Fabrication and characterization of remineralizing dental composites containing calcium type pre-reacted glass-ionomer (PRG-Ca) fillers

ObjectiveTo fabricate and characterize dental composites with calcium type pre-reacted glass-ionomer (PRG-Ca) fillers.MethodsPRG-Ca fillers were prepared by the reaction of calcium fluoroaluminosilicate glass with polyacrylic acid. Seven dental composites were produced from the same organic matrix (70/30 wt% Bis-GMA/TEGDMA), with partial replacement of barium borosilicate (BaBSi) fillers (60 wt%) by PRG-Ca fillers (wt%): E0 (0) – control, E1 (10), E2 (20), E3 (30), E4 (40), E5 (50) and E6 (60). Enamel remineralization was evaluated in caries-like enamel lesions induced by S. mutans biofilm using micro-CT. The following properties were characterized: degree of conversion (DC%), roughness (Ra), Knoop hardness (KHN), flexural strength (FS), flexural modulus (FM), water sorption (W_sp), water solubility (W_sl), and translucency (TP). Data were analyzed to one-way ANOVA and Tukey’s HSD test (α = 0.05).ResultsAll composites with PRG-Ca induced enamel remineralization. E0 and E1 presented similar and highest DC% than E2 = E3 = E4 = E5 = E6. Ra and KHN were not influenced by PRG-Ca fillers (p < 0.05). The higher the content of PRG-Ca, the lower FS, FM and TP (p < 0.05). W_sp increased linearly with the content of PRG-Ca fillers (p < 0.05). E6 presented the highest W_sl (p < 0.05), while the W_sl of the other composites were not different from each other (p > 0.05).SignificanceIncorporation of 10–40 wt.% of PRG-Ca fillers endowed remineralizing potential to dental composites without jeopardizing the overall behavior of their physicochemical properties. Dental composites with PRG-Ca fillers seems to be a good alternative for reinforcing the enamel against caries development.     

Influence of sucrose and xylitol on an early Streptococcus mutans biofilm in a dental simulator

ObjectivesIn vitro methods to study dental biofilms are useful in finding ways to support a healthy microbial balance in the oral cavity. The effects of sucrose, xylitol, and their combination on three strains of Streptococcus mutans and one strain of Streptococcus sobrinus were studied using a dental simulator.MethodsA simulator was used to mimic the oral cavity environment. It provided a continuous-flow system using artificial saliva (AS), constant temperature, mixing, and hydroxyapatite (HA) surface in which the influence of xylitol was studied. The quantities of planktonic and adhered bacteria were measured by real-time qPCR.ResultsCompared against the untreated AS, adding 1% sucrose increased the bacterial colonization of HA (p < 0.0001) whereas 2% xylitol decreased it (p < 0.05), with the exception of clinical S. mutans isolate 117. The combination of xylitol and sucrose decreased the bacterial quantities within the AS and the colonization on the HA by clinical S. mutans isolate 2366 was reduced (p < 0.05). Increasing the concentration (2%–5%) of xylitol caused a reduction in bacterial counts even in the presence of sucrose.ConclusionsThe continuous-culture biofilm model showed that within a young biofilm, sucrose significantly promotes whereas xylitol reduces bacterial colonization and proliferation. The results indicate that xylitol affects the ability of certain S. mutans strains to adhere to the HA. Clinical studies have also shown that xylitol consumption decreases caries incidence and reduces the amount of plaque. This study contributes to the understanding of the mechanism behind these clinical observations.     

Influence of a Brazilian wild green propolis on the enamel mineral loss and Streptococcus mutans’ count in dental biofilm

