Antibacterial activity of Brazilian red propolis and in vitro evaluation of free radical production.

OBJECTIVE: This study aimed to evaluate the antibacterial activity of crude Brazilian red propolis (BRP) extract against anaerobic bacteria involved in primary endodontic infection. Additionally, we evaluate the cell viability and free radical production of human dental pulp fibroblasts (HDPF) in direct contact with mineral trioxide aggregate (MTA) and BRP. DESIGN: The Minimum Inhibitory Concentration, Minimum Bactericidal Concentration (MIC, MBC) and Minimum Inhibitory Concentration of Biofilm (MICB50) of BRP against anaerobic endodontic pathogens were determined. HDPF were exposed to BRP10 (10 µg/mL), BRP50 (50 µg/mL), MTA extract (1:1, 1:2, 1:4 e 1:8), dimethyl sulfoxide 0.5% (DMSO), and cell culture medium (DMEM). The groups were tested for cell viability (MTT assay), and free radical production (reactive oxygen species - ROS, DCFH-DA probe and nitric oxide - NO, Griess reagent). The one-way ANOVA and Tukey's tests were employed at a significance level of 5%. RESULTS: MIC/MBC values of BRP performed antibacterial activity for Parvimonas micra (6.25/6.25 µg/mL), Fusobacterium nucleatum (25/25 µg/mL), Prevotella melaninogenica (50/100 µg/mL), Prevotella nigrescens (50/100 µg/mL), Prevotella intermedia (50/100 µg/mL), and Porphyromonas gingivalis (50/200 µg/mL). The MICB50 values ranged from 1.56 to 50 µg/mL. BRP and MTA stimulated cell viability, emphasizing BRP10 (p = 0.007). Furthermore, it was observed that MTA 1:1, MTA 1:2, and BRP50 slightly increased ROS (p < 0.001) and NO production (p = 0.008, p = 0.007, and p < 0.001 respectively) compared to DMEM group. CONCLUSIONS: BRP exhibits good antibacterial activity against endodontic pathogens, and both BRP and MTA promote the viability of HDPF without increasing NO and ROS production.

Photobiomodulation effect of red LED (630 nm) on the free radical levels produced by pulp cells under stress conditions.

The aim of this study was to assess the ability of red light emitting diodes (LED) to modulate oxidative stress in human dental pulp fibroblasts (HDPFs) when different irradiation parameters are employed. Cells from primary teeth were seeded (100,000 cells/well) in 24-well plates in culture medium (DMEM). At 24 h after incubation, the culture medium was replaced with DMEM containing 10 µg/mL lipopolysaccharide (LPS). Thereafter, the cells were irradiated (LED 630 nm, 0.04 W/cm2 and 0.08 W/cm2) at 0 J/cm2 (control group), 4 J/cm2, 15 J/cm2, and 30 J/cm2; and their viability (MTT assay), number (Trypan Blue), synthesis of nitric oxide (NO) (Griess reagent), and reactive oxygen species (ROS) (fluorescence probe, DCFH-DA) were assessed. The Kruskal-Wallis and Mann-Whitney statistical tests using Bonferroni correction were employed (significance level of 5%). Compared to that in control fibroblasts, increased viability was observed in HDPFs exposed to LPS and irradiated with 15 J/cm2 and 30 J/cm2 at 0.04 W/cm2 and 4 J/cm2 and 15 J/cm2 at 0.08 W/cm2 (p < 0.05). Exposure to 4 J/cm2 at 0.04 W/cm2 and 15 J/cm2 and 30 J/cm2 at 0.08 W/cm2 modulated the oxidative stress in cells relative to that observed in non-irradiated LPS-treated pulp cells (p < 0.05). It was concluded that the irradiation strategies of using red LED with radiant exposures of 15 J/cm2 and 30 J/cm2 at 0.04 W/cm2 and 15 J/cm2 at 0.08 W/cm2 were the best parameters to decrease NO and ROS concentration and to stimulate viability of HDPFs exposed to LPS challenge.

