Andrei C. Ionescu

Influence of surface properties of resin-based composites on in vitro Streptococcus mutans biofilm development

The aim of this in vitro study was to evaluate the influence of physicochemical surface properties of resin-based composites on Streptococcus mutans biofilm formation. Specimens were prepared from each of four resin-based composites by polymerization against Mylar strips. Half of the number of specimens received no further surface treatment, whereas the other half were subjected to a polishing treatment. Surface roughness (SR) and topography were assessed using profilometry and atomic force microscopy. Surface free-energy (SFE) was determined, and the chemical surface composition was analysed by X-ray photoelectron spectroscopy (XPS). S. mutans biofilms were formed on the surface of the resin-based composite specimens for either 48 or 96 h using an artificial mouth system (AMS). Polishing caused a significant decrease in SFE, and XPS analysis indicated an increase of surface silicon and a decrease of surface carbon. Only for Grandio was a significant increase in SR identified after polishing, which was probably related to the higher concentration of filler particles on its surface. Significantly less S. mutans biofilm formation was observed on polished resin-based composites than on unpolished resin-based composites. These results indicate that the proportions of resin matrix and filler particles on the surface of resin-based composites strongly influence S. mutans biofilm formation in vitro, suggesting that minimization of resin matrix exposure might be useful to reduce biofilm formation on the surface of resin-based composites.

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.

Biofilm formation on composite resins for dental restorations: an in situ study on the effect of chlorhexidine mouthrinses.

Purpose Biofilm formation on the surface of dental restorative materials by oral bacteria is considered an important step in the development of secondary caries. The aim of this study was to evaluate the in situ effect of a chlorhexidine (CHX)-containing mouthrinse on the biofilm formation occurring on the surface of human enamel and of two resin-based commercially available materials: a silorane-based material (Filtek Silorane®) and a methacrylate-based material (Filtek Supreme XT®). Methods 53 disks were obtained for each of the two composites and 37 disks for enamel. The surface was characterized by determining the surface roughness and the surface free energy of 5 samples for each of the three materials tested, then the remaining samples were mounted on splints worn by 16 volunteers. The participants were randomly divided into two groups: an experimental group that used 0.12% CHX-based mouthrinse and a control group that used a placebo mouthrinse. Biofilm formation on the different surfaces after a 24 h period was assessed using MTT assay. Results The two composites in the group treated with the placebo mouthrinse showed a similar biofilm formation, which was significantly higher than that occurring on enamel surfaces. The CHX-based mouthrinse significantly reduced biofilm formation on the surfaces of the two resin-based materials when compared with the placebo mouthrinse. The reduction was particularly relevant on the Filtek Silorane surfaces. Conclusions The new silorane-based material seems to interact with CHX in a promising way from the point of view of biofilm formation control.

Hydrophilicity of dentin bonding systems influences in vitro Streptococcus mutans biofilm formation

OBJECTIVE To evaluate in vitro Streptococcus mutans (S. mutans) biofilm formation on the surface of five light-curing experimental dental bonding systems (DBS) with increasing hydrophilicity. The null hypothesis tested was that resin chemical composition and hydrophilicity does not affect S. mutans biofilm formation. METHODS Five light-curing versions of experimental resin blends with increasing hydrophilicity were investigated (R1, R2, R3, R4 and R5). R1 and R2 contained ethoxylated BisGMA/TEGDMA or BisGMA/TEGDMA, respectively, and were very hydrophobic, were representative of pit-and-fissure bonding agents. R3 was representative of a typical two-step etch-and-rinse adhesive, while R4 and R5 were very hydrophilic resins analogous to self-etching adhesives. Twenty-eight disks were prepared for each resin blend. After a 24h-incubation at 37°C, a multilayer monospecific biofilm of S. mutans was obtained on the surface of each disk. The adherent biomass was determined using the MTT assay and evaluated morphologically with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). RESULTS R2 and R3 surfaces showed the highest biofilm formation while R1 and R4 showed a similar intermediate biofilm formation. R5 was more hydrophilic and acidic and was significantly less colonized than all the other resins. A significant quadratic relationship between biofilm formation and hydrophilicity of the resin blends was found. CLSM and SEM evaluation confirmed MTT assay results. CONCLUSIONS The null hypothesis was rejected since S. mutans biofilm formation was influenced by hydrophilicity, surface acidity and chemical composition of the experimental resins. Further studies using a bioreactor are needed to confirm the results and clarify the role of the single factors.