ObjectiveThis study investigated the anti-demineralizing and antibacterial effects of a propolis ethanolic extract (EEP) against Streptococcus mutans dental biofilm.DesignBlocks of sound bovine enamel (n = 24) were fixed on polystyrene plates. S. mutans inoculum (ATCC 25175) and culture media were added (48 h–37 °C) to form biofilm. Blocks with biofilm received daily treatment (30 μL/1 min), for 5 days, as following: G1 (EEP 33.3%); G2 (chlorhexidine digluconate 0.12%); G3 (ethanol 80%); and G4 (Milli-Q water). G5 and G6 were blocks without biofilm that received only EEP and Milli-Q water, respectively. Final surface hardness was evaluated and the percentage of hardness loss (%HL) was calculated. The EEP extract pH and total solids were determined. S. mutans count was expressed by log₁₀ scale of Colony-Forming Units (CFU/mL). One way ANOVA was used to compare results which differed at a 95% significance level.ResultsG2 presented the lowest average %HL value (68.44% ± 12.98) (p = 0.010), while G4 presented the highest (90.49% ± 5.38%HL) (p = 0.007). G1 showed %HL (84.41% ± 2.77) similar to G3 (87.80% ± 6.89) (p = 0.477). Groups G5 and G6 presented %HL = 16.11% ± 7.92 and 20.55% ± 10.65; respectively (p = 0.952). G1 and G4 differed as regards to S. mutans count: 7.26 ± 0.08 and 8.29 ± 0.17 CFU/mL, respectively (p = 0.001). The lowest bacterial count was observed in chlorhexidine group (G2 = 6.79 ± 0.10 CFU/mL) (p = 0.043). There was no difference between S. mutans count of G3 and G4 (p = 0.435). The EEP showed pH 4.8 and total soluble solids content = 25.9 Brix.ConclusionThe EEP seems to be a potent antibacterial substance against S. mutans dental biofilm, but presented no inhibitory action on the de-remineralization of caries process.     

Evaluation of the Candida albicans removal and mechanical properties of denture acrylics cleaned by a low-cost powered toothbrush

PurposeTo investigate the effects of using a low-cost powered toothbrush for cleaning on dental prostheses made of heat polymerized poly(methyl methacrylate), PMMA.MethodsHeat cured PMMA specimens beam with the dimensions of 45.0 mm × 6.5 mm × 4.5 mm were fabricated. The specimens were kept in water storage at 37 °C constant temperature for 0, 1, 7, 15, 30 and 60 days and randomly assigned for testing or control. Test specimens underwent brushing by using a powered toothbrush at an applied force of 2.00 N for 22 min with water as medium. Surface roughness measurement (R_a), flexural strength and efficacy of brushing to remove coated Candida albicans biofilm were investigated.ResultsThe results of the mean surface roughness value and the flexural strength were analysed by using two-way ANOVA and Tukey post hoc test at 5% significance level. In general, the specimens showed no significant changes in flexural strength after brushing. However, the flexural strength and the surface roughness value were significantly lower in specimens group after 7 days in water storage compared to the control. SEM micrographs of post-brushed specimens revealed satisfactory removal of C. albicans biofilm.ConclusionA low-cost powered toothbrush together with a liquid medium successfully removed C. albicans biofilm on dental acrylic PMMA-based prostheses, without compromising the mechanical properties.     

Antibacterial response of oral microcosm biofilm to nano-zinc oxide in adhesive resin

ObjectiveVarious nanoparticles are currently under investigation to impart biointeractivity for dental materials. This study aimed to: (1) formulate an experimental dental adhesive containing ZnO nanoparticles; (2) evaluate its chemical and mechanical properties; and (3) assess the antibacterial response against oral microcosm biofilm.MethodsNanosized ZnO was chemically and morphologically evaluated. ZnO was incorporated at 0 (G_CTRL), 2.5 (G_2.5%), 5 (G_5%) and 7.5 (G_5%) wt.% in an experimental dental adhesive. The adhesives were evaluated for the degree of conversion (DC), flexural strength (FS), and elastic modulus (E). The antibacterial activity was evaluated using a 48 h-microcosm biofilm model after the formation of acquired pellicle on samples’ surfaces. Colony-forming units (CFU), metabolic activity, and live/dead staining were assessed.ResultsNanosized ZnO presented characteristic peaks of Zn-O bonds, and the particles were arranged in agglomerates. The DC ranged from 62.21 (±1.05) % for G_Ctrl to 46.15 (±1.23) % for G_7.5% (p < 0.05). G_7.5% showed lower FS compared to all groups (p < 0.05). Despite achieving higher E (p < 0.05), G_2.5% did not show differences for G_Ctrl regarding the FS (p > 0.05). G_7.5% had lower CFU/mL compared to G_Ctrl for mutans streptococci (p < 0.05) and total microorganisms (p < 0.05), besides presenting lower metabolic activity (p < 0.05) and higher dead bacteria via biofilm staining.SignificanceThe dental adhesives' physicochemical properties were similar to commercial adhesives and in compliance with ISO recommendations. G_7.5% restricted the growth of oral microcosm biofilm without impairing the physicochemical performance.     

