The development of dental adhesive resins with long-lasting antibacterial properties is a possible solution to overcome the problem of secondary caries in modern adhesive dentistry. Objectives: Synthesis and characterization of nitrogen-doped titanium dioxide nanoparticles (N_TiO2), (ii) topographical, compositional and wettability characterization of thin-films (unaltered and experimental) and, (iii) antibacterial efficacy of N_TiO2-containing dental adhesives against Streptococcus mutans biofilms. Materials and methods: Nanoparticles were synthesized and characterized using different techniques. Specimens (diameter= 12 mm, thickness ≅ 15 μm) of OptiBond Solo Plus (Kerr Corp., USA) and experimental adhesives [50, 67 and 80% (v/v)] were fabricated, photopolymerized (1000 mW/cm2, 1 min) and UV-sterilized (254 nm, 800,000 μJ/cm2) for microscopy, spectroscopy, wettability and antibacterial testing. Wettability was assessed with a contact angle goniometer by dispensing water droplets (2 μL) onto four random locations of each specimen (16 drops/group). Drop profiles were recorded (1 min, 25 frames/sec, 37°C) and contact angles were calculated at time=0s (θINITIAL) and time=59s (θFINAL). Antibacterial testing was performed by growing S. mutans (UA159-ldh, JM10) biofilms for either 3 or 24 hours (anaerobic conditions, 37°C) with or without continuous light irradiation (410 ± 10 nm, 3h= 38.75 J/cm2, 24h= 310.07 J/cm2) against the surfaces of sterile specimens. Results: N_TiO2 was successfully prepared using solvothermal methods. Doped-nanoparticles displayed higher light absorption levels when compared to undoped titania. Experimental adhesives demonstrated superior antibacterial efficacy in dark conditions. Conclusions: The findings presented herein suggest that N_TiO2 is a feasible antibacterial agent against cariogenic biofilms.

Objectives The release of unpolymerized monomers and by-products of resin composites influences biofilm growth and confounds the measurement of metabolic activity. Current assays to measure biofilm viability have critical limitations and are typically not performed on relevant substrates. The objective of the present study was to determine the utility of firefly luciferase assay for quantification of the viability of intact biofilms on a resin composite substrate, and correlate the results with a standard method (viable colony counts). Methods Disk-shaped specimens of a dental resin composite were fabricated, wet-polished, UV-sterilized, and stored in water. Biofilms of S. mutans (strain UA159 modified by insertion of constitutively expressed firefly luc gene) were grown (1:500 dilution; anaerobic conditions, 24h, 37°C) in two media concentrations (0.35x and 0.65x THY medium supplemented with 0.1% sucrose; n=15/group). An additional group of specimens with biofilms grown in 0.65x + sucrose media was treated with chlorhexidine gluconate solution to serve as the control group. Bioluminescence measurements of non-disrupted biofilms were obtained after addition of D-luciferin substrate. The adherent biofilms were removed by sonication, and bioluminescence of sonicated bacteria was then measured. Viable colony counts were performed after plating sonicated bacteria on THY agar plates supplemented with spectinomycin. Bioluminescence values and cell counts were correlated using Spearman Correlation tests (α=0.05). Results Strong positive correlations between viable colony counts and bioluminescence values, both before- and after-sonication, validated the utility of this assay. Significance A novel non-disruptive, real-time bioluminescence assay is presented for quantification of intact S. mutans biofilms grown on a resin composite, and potentially on antibacterial materials and other types of dental biomaterials.

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This study demonstrated that aPDT has promising potential to be used as an additional method to control oral cariogenic biofilms.

Objective. The present work shows the optimization of a high-throughput bioluminescence assay to assess the metabolism of intact Streptococcus mutans biofilms and its utility as a screening method for nanofilled antibacterial dental materials. Methods. The assay was optimized by monitoring changes in bioluminescence mediated by variation of the experimental parameters investigated (growth media and sucrose concentration, inoculum:D-Luciferin ratio, dilution factor, inoculum volume, luminescence wavelength, replicate and luciferase metabolic activity). Confocal microscopy was then used to demonstrate the impact of biofilm growth conditions on the 3-D distribution of extracellular polymeric substance (EPS) within Streptococcus mutans biofilms and its implications as confounding factors in high-throughput studies (HTS). Results. Relative Luminescence Unit (RLU) values from the HTS optimization were analyzed by multivariate ANOVA ( α = 0.05) and coefficients of variation, whereas data from 3-D structural parameters and RLU values of biofilms grown on experimental antibacterial dental adhesive resins were analyzed using General Linear Models and Student–Newman–Keuls post hoc tests ( α = 0.05). Confocal microscopy demonstrated that biofilm growth conditions significantly influenced the quantity and distribution of EPS within the 3-D structures of the biofilms. An optimized HTS bioluminescence assay was developed and its applicability as a screening method in dentistry was demonstrated using nanofilled experimental antibacterial dental adhesive resins. Significance. The present study is anticipated to positively impact the direction of future biofilm research in dentistry, because it offers fundamental information for the design of metabolic-based assays, increases the current levels of standardization and reproducibility while offering a tool to decrease intra-study variability.

