Substituted Nano-Hydroxyapatite Toothpastes Reduce Biofilm Formation on Enamel and Resin-Based Composite Surfaces.

Background: Toothpastes containing nano-hydroxyapatite (n-HAp) substituted with metal ions provide calcium and phosphate ions to dental hard tissues, reducing demineralization, and promoting remineralization. Few data are available about the effect of these bioactive compounds on oral microbiota. Methods: This in vitro study evaluated the influence of two commercially-available substituted n-HAp-based toothpastes (α: Zn-carbonate substituted n-HAp; ß: F, Mg, Sr-carbonate substituted n-HAp) on early colonization (EC, 12 h) and biofilm formation (BF, 24 h) by oral microbiota. Controls were brushed with distilled water. Artificial oral microcosm and Streptococcus mutans biofilms were developed using human enamel and a resin-based composite (RBC) as adherence surfaces. Two test setups, a shaking multiwell plate and a modified drip-flow reactor (MDFR), were used to simulate clinical conditions during the night (low salivary flow and clearance) and daytime, respectively. Energy-dispersive X-ray spectrometry (EDS) was used to evaluate specimens' surfaces after toothpaste treatment. Fluoride release from ß toothpaste was evaluated. Viable adherent biomass was quantified by MTT assay, and biofilms' morphology was highlighted using confocal microscopy. Results: EDS showed the presence of remnants from the tested toothpastes on both adherence surfaces. ß toothpaste showed significantly lower EC and BF compared to control using the artificial oral microcosm model, while α toothpaste showed lower EC and BF compared to control, but higher EC and BF compared to ß toothpaste. The effect shown by ß toothpaste was, to a minimal extent, due to fluoride release. Interestingly, this result was seen on both adherence surfaces, meaning that the tested toothpastes significantly influenced EC and BF even on RBC surfaces. Furthermore, the effect of toothpaste treatments was higher after 12 h than 24 h, suggesting that toothbrushing twice a day is more effective than brushing once. Conclusions: The efficacy of these treatments in reducing microbial colonization of RBC surfaces may represent a promising possibility in the prevention of secondary caries.

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.

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.

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.

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.

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 matrix and filler fraction on biofilm formation on the surface of experimental resin-based composites.

The aim of this study was to investigate the impact of resin matrix chemistry and filler fraction on biofilm formation on the surface of experimental resin-based composites (RBCs). Specimens were prepared from eight experimental RBC formulations differing in resin matrix blend (BisGMA/TEGDMA in a 7:3 wt% ratio or UDMA/aliphatic dimethacrylate in a 1:1 wt% ratio) and filler fraction (no fillers; 65 wt% dental glass with an average diameter of 7 or 0.7 µm or 65 wt% SiO2 with an average diameter of 20 nm). Surface roughness, surface free energy, and chemical surface composition were determined; surface topography was visualized using atomic force microscopy. Biofilm formation was simulated under continuous flow conditions for a 48 h period using a monospecies Streptococcus mutans and a multispecies biofilm model. In the monospecies biofilm model, the impact of the filler fraction overruled the influence of the resin matrix, indicating lowest biofilm formation on RBCs with nano-scaled filler particles and those manufactured from the neat resin blends. The multispecies model suggested a more pronounced effect of the resin matrix blend, as significantly higher biofilm formation was identified on RBCs with a UDMA/dimethacrylate matrix blend than on those including a BisGMA/TEGDMA matrix blend but analogous filler fractions. Although significant differences in surface properties between the various materials were identified, correlations between the surface properties and biofilm formation were poor, which highlights the relevance of surface topography and chemistry. These results may help to tailor novel RBC formulations which feature reduced biofilm formation on their surface.

Surface properties of resin-based composite materials and biofilm formation: A review of the current literature.