Control of microorganisms of oral health interest with Arctium lappa L. (burdock) extract non-cytotoxic to cell culture of macrophages (RAW 264.7)

ObjectivesTo evaluate the antimicrobial activity of Arctium lappa L. extract on Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Candida albicans, C. tropicalis and C. glabrata. In addition, the cytotoxicity of this extract was analyzed on macrophages (RAW 264.7).DesignBy broth microdilution method, different concentrations of the extract (250–0.4 mg/mL) were used in order to determine the minimum microbicidal concentration (MMC) in planktonic cultures and the most effective concentration was used on biofilms on discs made of acrylic resin. The cytotoxicity A. lappa L. extract MMC was evaluated on RAW 264.7 by MTT assay and the quantification of IL-1β and TNF-α by ELISA.ResultsThe most effective concentration was 250 mg/mL and also promoted significant reduction (log₁₀) in the biofilms of S. aureus (0.438 ± 0.269), S. epidermidis (0.377 ± 0.298), S. mutans (0.244 ± 0.161) and C. albicans (0.746 ± 0.209). Cell viability was similar to 100%. The production of IL-1β was similar to the control group (p > 0.05) and there was inhibition of TNF-α (p < 0.01).ConclusionsA. lappa L. extract was microbicidal for all the evaluated strains in planktonic cultures, microbiostatic for biofilms and not cytotoxic to the macrophages.     

The effect of antifungal agents on surface properties of poly(methyl methacrylate) and its relation to adherence of Candida albicans

PurposeCandida-associated denture stomatitis is the most prevalent form of oral candidosis affecting 65% of denture wearers. Failure of therapy and recurrence of infection are not uncommon and the continuous use of antifungal agents may affect the surface properties of the denture material and may contribute to Candida adhesion. This study aimed to investigate surface properties of poly(methyl methacrylate) PMMA denture material before and after exposure to antifungal agents and its relation to in vitro adhesion of Candida albicans.MethodsFour groups of acrylic specimens (20 mm × 20 mm × 2.5 mm) were prepared (25 specimens in each group). Specimens were immersed in nystatin (group 1), fluconazole (group 2), distilled water (group 3) and group 4 was not exposed. Specimens were tested for surface roughness, contact angle, surface hardness and in vitro Candida adherence to PMMA.ResultsThe results showed that nystatin had no statistically significant effect on surface hardness (P > 0.05), but had a statistically significant effect on surface roughness, contact angle, and Candida adhesion to PMMA (P < 0.05). On the other hand, fluconazole had no statistically significant effect on surface hardness or roughness (P > 0.05), but had a statistically significant effect on contact angle, and Candida adhesion to PMMA (P < 0.05). Distilled water had no statistically significant effect on surface hardness, roughness, contact angle, or Candida adhesion to PMMA (P > 0.05).ConclusionsExposure of PMMA to nystatin may induce changes in roughness, wettability while exposure to fluconazole may affect surface free energy and therefore may increase Candida adhesion to it.     

Effect of arginine on the growth and biofilm formation of oral bacteria

BackgroundAlkali production via arginine deiminase system (ADS) of oral bacteria plays a significant role in oral ecology, pH homeostasis and inhibition of dental caries. ADS activity in dental plaque varies greatly between individuals, which may profoundly affect their susceptibility to caries.ObjectiveTo investigate the effect of arginine on the growth and biofilm formation of oral bacteria.Methods and resultsPolymicrobial dental biofilms derived from saliva were formed in a high-throughput active attachment biofilm model and l-arginine (Arg) was shown to reduce the colony forming units (CFU) counts of such biofilms grown for various periods or biofilms derived from saliva of subjects with different caries status. Arg hardly disturbed bacterial growth of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus gordonii in BHI medium, but only inhibited biofilm formation of S. mutans. Scanning electron microscope (SEM) showed S. mutans biofilms harboured fewer cells grown with Arg than that without Arg, even in the initial 2 h and 8 h phase. Confocal laser scanning microscope (CLSM) images of poly-microbial dental and S. mutans biofilms revealed the biofilms grown with Arg had lower exopolysaccharide (EPS)/bacteria ratios than those without Arg (P = 0.004, 0.002, respectively). Arg could significantly reduce the production of water-insoluble EPS in S. mutans biofilms (P < 0.001); however, quantitative real-time PCR (qRT-PCR) did not show significantly influence in gene expression of gtfB, gtfC or gtfD (P = 0.32, 0.06, 0.44 respectively).ConclusionsArg could reduce the biomass of poly-microbial dental biofilms and S. mutans biofilms, which may be due to the impact of Arg on water-insoluble EPS. Considering the contribution to pH homeostasis in dental biofilms, Arg may serve as an important agent keeping oral biofilms healthy thus prevent dental caries.     

