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Objective: The present work demonstrates the optimization of a renilla-based real-time, ultra-bright, non-disruptive, high-throughput bioluminescence assay (HTS) to assess the metabolism of intact Streptococcus mutans biofilms and its utility in screening the antibacterial efficacy of experimental nanofilled dental adhesive resins containing varying concentrations of nitrogen-doped titanium dioxide nanoparticles (N_TiO 2 )., Methods: Optimization of the assay was achieved by screening real-time bioluminescence changes in intact Streptococcus mutans biofilms imposed by the various experimental biofilm growth parameters investigated (bacterial strain, growth media, sucrose concentration, dilution factor, and inoculum volume). The optimized assay was then used to characterize the antibacterial efficacy of experimental nanofilled dental adhesive resins. The assay's ability to discriminate between bacteriostatic and bactericidal approaches was also investigated., Results: Relative Light Units (RLU) values from the HTS optimization were analyzed by multivariate ANOVA (α = 0.05) and coefficients of variation. An optimized HTS bioluminescence assay was developed displaying RLUs values (brightness) that are much more intense when comparing to other previously reported bioluminescence assays, thereby decreasing the error associated with bioluminescence assays and displaying better utility while investigating the functionalities of antimicrobial nanofilled experimental dental adhesive resins with proven long-term properties., Significance: The present study is anticipated to positively impact subsequent research on dental materials and oral microbiology because it serves as a valuable screening tool in metabolic-based assays with increased sensitivity and robustness. The assay reported is anticipated to be further optimized to be used as a co-reporter for other Luc based assays., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Objective: 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 Streptococcus 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., (Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objectives: Thermal expansion measurement of glassy materials is complicated by thermal history effects. Excess volume--trapped in quenched dental porcelains after firing--collapses via structural relaxation on first slow heating during conventional dilatometry, making the thermal expansion coefficient (alpha) obtained on first heating unreliable. The purpose of this study was to determine whether porcelain thermal expansion measurement at high thermal rates could minimize the influence of thermal history., Methods: Eight thermal expansion specimens each of six body porcelains and the Component No. 1 (leucite-containing) frit prepared according to the patent by Weinstein et al. (US Patent No. 3,052,982) were subjected to three heat-cool conventional dilatometry runs at 3 degrees C/min, while eight thermal expansion specimens of each porcelain were reserved as untreated controls. Eight hollow, cylindrical specimens of the same brands were subjected to three heat-cool laser dilatometer thermal expansion runs at 600 degrees C/min, while eight cylindrical specimens of each porcelain were reserved as untreated controls. Thermal expansion data (25-500 degrees C) of all specimens were subjected to repeated measures analysis of variance., Results: The alpha obtained on first slow heating was significantly lower than values for succeeding slow heat and cool runs in all porcelains (P < 0.001). High-rate alpha obtained on first heating was not significantly different from values of succeeding heat and cool runs in all porcelains (P > 0.05)., Significance: Conventional dilatometer measurements demonstrated occurrence of structural relaxation, as evidenced by the significant difference in the first heating and subsequent runs. High-rate laser dilatometry eliminated structural relaxation, thereby providing a thermal expansion measurement that is free of interference from thermal history effects.

Objectives: The fracture toughness (KIC) and load relaxation of four dentin bonding resins were determined to characterize some of the mechanical properties of these materials after polymerization., Methods: A total of 40 single-edge notch bar specimens were fabricated, 10 each of four commercially available brands, and subjected to three-point bending until fracture, as described in ASTM Standard E399-83 (1991a). The critical stress intensification factor, KIC, was derived for each specimen and compared by analysis of variance and Scheffé's multiple comparisons test (p < 0.01). To study the load relaxation characteristics, five rectangular specimens (without notches) of each brand were subjected to three-point loading until a predetermined limiting load value was reached. The test load was allowed to relax for 4 min, after which the specimen was unloaded to the zero load condition, and the load was allowed to build up on its own accord for 3 min. Load relaxation values were measured from the chart, and the mean percent load drop was calculated. The load relaxation data were compared using analysis of variance and Scheffé's multiple comparisons test (p < 0.05)., Results: The fracture toughness (KIC) values of the four adhesive resins studied in this investigation ranged from 0.37-0.94 MPa.m0.5 and were statistically different from each other (p < 0.001). The load relaxation values were found to be greatest within the first 0.5 min, with the total load relaxation of the four bonding agents ranging from 16%-30%. Two of the materials studied showed significantly different short-term load relaxation behavior than the other two resins (p < 0.05)., Significance: Bonding agents can be implicated as one of the factors that weaken the interface between the dentin and the composite restorative material. These materials are capable of a rapid short-term response, demonstrating significant load relaxation in the first 0.5 min after loading.

Objectives: The elevated temperatures and slow heating and cooling rates associated with a thermal expansion run provide a thermal treatment comparable to those previously shown to produce leucite crystallization in dental PFM porcelains. The purpose of this study was to determine whether the exposure of dental porcelains to the time-temperature profile of a thermal expansion run could result in leucite crystallization, a potential interference with the accurate determination of the thermal expansion coefficient., Methods: A total of 112 thermal expansion specimens were prepared, 16 each of six commercially available body porcelains, as well as the "Component No. 1" (leucite-containing) frit of the Weinstein et al. (1962) patent. In a randomized design, the leucite contents of specimens subjected to a 3 degrees C/min thermal expansion run were compared to those of untreated controls. The leucite content of each specimen was determined via quantitative X-ray powder diffraction using an Al2O3 internal standard. One-tailed t-tests were applied to determine whether the dilatometer thermal treatment produced a statistically significant increase in leucite content., Results: The heat treatment afforded by a 3 degrees C/min dilatometer run was not sufficient to induce measurable leucite crystallization in any of the porcelains examined in this study., Significance: The crystallization of leucite during a dilatometer heating run can be ruled out as a possible interference with the determination of the coefficient of thermal expansion of dental porcelain.