Marco Ottobelli

Streptococcus mutans adherence and biofilm formation on experimental composites containing dicalcium phosphate dihydrate nanoparticles

This study aimed at evaluating bacterial adhesion and biofilm formation on resin-based composites (RBC) including dicalcium phosphate dihydrate nanoparticles (nDCPD). Methods: Specimens were prepared from experimental RBCs with BisGMA/TEGDMA resin matrix including 20 vol% of either nDCPD (nDCPD-RBC), TEGDMA-functionalized nDPCD (F-nDCPD-RBC) or silanized silica (SiO2-RBC). Neat resin blend (control-Resin), conventional nanohybrid RBC (control-RBC) and human enamel were used for reference. Characterization of the specimens included surface roughness (SR), surface free energy (SFE), chemical surface composition (EDS, XPS), and buffering ability of a pH = 4.00 solution. Streptococcus mutans adherence was assessed after 2 h; biofilm formation was simulated for 48 h using a bioreactor. Adherent, viable biomass was determined using tetrazolium salt assay (MTT). Results: nDCPD-RBC yielded highest roughness and showed higher polar and lower disperse component to total SFE. EDS and XPS indicated higher amounts of calcium and phosphate on the surface of nDCPD-RBC than on F-nDCPD-RBC. nDCPD buffered the acidic solution to 5.74, while functionalization almost prevented buffering (pH = 4.26). F-nDCPD-RBC reduced adherence and biofilm formation in comparison to nDCPD-RBC. Regardless of functionalization, biofilm formation on nDCPD-containing RBCs was not significantly different from SiO2-RBC. Control-Resin, control-RBC, and enamel surfaces showed similar adherence values as F-nDCPD-RBC, but lower biofilm formation compared to both nDCPD-containing RBCs. In conclusion, the incorporation of nDCPD did not minimize S. mutans adherence and biofilm formation as a function of the materials´ surface properties. However, results observed for the buffering capacity indicated that optimized formulations of biomimetic RBCs may be useful for modulating their interaction with microorganisms.Graphical Abstract

Biofilm formation and release of fluoride from dental restorative materials in relation to their surface properties

OBJECTIVES To elucidate the impact of surface properties and the release of fluoride from different glass ionomer cements on biofilm formation. METHODS Standardized specimens manufactured from various classes of glass ionomer cements (GICs), a resin-based composite (RBC), and human enamel were subjected to surface analyses. Subsequent to simulation of salivary pellicle formation, Streptococcus mutans biofilm formation was initiated using a drip flow reactor for 48h and 96h. Biofilms were characterized by determining viable bacterial biomass and 3D biofilm architecture using SEM and CLSM; the release of fluoride from the specimens was measured using the ion selective micro method in dependence on various experimental conditions (incubation with sterile broth/bacteria/acid). RESULTS Surface properties and biofilm formation correlated poorly, while the release of fluoride correlated well with viable streptococcal biomass and SEM/CLSM analyses. For all investigated materials, biofilm formation was lower than on enamel. The release of fluoride showed a significant dependency on the experimental conditions applied; the presence of biofilms reduced fluoride release in comparison to sterile incubation conditions. CONCLUSIONS Within the limitations of a laboratory study, the results suggest that biofilm formation on GICs cannot be easily predicted as a function of substratum surface parameters. The release of fluoride from glass ionomer cements contributes to control biofilm formation particularly in its early phases. CLINICAL SIGNIFICANCE Glass ionomer cements can actively control microbial biofilm formation, while biofilms modulate the release of fluoride from GIC materials.