Self-cured resin modified by quaternary ammonium methacrylates and chlorhexidine: Cytotoxicity,antimicrobial, physical,and mechanical properties

ObjectiveTo evaluate the addition of dimethylaminohexadecyl methacrylate (DMAHDM) and chlorhexidine diacetate on cytotoxicity, antimicrobial activity, physical, and mechanical properties of a self-cured resin.Methods132 disk-shaped and 48 rectangular specimens were divided into four experimental groups as described: Control Group (CG – no addition), dCHX (1%), DMAHDM (5%), and DMAHDM + dCHX (5% + 1%). The biofilm viability, flexural strength (FS - ISO 20795-1:2013), surface roughness (SR), and color stability (ΔE) were analyzed after being stored for 4 weeks in distilled water and immersed for 72 h in coffee. Cytotoxicity was measured after 24 h, 3, and 7 days of elution using an MTT test on L929 cells (ISO 10993-5:2009). SR and ΔE were measured by a contact profilometer and a spectrophotometer using the CIELab parameter. Data were submitted to ANOVA and Bonferroni’s/Tukey’s tests (p ≤ 0.05).ResultsSignificant antimicrobial activity against Streptococcus mutans and Candida albicans was detected in all groups when compared to the CG (p < 0.05). Only the dCHX group, in 24 h of elution, demonstrated no cytotoxicity effects. There was a statistical difference for FS on the tested groups (p < 0.05). No differences were detected in the initial roughness’ measurements among the groups (p > 0.05). However, after storage and immersion in coffee, the groups containing DMAHDM presented with rougher surfaces and significantly lower color stability compared to the control (p < 0.05).SignificanceThe addition of dCHX and DMAHDM in self-cured resin presented antimicrobial properties; however, cytotoxicity, physical, and mechanical properties were compromised.     

Titanium dioxide and modified titanium dioxide by silver nanoparticles as an anti biofilm filler content for composite resins

Objective

The aim of this study was to evaluate the antibacterial activity of a composite resin modified by TiO₂ and TiO₂/Ag nanoparticles and their influence over different properties.

Pre-heating time and exposure duration: Effects on post-irradiation properties of a thermo-viscous resin-composite

ObjectiveTo evaluate the effects of pre-heating time and exposure duration on the degree of conversion (DC), maximum rate of polymerization (RP_max), polymerization shrinkage strain (PS) and surface micro-hardness (VHN) of Viscalor.MethodsViscalor syringes were pre-heated using a Caps Warmer (VOCO, Germany) in T3 mode (at 68 °C) for 30 s (T3-30s) and 3 min (T3-3min) and then the composite paste was extruded into appropriately sized molds. Light irradiation was applied at zero distance from the upper surface with a LED-LCU of mean irradiance 1200 mW/cm² for either 20 or 40 s. The real-time polymerization kinetics and DC at 5 min and 24 h post-irradiation (DC_5min and DC_24h) were measured using ATR-FTIR (n = 3). PS was obtained with the bonded-disk technique (n = 3). Top and bottom Vickers micro-hardness (VHN_top and VHN_bottom) were measured at 5 min post-irradiation and after 24 h dry storage (n = 5). Data were analysed using one-way ANOVA, two-way ANOVA, independent t-test and Tukey post hoc tests (p < 0.05).ResultsPolymerization kinetic curves of Viscalor from 0 to 15 min were similar for different pre-heating times and exposure durations. Pre-heated Viscalor (T3-30s and T3-3min) with 40 s exposure had greater VHN_top and VHN_bottom than for Viscalor (no heat) (p < 0.05). Exposure duration did not significantly affect DC, RP_max and PS (p > 0.05). After 24 h storage, DC and VHN increased. Pre-heating did not increase the DC_24h, relative to no pre-heating (p > 0.05). Two-way ANOVA showed that there was no significant interaction between pre-heating time and exposure duration (p > 0.05).SignificanceIncreasing irradiation time from 20 to 40 s did not affect DC, RP_max or PS, but increased VHN_top. Composite pre-heating had no adverse effect through any premature polymerization. For Viscalor, 3 min pre-heating and 20 s irradiation were sufficient to provide adequate hardness, without increasing PS or compromising polymerization kinetics.     