Dental adhesives hydrolyze in the mouth. this study investigated the water sorption (SoR), solubility (SoL) and cytotoxicity (cYto) of experimental adhesives containing nitrogen-doped titanium dioxide nanoparticles (n_tio 2). Specimens (n = 15/group [SOR, SOL]; n = 10/group [CYTO]) of unaltered Clearfil SE Protect (CSP), OptiBond Solo Plus (OSP), Adper Scotchbond (ASB) and experimental adhesives (OSP + 25% or 30% of N_TiO 2) were fabricated, desiccated (37 °C) and tested for SOR and SOL according to ISO Specification 4049 (2009). CYTO specimens were UV-sterilized (8 J/cm 2) and monomer extracted in growth medium (1, 3 or 7 days). Human pulp cells were isolated and seeded (0.5 × 10 4) for MTT assay. SOR and SOL data was analyzed using GLM and SNK (α = 0.05) and CYTO data was analyzed with Kruskal-Wallis and SNK tests (α = 0.05). SOR and SOL values ranged from 25.80 μg/mm 3 (30% N_TiO 2) to 28.01 μg/mm 3 (OSP) and 23.88 μg/mm 3 (30% N_TiO 2) to 25.39 μg/mm 3 (25% N_TiO 2). cYto results indicated that pulp cells exposed to experimental materials displayed comparable viabilities (p > 0.05) to those of OSP. Experimental materials displayed comparable SOR, SOL and CYTO values (p > 0.05) when compared to unaltered materials. N_TiO 2 incorporation have not adversely impacted SoR, SoL and cYto properties of unaltered adhesives. Resin composite restorations are currently the most prevalent medical intervention in human beings with more than five hundred million restorations placed globally every year 1. Such popularity amongst patients and clinicians precipitates from their mercury-free compositions 2 , outstanding esthetic properties, and their minimally invasive and ultra-conservative restorative techniques 3. Their clinical use involves the removal of demineralized and bacteria-contaminated tooth structure, application of phosphoric acid (37%, 15-30 s), and the subsequent application of a primer and a dental adhesive resin in preparation for the intraoral fabrication of the resin composite restoration 4. The formation of the hybrid layer starts with the penetration of uncured monomers into water-rich, mineraldepleted areas of dentin, followed by the envelopment of exposed collagen fibrils, and the subsequent in situ polymerization by on-demand visible light irradiation 5. Its successful completion 6 should allow for the establishment of a crosslinked 7 and hermetically sealed 3-dimensional polymer-collagen network 6 capable of reducing microleakage, bacterial invasion, marginal staining, secondary caries and pulpal irritation 8. However, because these materials contain both hydrophilic and hydrophobic moieties in a single product 9 , they tend to become chemically unstable when placed in contact with moist dentin 10. The physical manifestation of such instability is translated into materials that phase-separate 11 and display inadequate degrees of conversion 12 .

For the advancement of Class V restoratives, our goal was to evaluate the physicochemical and mechanical properties, antimicrobial functionality, and cytotoxic potential of novel antimicrobial copolymers. 5-Carboxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethylpentan-1-aminium bromide (AMadh1) and 10-carboxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyldecan-1-aminium bromide (AMadh2) were incorporated into light-curable urethane dimethacrylate, polyethylene glycol–extended urethane dimethacrylate, ethyl 2-(hydroxymethyl) acrylate resin (UPE resin). In the AMadhs-UPE resin, the hydrophobic/hydrophilic balance, degree of vinyl conversion, flexural strength, elastic modulus, and shear bond strength were assessed. Antimicrobial properties were measured using Streptococcus mutans (planktonic and biofilm). Cytotoxicity was tested using human gingival fibroblasts and mouse connective tissue fibroblasts (ATCC® CCL-1™) exposed to two-fold serial dilutions (≤10.6 mmol/L AMadh1 or ≤8.8 mmol/L AMadh2). At 10% mass of AMadh, the attained degree of vinyl conversion values (AMadh1 = 90.1% and AMadh2 = 88.5%) were not statistically different from the UPE resin (88.1%). At both AMadh levels, the flexural strength was reduced in a dose-dependent manner. Elastic modulus and contact angle were not significantly affected by AMadh1. Variations in elastic modulus and contact angle were observed with AMadh2; however, this does not disqualify it in future design of Class V restoratives. Compared to UPE resin, AMadh1-UPE and AMadh2-UPE (10% mass) copolymers reduced S. mutans biofilm 4.2- and 1.6-fold, respectively (p ≤ 0.006). In direct contact with human gingival fibroblasts or ATCC CCL-1 cells, at biologically relevant concentrations, the AMadhs did not adversely affect cell viability or their metabolic activity. This effort addresses a significant oral health issue associated with elderly populations. Its successful completion is expected to yield dental restoratives with well-controlled biofunction.

The aim of this study was to evaluate the antimicrobial efficacy of non-thermal atmospheric plasma (NTAP) against Streptococcus mutans biofilms. Resin discs were fabricated, wet-polished, UV sterilized, and immersed in water for monomer extraction (37 °C, 24 h). Biofilms of bioluminescent S. mutans strain JM10 was grown on resin discs in anaerobic conditions for (37 °C, 24 h). Discs were divided into seven groups: control (CON), 2% chlorhexidine (CHX), only argon gas 150 s (ARG) and four NTAP treatments (30 s, 90 s, 120 s, 150 s). NTAP was applied using a plasma jet device. After treatment, biofilms were analyzed through the counting of viable colonies (CFU), bioluminescence assay (BL), scanning electron microscopy (SEM), and polymerase chain reaction (PCR). All NTAP-treated biofilm yielded a significant CFU reduction when compared to ARG and CON. BL values showed that NTAP treatment for 90 s, 120 s or 150 s resulted in statistically significantly lower metabolic activity when compared to the other groups. CHX displayed the lowest means of CFU and BL. SEM showed significant morphological changes in NTAP-treated biofilm. PCR indicated damage to the DNA structure after NTAP treatment. NTAP treatment was effective in lowering the viability and metabolism of S. mutans in a time-dependent manner, suggesting its use as an intraoral surface-decontamination strategy.