PURPOSE: To evaluate the state of art on the relations between surface properties (surface roughness, topography, surface free energy and chemistry) of resin-based composite materials and microbial adhesion and biofilm formation. METHODS: An electronic search using Scopus and PubMed (until May 2015) was conducted applying the following search items: "Plaque OR Biofilm AND Surface chemistry", "Plaque OR Biofilm AND Surface-free energy", "Plaque OR Biofilm AND Roughness", "Surface characteristics AND Composites", "Biofilm AND Surface characteristics". RESULTS: Surface properties of resin-based composite materials as well as surface treatments can strongly affect bacterial adhesion and biofilm formation, although the "ideal" surface features have not been identified yet. Moreover, investigations highlighted that cariogenic biofilm formation may alter materials' surface properties, thus encouraging bacterial adhesion and biofilm formation, starting a "vicious cycle" which might compromise restoration longevity.

The influence of antibacterial toothpastes on in vitro Streptococcus mutans biofilm formation: a continuous culture study.

PURPOSE: To evaluate the in vitro effect of five toothpastes containing antimicrobial compounds including fluoride, triclosan or hydroxyapatite nano-particles on Streptococcus mutans (S. mutans) biofilm formation. Fluoride uptake by enamel after bacterial challenge was also evaluated. METHODS: Human enamel disks (n= 192) were randomly divided into six groups and brushed with five different toothpastes while the control group was brushed with distilled water. Each group was incubated for 24 and 72 hours with a S. mutans biofilm growing on a modified drip-flow reactor (MDFR). Biofilm formation was determined using a viable biomass assay based on a tetrazolium salt (MTT) and evaluated morphologically with confocal laser-scanning microscopy (CLSM) and scanning electron-microscopy (SEM). Fluoride uptake was evaluated using the enamel biopsy technique. Biofilm formation was also evaluated using 120 disks randomly divided into the same six groups. The number of viable bacteria was determined through plate count on Mitis Salivarius Bacitracin agar (MSB agar). RESULTS: Data from plate count showed the same overall trend of MTT assay. The latter showed that after 24 hours the effect of the tested toothpastes was significantly higher in reducing biofilm formation than after 72 hours. The toothpaste containing a high concentration of amine fluoride (AmF) had the highest performance in reducing biofilm formation. Fluoride uptake of enamel showed a positive trend related to the fluoride concentration in both incubation times.

Streptococcus mutans biofilm formation and release of fluoride from experimental resin-based composites depending on surface treatment and S-PRG filler particle fraction.

PURPOSE: To evaluate fluoride release and biofilm formation on resin-based composites (RBCs) including surface pre-reacted glass ionomer (S-PRG) filler particles. MATERIALS AND METHODS: Specimens were prepared from experimental RBCs including different fractions of S-PRG fillers (0/10/30/50/70% w/v). RBCs were light cured against mylar strips (MYL), and 50% of the specimens were additionally polished to a high gloss (POL). Surface roughness (SR), surface free energy (SFE) and fluoride release were determined. Streptococcus mutans biofilm formation (SMBF) was simulated for 48 h and 120 h; adherent viable biomass was assessed using an MTT-based assay. RESULTS: The highest SR was identified for POL specimens manufactured from the RBC with a filler fraction of 70%. For all specimens and surface treatments, polishing caused an increase in surface free energy. For both MYL and POL specimens, increasing the filler fraction coincided with an increased release of fluoride; a higher release of fluoride was identified for POL specimens with filler fractions of 50% and 70% in comparison to their MYL counterparts. Release of fluoride was lower after 120 h than after 48 h. No differences in SMBF were identified between MYL and POL specimens with identical filler fractions after 48 h of biofilm formation; with increasing filler fractions, a tendency towards decreasing SMBF was observed. After 120 h, less SMBF was identified for POL specimens with filler fractions of 30%, 50% and 70% in comparison to corresponding MYL specimens. CONCLUSION: The inclusion of S-PRG fillers and an effective surface treatment may reduce biofilm formation on RBCs.