambio de color a rosado oscuro. Se realizó el tratamiento de conductos seguido de aclaramiento interno en ambas piezas dentarias. Fueron necesarias tres sesiones para lograr un cambio de color significativo. El seguimiento de dos años mostró que ambos dientess conservaron un color aceptable. En conclusión, la pigmentación dentaria después de la cirugía ortognática es una posible complicación, sin embargo, puede ser tratada por medio de un enfoque conservador. Además, los pacientes deben ser informados antes de la cirugía.

Bioactivity effects of extracellular matrix proteins on apical papilla cells

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans

Abstract Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Increased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel

Abstract Objective This study was designed for the chemical activation of a 35% hydrogen peroxide (H2O2) bleaching gel to increase its whitening effectiveness and reduce its toxicity. Methodology First, the bleaching gel - associated or not with ferrous sulfate (FS), manganese chloride (MC), peroxidase (PR), or catalase (CT) - was applied (3x 15 min) to enamel/dentin discs adapted to artificial pulp chambers. Then, odontoblast-like MDPC-23 cells were exposed for 1 h to the extracts (culture medium + components released from the product), for the assessment of viability (MTT assay) and oxidative stress (H2DCFDA). Residual H2O2 and bleaching effectiveness (DE) were also evaluated. Data were analyzed with one-way ANOVA complemented with Tukey's test (n=8. p<0.05). Results All chemically activated groups minimized MDPC-23 oxidative stress generation; however, significantly higher cell viability was detected for MC, PR, and CT than for plain 35% H2O2 gel. Nevertheless, FS, MC, PR, and CT reduced the amount of residual H2O2 and increased bleaching effectiveness. Conclusion Chemical activation of 35% H2O2 gel with MC, PR, and CT minimized residual H2O2 and pulp cell toxicity; but PR duplicated the whitening potential of the bleaching gel after a single 45-minute session.

Interactions between Terpinen-4-ol and Nystatin on biofilm of Candida albicans and Candida tropicalis

Abstract The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity ("checkerboard" method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.

Resumo O objetivo desse estudo foi avaliar a atividade antifúngica do Terpinen4-ol associado à nistatina em biofilmes simples e misto, formados por Candida albicans e Candida tropicalis, bem como o efeito do terpinen-4-ol na adesão em células orais e atividade enzimática. As concentrações inibitórias mínimas e as concentrações fungicidas mínimas do terpinen-4-ol e da nistatina em Candida albicans e Candida tropicalis foram determinadas pelo método de microdiluição em caldo, juntamente com a atividade sinérgica (método do tabuleiro de "xadrez"). Biofilmes simples e misto foram preparados usando o modelo de placa de microtitulação estática e quantificados por unidades formadoras de colônias (CFU/mL). O efeito do Terpinen-4-ol na adesão de Candida albicans e Candida tropicalis em co-cultura com queratinócitos orais (NOK Si) foi avaliado, bem como a atividade enzimática, medindo o tamanho da zona de precipitação, após o crescimento em ágar fosfolipase, protease e hemolisina. O terpinen-4-ol (4.53 mg mL-1) e a nistatina (0,008 mg mL-1) conseguiram inibir o crescimento de biofilmes e um efeito antifúngico sinérgico foi demonstrado com a associação de fármaco, reduzindo a concentração inibidora de nistatina até 8 vezes em biofilme simpes de Candida albicans e 2 vezes em biofilme misto. Uma pequena diminuição na adesão de Candida tropicalis em células NOK Si foi mostrada após o tratamento com terpinen-4-ol e a nistatina teve um efeito maior para ambas as espécies. Para a atividade enzimática, as drogas não apresentaram ação. O efeito potencializado pela combinação de terpinen-4-ol e nistatina e a redução de adesão evidenciam seu potencial como agente anti-fúngico.

Influência do gluconato de cálcio sobre a citotoxicidade trans-amelodentinária de um agente clareador com 20% de peróxido de hidrogênio / Influence of calcium gluconate on the trans-enamel and trans-dentinal cytotoxicity of a bleaching agent with 20% hydrogen peroxide