Pre-heating effects on extrusion force,stickiness and packability of resin-based composite

ObjectivesTo measure temperature effects on stickiness and packability of representative resin-based composites and the effect of pre-heating time on pre-cure properties of Viscalor, including extrusion force.MethodsFive resin-based composites (RBC) and an additional RBC, Viscalor, used with a Caps Warmer (VOCO, Germany) were studied. The extrusion force (N) and extruded mass (g) were measured from Viscalor compules heated in T3 mode for 30 s (T3-30 s) and 3 min (T3-3 min). For stickiness and packability measurements, RBCs were packed into a brass cylindrical cavity controlled at 22 and 37 °C. A flat-ended probe was lowered into the RBC pastes at constant speed. Stickiness: F_max (N) and W_s (N mm), and packability: F_p (N), were measured. Viscalor was LED photo-cured at 1200 mW/cm² for 40 s. The degrees of conversion at 5 min and 24 h post cure (DC_5min and DC_24h) of Viscalor (no heat, T3-30 s and T3-3 min) were measured by ATR-FTIR. Data were analysed by one-way ANOVA, independent t-test and Tukey post-hoc tests (p < 0.05).ResultsThe maximum temperature of the Caps Warmer, in T3 mode, reached 68 °C in 20 min. Viscalor temperatures of 34.5 °C and 60.6 °C were recorded after 30 s and 3 min pre-heating, respectively. Pre-heating significantly reduced extrusion force and increased extruded mass, especially after 3 min. RBCs varied in F_max, W_s and F_p (p < 0.05). Temperature also affected F_max (p = 0.000), W_s (p = 0.002) and F_p (p = 0.000). Pre-heating Viscalor for either 30 s or 3 min did not increase the post-cure DC at either 5 min or 24 h, relative to no pre-heating (p > 0.05).SignificanceThe composites varied to an extent in stickiness and packability but the overall magnitudes remained within a clinically acceptable range. Pre-heating was beneficial in placement of Viscalor and caused no adverse effects through premature polymerization.     

Effect of honey in preventing gingivitis and dental caries in patients undergoing orthodontic treatment

ObjectivesThis study was conducted to investigate the following: (1) the effects of chewing honey on plaque formation in orthodontic patients, (2) the effect of chewing honey on dental plaque bacterial counts, (3) determine if honey possesses antibacterial effects on bacteria recovered from plaques.MethodsFemale orthodontic patients (n = 20, 12–18 years of age) participated in this randomized controlled study. The effects of honey were compared to treatment with either 10% sucrose or 10% sorbitol that served as positive and negative controls, respectively. The pH of plaque was measured using a digital pH meter prior to baseline and at 2, 5, 10, 20, and 30 min after chewing honey or rinsing with control solutions and the numbers of Streptococcus mutans, Lactobacilli, and Prophymonas gingivalis in respective plaques were determined. The antibacterial activity of honey was tested against commonly used antibiotics using the disk diffusion method.ResultsSignificant differences in pH were observed in the honey and sucrose groups compared to the pH observed in the sorbitol group (p ? 0.001). The maximum pH drop occurred at 5 min in both the honey and sucrose groups; however the pH in the honey group rapidly recovered 10–20 min after exposure and did not drop below the critical decalcification pH of 5.5. On the other hand, the pH following sucrose exposure fell <5.5 and was associated with a 30 min recovery time. The pH observed for the sorbitol group did not change over time. Bacterial counts were significantly reduced in the honey group compared to the other treatment groups (p ? 0.001) and honey significantly inhibited the growth of all studied strains compared to inhibition observed with antibiotics (p ? 0.001).ConclusionsHoney can be used as an alternative to traditional remedies for the prevention of dental caries and gingivitis following orthodontic treatment.     