In vitro biofilm formation on resin-based composites after different finishing and polishing procedures

OBJECTIVES To evaluate the influence of surface treatments of different resin-based composites (RBCs) on S. mutans biofilm formation. METHODS 4 RBCs (microhybrid, nanohybrid, nanofilled, bulk-filled) and 6 finishing-polishing (F/P) procedures (open-air light-curing, light-curing against Mylar strip, aluminum oxide discs, one-step rubber point, diamond bur, multi-blade carbide bur) were evaluated. Surface roughness (SR) (n=5/group), gloss (n=5/group), scanning electron microscopy morphological analysis (SEM), energy-dispersive X-ray spectrometry (EDS) (n=3/group), and S. mutans biofilm formation (n=16/group) were assessed. EDS analysis was repeated after the biofilm assay. A morphological evaluation of S. mutans biofilm was also performed using confocal laser-scanning microscopy (CLSM) (n=2/group). The data were analyzed using Wilcoxon (SR, gloss) and two-way ANOVA with Tukey as post-hoc tests (EDS, biofilm formation). RESULTS F/P procedures as well as RBCs significantly influenced SR and gloss. While F/P procedures did not significantly influence S. mutans biofilm formation, a significant influence of RBCs on the same parameter was found. Different RBCs showed different surface elemental composition. Both F/P procedures and S. mutans biofilm formation significantly modified this parameter. CONCLUSIONS The tested F/P procedures significantly influenced RBCs surface properties but did not significantly affect S. mutans biofilm formation. The significant influence of the different RBCs tested on S. mutans biofilm formation suggests that material characteristics and composition play a greater role than SR. CLINICAL SIGNIFICANCE F/P procedures of RBCs may unexpectedly play a minor role compared to that of the restoration material itself in bacterial colonization.

Levorotatory carbohydrates and xylitol subdue Streptococcus mutans and Candida albicans adhesion and biofilm formation

Dietary carbohydrates and polyols affect the microbial colonization of oral surfaces by modulating adhesion and biofilm formation. The aim of this study was to evaluate the influence of a select group of l‐carbohydrates and polyols on either Streptococcus mutans or Candida albicans adhesion and biofilm formation in vitro. S. mutans or C. albicans suspensions were inoculated on polystyrene substrata in the presence of Tryptic soy broth containing 5% of the following compounds: d‐glucose, d‐mannose, l‐glucose, l‐mannose, d‐ and l‐glucose (raceme), d‐ and l‐mannose (raceme), l‐glucose and l‐mannose, sorbitol, mannitol, and xylitol. Microbial adhesion (2 h) and biofilm formation (24 h) were evaluated using MTT‐test and Scanning Electron Microscopy (SEM). Xylitol and l‐carbohydrates induced the lowest adhesion and biofilm formation in both the tested species, while sorbitol and mannitol did not promote C. albicans biofilm formation. Higher adhesion and biofilm formation was noted in both organisms in the presence of d‐carbohydrates relative to their l‐carbohydrate counterparts. These results elucidate, hitherto undescribed, interactions of the individually tested strains with l‐ and d‐carbohydrates, and how they impact fungal and bacterial colonization. In translational terms, our data raise the possibility of using l‐form of carbohydrates and xylitol for dietary control of oral plaque biofilms.

Influence of Light-curing Parameters on Biofilm Development and Flexural Strength of a Silorane-based Composite

OBJECTIVES The aim of this study was to evaluate the differences in biological and mechanical performances of a silorane-based and a methacrylate-based composite. Another aim was to assess the influence of light-curing time and light-curing intensity on in vitro biofilm formation and flexural strength of the two tested composites. METHODS Experiment 1: 432 specimens obtained from a silorane-based composite and from a standard methacrylate-based composite were divided into six groups and light-cured for 10, 20, 30, 40, 60, or 80 seconds, using one of two light-curing intensities, 400 mW/cm(2) or 800 mW/cm(2). At 24 hours, a monospecific Streptococcus mutans biofilm adherent to the surfaces of the samples was obtained. Then, a colorimetric technique (MTT assay) was used to evaluate the adherent viable biomass. Two samples per group were observed using confocal laser scanning microscopy. Analysis of variance (ANOVA) and Tukey tests were used to analyze the results (p<0.05). Experiment 2: 192 bar-shaped specimens were obtained and light-cured as in the previous experiment. A three-point bend test using a universal testing machine was performed to obtain flexural strength values. ANOVA and Tukey tests were used to analyze the results (p<0.05). RESULTS In experiment 1, a highly significant difference (p<0.0001) in biofilm development was shown between silorane-based and methacrylate-based composites. In fact, the silorane-based composite exhibited better biological performance. Significant differences were also found between the two light-curing intensities (p<0.018) and for curing times (p<0.0001): silorane-based composite light-cured for 80 seconds at 800 mW/cm(2) light-curing intensity showed the lowest biofilm development. In experiment 2, a significant difference in flexural strength (p<0.0318) was only found between the different composites. Nevertheless, both resin composites showed flexural strength values in accordance with International Organization for Standardization guidelines even after 10 seconds of light-curing time. CONCLUSIONS Silorane-based composite was less prone to biofilm development compared with a methacrylate-based composite. Acceptable flexural strength values for both composites were obtained after 10 seconds of light-curing time.