Objetivo: Avaliar a citotoxicidade de um agente clareador contendo 2% de gluconato de cálcio (GC) sobre células pulpares humanas (HDPCs). Materiais e Métodos: Discos de esmalte-dentina adaptados em câmaras pulpares artificiais (CPAs) foram posicionados em compartimentos de forma que a dentina permaneceu imersa em meio de cultura, enquanto que o esmalte foi submetido ao clareamento com géis a 20% de H2O2 contendo ou não GC, durante 1x 45, 1x15 ou 1x5 minutos. No controle positivo foi realizado clareamento com 35% de H2O2 aplicado por 1x 45 minutos, sendo que no controle negativo nenhum tratamento foi realizado sobre o esmalte. A viabilidade celular (teste do MTT) e a difusão trans-amelodentinária de H2O2 (violeta leuco- -cristal/peroxidase) foram avaliadas (ANOVA/Tukey α = 5%; n = 8). Resultados: Foi observada redução significativa na viabilidade celular em todos os grupos clareados quando comparados ao controle negativo (p < 0,05); no entanto, os grupos expostos aos géis contendo 20% de H2O2, com ou sem GC, apresentaram valores de viabilidade celular significativamente superiores ao controle positivo (p < 0,05). A redução da viabilidade celular e a difusão de H2O2 residual para os grupos clareados com 20% de H2O2 foi proporcional ao tempo de contato dos produtos com a superfície dental, sendo que a presença de GC resultou em minimização significativo do efeito tóxico/difusão de H2O2 para os protocolos 1x 15 e 1x 5 min (p < 0,05). Conclusão: A presença de 2% de GC nos géis com 20% de H2O2 resulta em redução da difusão de H2O2 residual pela estrutura dental e do efeito citotóxico sobre células pulpares humanas, quando o produto é aplicado por curtos períodos sobre a superfície dental.

Objective: To evaluate the cytotoxicity of a bleaching agent containing 2% calcium gluconate (CG) on human pulp cells (HDPCs). Materials and Methods: Enamel-dentin disks adapted in artificial pulp chambers (CPAs) were placed in compartments so that dentin remained immersed in culture medium, while the enamel was subjected to bleaching with 20% H2O2 gels containing or not CG for 1x 45, 1x15 or 1x5 minutes. In the positive control, bleaching was performed with 35% H2O2 applied for 1x 45 minutes, and in the negative control no treatment was performed on the enamel. Cell viability (MTT test) and transenamel and trans-dentinal diffusion of H2O2 (leuco-crystal violet / peroxidase) were evaluated (ANOVA / Tukey α = 5%, n = 8). Results: A significant reduction in cell viability was observed in all bleached groups when compared to the negative control (p <0.05); However, groups exposed to gels containing 20% H2O2, with or without CG, had significantly higher values of cell viability than the positive control (p <0.05). The reduction of cell viability and the diffusion of residual H2O2 to the bleached groups with 20% H2O2 was proportional to the contact time of the products with the dental surface, and the presence of CG resulted in a significant minimization of the toxic /diffusion effect of H2O2 For the 1x15 and 1x5min protocols (p <0.05). Conclusion: The presence of 2% GC in gels with 20% H2O2 results in reduction of residual H2O2 diffusion by dental structure and cytotoxic effect on human pulp cells when the product is applied for short periods on the dental surface.

Bioestimulação de células pulpares humanas expostas a sinvastatina / Biostimulation of human pulp cells exposed to simvastatin

O objetivo desse estudo foi avaliar o potencial bioativo da sinvastatina (SV), aplicada por diferentes períodos sobre células da polpa dental humana (HDPCs). Para isto, HDPCs em 80% de confluência (n=6) foram tratadas com meio osteogênico suplementado com 0,01 µM de SV pelos períodos de 24 h, 72 h ou continuamente por até 21 dias. No controle negativo, as células foram mantidas em meio osteogênico. A viabilidade celular (MTT) foi avaliada em períodos de 1, 3, 7, 14 e 21 dias, e a deposição de matriz mineralizada (alizarin red) após 14 e 21 dias de cultivo celular. Os dados foram submetidos aos testes ANOVA e Tukey (α=5%). Foi observado que nos períodos de 1, 3, 7 e 14 dias não houve diferença significativa na viabilidade das células submetidas aos tratamentos com SV em comparação ao controle (p<0,05); no entanto, redução tardia foi observada aos 21 dias para as células tratadas com SV por 72 h ou de modo contínuo (p<0,05). Em contrapartida, aumento na deposição de matriz mineralizada foi observado para o tratamento contínuo com SV aos 21 dias, quando comparado ao controle (p<0,05). Foi possível concluir que o tratamento contínuo de células pulpares humanas com 0,01µM de SV foi capaz de bioestimular a deposição de matriz mineralizada in vitro.

The objective of this study was to evaluate the bioactive potential of simvastatin (SV), applied during different periods on human dental pulp cells (HDPCs). For this, HDPCs at 80% confluency (n = 6) were treated with osteogenic medium supplemented with 0.01 µM SV for periods of 24 h, 72 h or continuously up to 21 days. In the negative control group, the cells were cultivated in osteogenic medium. The cell viability (MTT) was evaluated after 1, 3, 7, 14 and 21 days, and the mineralized matrix deposition (alizarin red) was assessed at 14 and 21 days of cell culture. Data were submitted to ANOVA and Tukey's test (α=5%). No significant difference in cell viability was observed at 1, 3, 7 and 14 days for the cells exposed to SV compared to negative control (p<0.05); however, significant reduction was observed at 21 days for cells treated with SV during 72 h or continuously (p<0.05). On the other hand, increase in mineralized matrix deposition at 21 days was observed for cells treated continuously with SV when compared to control (p<0.05). It was possible to conclude that the continuous treatment of human pulp cells with 0.01 µM of SV was able to biostimulate mineralized matrix deposition in vitro.