Development and characterisation of dental composites containing anisotropic fluorapatite bundles and rods

ObjectivesTo develop dental composites incorporating fluorapatite (FA) crystals as a secondary filler and to characterise degree of conversion, key mechanical properties and fluoride release.MethodsFA rod-like crystals and bundles were hydrothermally synthesised and characterised by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS), X-ray diffraction (XRD) and ¹⁹F MAS-NMR. Composites were formulated containing BisGMA/TEGDMA/BisEMA and barium-aluminium-silicate glass (0FA). FA crystals were incorporated at 10 (10FA), 20 (20FA), 30 (30FA) and 40 wt% (40FA) maintaining a filler content of 80 wt% (63–67 vol%). Degree of conversion (DC), flexural strength (FS), flexural modulus (FM), fracture toughness (K_1C), Vickers hardness (HV) and 2-body wear were measured. Fluoride release was measured in neutral and acidic buffers.ResultsXRD and ¹⁹F MAS-NMR confirmed that only FA was formed, whilst SEM revealed the presence of single rods and bundles of nano-rods. DC ranged between 56–60% (p > 0.05). FA composites showed lower FM and lower FS (p < 0.05), but comparable wear resistance and HV (p > 0.05) to 0FA. 30FA and 40FA showed similar K_1C to 0FA (p > 0.05), with SEM showing evidence of toughening mechanisms, whereas 10FA and 20FA showed lower K_1C (p < 0.05). FA containing composites released fluoride that was proportional to the amount of FA incorporated (p < 0.05) but only under acidic conditions.SignificanceThe addition of FA to the experimental composites reduced strength and stiffness but not the DC, hardness or wear rate. 30FA and 40FA had a higher K_1C compared to other FA groups. Fluoride release occurred under an accelerated acidic regime, suggesting potential as a bioactive ‘smart’ composite.     

Effects of S. mutans gene-modification and antibacterial monomer dimethylaminohexadecyl methacrylate on biofilm growth and acid production

ObjectivesAntibacterial quaternary ammonium monomers (QAMs) are used in resins. The rnc gene in Streptococcus mutans (S. mutans) plays a key role in resisting antibiotics. The objectives of this study were to investigate for the first time: (1) the effects of rnc deletion on S. mutans biofilms and acid production; (2) the combined effects of rnc deletion with dimethylaminohexadecyl methacrylate (DMAHDM) on biofilm-inhibition efficacy.MethodsParent S. mutans strain UA159 (ATCC 700610) and the rnc-deleted S. mutans were used. Bacterial growth, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) were measured to analyze the bacterial susceptibility of the parent and rnc-deleted S. mutans against DMAHDM, with the gold-standard chlorhexidine (CHX) as control. Biofilm biomass, polysaccharide and lactic acid production were measured.ResultsThe drug-susceptibility of the rnc-deleted S. mutans to DMAHDM or CHX was 2-fold higher than parent S. mutans. The drug-susceptibility did not increase after 10 passages (p < 0.05). Deleting the rnc gene increased the biofilm susceptibility to DMAHDM or CHX by 2-fold. The rnc-deletion in S. mutans reduced biofilm biomass, polysaccharide and lactic acid production, even at no drugs. DMAHDM was nearly 40 % more potent than the gold-standard CHX. The combination of rnc deletion + DMAHDM treatment achieved the greatest reduction in biofilm biomass, polysaccharide synthesis, and lactic acid production.SignificanceGene modification by deleting the rnc in S. mutans reduced the biofilm growth and acid production, and the rnc deletion + DMAHDM method showed the greatest biofilm-inhibition efficacy, for the first time. The dual strategy of antibacterial monomer + bacterial gene modification shows great potential to control biofilms and inhibit caries.     