Laser microtextured titanium implant surfaces reduce in vitro and in situ oral biofilm formation

Introduction Micro- or nano-topography can both provide antimicrobial properties and improve osseointegration of dental implant titanium surfaces. Laser treatment is one of the best surface microtexturing techniques. The aim of this study was to evaluate in vitro and in situ biofilm formation on a laser-treated titanium surface, comparing it with two conventional surfaces, machined and grit-blasted. Methods For the in vitro experiment, an oral microcosm biofilm model was developed on the surface of titanium disks and reference human enamel using a bioreactor for 48 h. For the in situ experiment, titanium implants with laser-treated, machined and grit-blasted surfaces were mounted on intraoral trays and worn by ten volunteers for 48 h. Biofilm formation was quantitatively evaluated, and surfaces were analyzed using confocal laser scanning microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Results–in vitro study Biofilm structures with a prevalence of viable cells covered most of the machined, grit-blasted and human enamel surfaces, whereas less dense biofilm structures with non-confluent microcolonies were observed on the laser-treated titanium. Laser-treated titanium showed the lowest biofilm formation, where microorganisms colonized the edges of the laser-created pits, with very few or no biofilm formation observed inside the pits. Results–in situ study The biofilm formation pattern observed was similar to that in the in vitro experiment. Confocal laser scanning microscopy showed complete coverage of the implant threads, with mostly viable cells in grit-blasted and machined specimens. Unexpectedly, laser-treated specimens showed few dead microbial cells colonizing the bottom of the threads, while an intense colonization was found on the threading sides. Conclusion This data suggests that laser-created microtopography can reduce biofilm formation, with a maximum effect when the surface is blasted orthogonally by the laser beam. In this sense the orientation of the laser beam seems to be relevant for the biological interaction with biofilms.

In vitro biofilm formation on resin-based composites cured under different surface conditions

OBJECTIVES The interfacial conditions occurring during light-curing procedures of resin-based composites (RBCs) influence their surface properties and therefore the biological behavior of the material. This study aimed to evaluate the influence of different surface curing conditions on in vitro biofilm formation by Streptococcus mutans and mixed oral microflora, in the presence or absence of surface salivary pre-conditioning. METHODS Two nanohybrid RBCs and four interfacial curing conditions (open air, argon, nitrogen and glycerin) were evaluated. Surface roughness (SR), surface elemental composition (energy-dispersive X-ray spectrometry, EDS) and biofilm formation (S. mutans and oral microcosm) were assessed. Surfaces were observed using scanning electron microscopy (SEM). Microbiological tests were performed with and without saliva pre-conditioning of the surfaces. EDS analysis was performed before and after biofilm formation, and biofilm morphology was evaluated using confocal laser scanning microscopy (CLSM). Data were analyzed using multi-way ANOVA and Tukey post-hoc test (p < 0.05). RESULTS Interfacial curing conditions significantly influenced SR depending on the tested RBC. EDS analysis showed that surface elemental composition was significantly influenced by the interfacial curing condition depending on the tested RBC. Interfacial curing conditions significantly influenced biofilm formation in both microbiological models in the absence of saliva pre-conditioning, depending on the tested RBC, whereas saliva pre-conditioning abrogated these effects. CONCLUSIONS Surface curing conditions significantly impacted biofilm formation in a material-dependent manner, which was abrogated when surfaces were pre-conditioned with saliva. CLINICAL SIGNIFICANCE Curing under glycerin did not improve the microbiological performances of the tested RBCs. These results, needing to be confirmed by in vivo data, have the potential to simplify operative procedures in restorative dentistry.