Cytotoxicity and dentin composition alterations promoted by different chemomechanical caries removal agents: A preliminary in vitro study.

BACKGROUND: The use of chemomechanical agents for caries removal has been indicated as a non-invasive treatment option; however, their possible deleterious effects on the dental-pulp complex have been insufficiently studied. This study assessed the direct cytotoxicity of two chemomechanical caries removal agents (Brix 3000™ - BX and Papacarie Duo™ - PD) on pulp cells from deciduous teeth, as well as to assess the morphology and chemical compositions of the dentin surface after the application of these materials. MATERIAL AND METHODS: The cells were seeded (50,000 cells/cm²) in a culture medium (DMEM with 10% fetal bovine serum - FBS). After 24 hours, the BX and PD materials were added to 1:20, 1:100, and 1:1000 dilutions. Non-exposed cells were considered as the control group. The viability test (MTT), Trypan Blue assay (TB), and cell morphology (Scanning Electron Microscopy - SEM) were performed after 24 hours of agent application. For the SEM and chemical (energy-dispersive X-ray spectrometry - EDS) dentin evaluation, 0.3-mm-thick dentin discs were obtained and divided into control group (no treatment) and surfaces covered with 37% phosphoric acid, BX, or PD. Data were compared by one-way ANOVA and Tukey's test (p<0.05). RESULTS: Decreases in cell viability and numbers of viable cells were observed for both materials, at all dilutions, when compared with the control group (p<0.05). The BX and PD materials did not cause visually perceptible changes, according to SEM, on the surfaces of dentin discs. The EDS analysis did not indicate a statistically significant difference in the levels of calcium (Ca) and phosphorus (P) between the materials and the control group (p>0.05). CONCLUSIONS: Both materials showed cytotoxicity when in direct contact with the pulp cells from deciduous teeth, and the BX material presented lower cytotoxicity than the PD material. Moreover, both materials did not significantly change the dentin composition. Key words:Cell culture, cytotoxicity, dental pulp, papacarie, primary teeth.

Effects of zinc oxide and calcium-doped zinc oxide nanocrystals on cytotoxicity and reactive oxygen species production in different cell culture models.

OBJECTIVES: This study aimed to synthesize nanocrystals (NCs) of zinc oxide (ZnO) and calcium ion (Ca2+)-doped ZnO with different percentages of calcium oxide (CaO), to evaluate cytotoxicity and to assess the effects of the most promising NCs on cytotoxicity depending on lipopolysaccharide (LPS) stimulation. MATERIALS AND METHODS: Nanomaterials were synthesized (ZnO and ZnO:xCa, x = 0.7; 1.0; 5.0; 9.0) and characterized using X-ray diffractometry, scanning electron microscopy, and methylene blue degradation. SAOS-2 and RAW 264.7 were treated with NCs, and evaluated for viability using the MTT assay. NCs with lower cytotoxicity were maintained in contact with LPS-stimulated (+LPS) and nonstimulated (-LPS) human dental pulp cells (hDPCs). Cell viability, nitric oxide (NO), and reactive oxygen species (ROS) production were evaluated. Cells kept in culture medium or LPS served as negative and positive controls, respectively. One-way analysis of variance and the Dunnett test (α = 0.05) were used for statistical testing. RESULTS: ZnO:0.7Ca and ZnO:1.0Ca at 10 µg/mL were not cytotoxic to SAOS-2 and RAW 264.7. +LPS and -LPS hDPCs treated with ZnO, ZnO:0.7Ca, and ZnO:1.0Ca presented similar NO production to negative control (p > 0.05) and lower production compared to positive control (p < 0.05). All NCs showed reduced ROS production compared with the positive control group both in +LPS and -LPS cells (p < 0.05). CONCLUSIONS: NCs were successfully synthesized. ZnO, ZnO:0.7Ca and ZnO:1.0Ca presented the highest percentages of cell viability, decreased ROS and NO production in +LPS cells, and maintenance of NO production at basal levels.