Cytotoxicity and effect on protease activity of copolymer extracts containing catechin

ObjectiveTo evaluate cytotoxicity and effect on protease activity of epigallocatechin-gallate extracted from experimental restorative dental copolymers in comparison to the control compound chlorhexidine.MethodsCopolymer disks were prepared from bis-GMA/TEGDMA (70/30 mol%) containing no compound (control) or 1% w/w of either epigallocatechin-gallate or chlorhexidine. MDPC-23 odontoblast-like cells were seeded with the copolymer extracts leached out into deionized water. Cell metabolic activity was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24, 48, 72 h. Inhibition of protease activity by resin extracts was measured by a collagenolytic/genatinolytic enzyme activity assay and gelatin zymography. Data for MTT and protease inhibition were analyzed using two-way ANOVA followed by Tukey or Bonferroni post hoc tests (α = 0.05).ResultsThe MTT revealed that at 72 h, extracts from control (16.7%) and chlorhexidine (22.3%) copolymers induced significant reduction in cell metabolism (p < 0.05). All copolymer extracts caused enzymatic inhibition in a dose dependent manner (p < 0.01). Even when highly diluted, epigallocatechin-gallate extract had a significant antiproteolytic activity (p < 0.05). Zymograms showed that all extracts reduced activity of MMP-2 and MMP-9 (pro- and active forms), with MMP-9 exhibiting the highest percentage inhibition revealed by densitometry.ConclusionsEpigallocatechin-gallate and chlorhexidine extracts did not exert cytotoxicity on evaluated cells when compared to control extracts. Both compounds retained antiproteolytic activity after extraction from a dental copolymer.Clinical significanceOnce extracted from a dental copolymer, epigallocatechin-gallate is not cytotoxic and retains antiproteolytic activity. These results may allow incorporation of epigallocatechin-gallate as a natural-safe alternative to chlorhexidine in functionalized restorative materials.     

Cell migration,viability and tissue reaction of calcium hypochlorite based-solutions irrigants: An in vitro and in vivo study

ObjectiveThis study aimed to analyze in vitro cytotoxicity to cultured 3T3 fibroblasts and in vivo inflammatory reaction in rats by calcium hypochlorite (Ca(OCl)₂) solutions compared with sodium hypochlorite (NaOCl) solutions.DesignCultured 3T3 fibroblasts were exposed to different concentrations of (Ca(OCl)₂) and NaOCl solutions, and a scratch assay was performed. The viability rate was analyzed with trypan blue assay. Both solutions of 1% and 2.5% concentrations were injected into the subcutaneous tissue of 18 male Wistar rats aged 18 weeks. The inflammatory tissue reaction was evaluated at 2 h, 24 h, and 14 days after the injections. The samples were qualitatively analyzed using a light microscope. Statistical analysis was performed with ANOVA and Tukey post hoc tests for in vitro assays and Kruskal–Wallis and Dunn post hoc tests for in vivo assays (α = 0.05).ResultsIn the scratch assay, Ca(OCl)₂ showed no significant difference compared with the control group (culture medium) at 24 h (p < 0.05). Solutions of 0.0075% and 0.005% NaOCl and Ca(OCl)₂ concentrations presented similar results compared with those in the positive control group (hydrogen peroxide) (p > 0.05) in the trypan blue assay. In the in vivo assay, 1% Ca(OCl)₂ group showed a significant decrease in neutrophils at 2 h and 24 h (p = 0.041) and 2 h and 14 days (p = 0.017). There was no statistically significant difference for lymphocyte/plasmocyte and macrophage counts among the different concentration groups.ConclusionsCa(OCl)₂ showed favorable results of viability and induced a low-level inflammatory response. Ca(OCl)₂ presented acceptable cytotoxicity and biocompatibility as an irrigant solution.     

Thymus vulgaris L. extract has antimicrobial and anti-inflammatory effects in the absence of cytotoxicity and genotoxicity

ObjectivesThis study evaluated the biological effects of the T. vulgaris L. extract., such as antimicrobial activity on planktonic cultures and mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory activity and genotoxicity.MethodsMonomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed by C. albicans with each bacterium were formed for 48 h and exposed for 5 min to the plant extract. Murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7) and cervical carcinoma cells (HeLa) were also exposed to the plant extract for 5 min and the cell viability were analyzed by MTT, neutral red (NR) and crystal violet (CV) assays. Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) produced by RAW 264.7 was quantified by ELISA, after 24 h exposure to the plant extract, both in the absence and presence of lipopolysaccharide (LPS) from Escherichia coli. Genotoxicity of the plant extract was evaluated by micronucleus formation (MN) in 1000 cells. The results were analyzed by T-Test or ANOVA and Tukey’s Test (P ≤ 0.05).ResultsAll biofilms showed significant reductions in CFU/mL (colony-forming units per milliliter). Cell viability was above 50% for all cell lines. Anti-inflammatory effect on the synthesis of IL-1β and TNF-α was observed. The MN was similar or lower than the control group in all cells.ConclusionsT. vulgaris L. extract was effective against all biofilms, promoted high cell viability, anti-inflammatory effect and presented no genotoxicity.     

The effect of dietary intervention on paraffin-stimulated saliva and dental health of children participating in a randomized controlled trial

ObjectivesThe aim was to study the impact of dietary intervention on the properties of paraffin-stimulated saliva, and on dental caries.Study designAt 7 months of age 1062 infants (540 intervention; 522 controls) started in the prospective, randomized Special Turku Intervention Project (STRIP) aimed at restricting the child's saturated fat and cholesterol intake to prevent atherosclerosis of adult age (www.clinicaltrials.gov NCT 00223600). At 3 years of age, every fifth child was invited to an oral sub-study, and 148 (78 boys) children attended. At 6, 9, 12 and 16 years of age 135, 127, 114 and 88 children were restudied, respectively. Dietary intakes of carbohydrates, protein, saturated fat, calcium, phosphate, and fibre were regularly recorded using 4-day food records. Height and weight were regularly monitored. Paraffin-stimulated saliva samples were collected at 6, 9, 12 and 16 years of age, and analyzed for flow rate, buffer capacity, calcium, phosphate and proteins. Dental health was recorded and expressed as d₃mft/D₃MFT, and as time of caries onset.ResultsDietary intakes of calcium, phosphate and fibre, and salivary flow rate increased with time in both groups (p < 0.001, GLM for repeated measures). Fibre intake and salivary flow rate were higher in the intervention than in the control group (p = 0.042 and p = 0.0394, respectively, GLM for repeated measures). There were no correlations between dietary intakes and salivary concentrations of calcium or phosphate. Children who did not have caries experience (d₃mft/D₃MFT = 0) during the entire follow-up had higher salivary calcium than those who had caries already at 3 years of age. The association between salivary calcium and caries onset was significant up to 12 years of age. Toothbrushing frequency was statistically significantly associated with caries-onset at ages 6 (gamma statistic 0.457, p = 0.046) and 12 years (gamma statistic 0.473, p = 0.019).ConclusionsThe current long-term dietary intervention increased children's paraffin-stimulated salivary flow rate. The concentration of salivary calcium was directly correlated to dental health. Higher salivary flow rate in the intervention group is believed to be due to higher fibre intake in the intervention group.     

Hypoxia inhibits mineralization ability of human dental pulp cells treated with TEGDMA but increases cell survival in accordance with the culture time

ObjectiveTo evaluate the cytotoxicity and mineralization effects of TEGDMA in human dental pulp cells (hDPCs) under hypoxic and normoxic culture conditions.DesignCell viability was evaluated using XTT assay after incubation periods of 24, 48, or 72 h. The expression of mineralization-related genes (osteonectin, osteopontin, dentin sialophosphoprotein, collagen type 1) and heme oxygenase 1 (HO-1) was assessed by quantitative real-time polymerase chain reaction at 24 and 72 h.ResultsIn XTT assay, viability was higher in 0.3, 1, 2, 4, and 5 mM groups in the presence of 21% O₂ after 24 h (p < 0.05). Additionally, while 0.3, 1, 2 mM groups had higher cell viability in the presence of 21% O₂ after 48 h (p < 0.05), in 3 mM groups cell viability was higher under 3% O₂ than 21% O₂ after both 24 and 48 h (p < 0.05). 1–3 mM groups had higher cell viability under 3% O₂ after 72 h (p < 0.05). There was no difference between 4 and 5 mM groups with regards to cell viability after 48 or 72 h (p > 0.05). In the gene expression study, TEGDMA-treated hDPCs showed lower mineralization potential in the presence of 3% than with 21% O₂ (p < 0.05). hDPCs revealed higher HO 1 expression in 0.3 and 1 mM groups under hypoxic than under normoxic conditions after a 72-h time period (p < 0.001).ConclusionsHypoxic conditions increased cell survival in accordance with the culture period but inhibited the odontoblastic differentiation of hDPCs treated with TEGDMA.