Influence of MDPB-containing Primer on Streptococcus Mutans Biofilm Formation in Simulated Class I Restorations

PURPOSE To evaluate the activity of a methacryloyloxydodecylpyridinium bromide (MDPB)-containing self-etching primer (Clearfil Protect Bond) against Streptococcus mutans and its ability to reduce biofilm formation on standardized experimental Class I restorations in vitro. MATERIALS AND METHODS Forty experimental Class I round restorations were prepared on enamel-dentin slabs using different adhesive strategies: group 1 = MDPB-containing adhesive system (Clearfil Protect Bond); group 2 = MDPB-free self-etching adhesive system (Clearfil SE Bond); group 3: MDPB-containing self-etching primer in combination with a fluoride-free bonding agent; group 4: MDPB-free self-etching primer in combination with a fluoride-containing bonding agent; group 5: a three-step etch-and-rinse adhesive system (Adper Scotchbond Multi Purpose). A Streptococcus mutans biofilm was grown for 48 h on the restoration surfaces and subsequently evaluated using scanning electron microscopy on three different areas: enamel, composite, and interface surfaces. Statistical analysis was performed by multiple ANOVA after data transformation. RESULTS Specimens in groups 2, 4 and 5 showed greater biofilm formation than those in groups 1 and 3 (p < 0.001) on all investigated substrates (enamel, composite, and interface areas). CONCLUSIONS Specimens prepared with an MDPB-containing primer exhibited significant decreases in biofilm formation on Class I restorations in vitro. Further in vitro and in vivo studies are required to clarify the role of quaternary ammonium compounds in reducing bacterial biofilm formation on restoration surfaces.

Silver–polysaccharide antimicrobial nanocomposite coating for methacrylic surfaces reduces Streptococcus mutans biofilm formation in vitro

OBJECTIVES The aim of this study was to determine the in vitro microbiological performances of a lactose-modified chitosan (Chitlac) coating inside which silver nanoparticles were embedded (Chitlac-nAg) for BisGMA/TEGDMA methacrylic specimens. METHODS Different concentrations of nAg inside Chitlac coating were tested (1 mM, 2 mM, 5 mM). Specimen surface was analyzed by means of field-emission scanning electron microscopy (FEISEM) and energy-dispersive X-ray spectroscopy (EDS). A 48 h monospecific Streptococcus mutans biofilm was developed over the specimen surfaces using a modified drip-flow bioreactor; adherent viable biomass was assessed by MTT test and biofilm was imaged by confocal laser-scanning microscopy (CLSM). RESULTS The presence of finely dispersed nanoparticles inside the Chitlac coating was confirmed by FEISEM and EDS analysis. All nanoparticles were embedded in the Chitlac coating layer. Chitlac-nAg coatings were able to significantly decrease biofilm formation depending on the nAg concentration, reaching a -80% viable biomass decrease when the 5 mM nAg-Chitlac group was confronted to non-coated control specimens. CLSM analysis did not provide evidence of a contact-killing activity, however the antibacterial Chitlac-nAg coating was able to alter biofilm morphology preventing the development of mature biofilm structures. CONCLUSIONS The microbiological model applied in this study helped in assessing the antibacterial properties of a coating designed for methacrylate surfaces. CLINICAL SIGNIFICANCE A microbiological model based on a bioreactor-grown biofilm is useful for preliminary in vitro tests of dental materials. In translational terms, an antibacterial nanocomposite coating based on Chitlac-nAg and designed to be applied to methacrylic surfaces may be a promising way to obtain dental materials able to actively prevent secondary caries.

Streptococcus mutans and Streptococcus sobrinus biofilm formation and metabolic activity on dental materials.

Abstract Objectives. To examine potential correlations between streptococcal biofilm formation and lactate production in streptococcal biofilms formed on the surface of dental materials with different surface characteristics. Materials and methods. Samples of a glass-ionomer cement (Ketac Molar) and a ceramic (Empress 2) were incubated with whole saliva and suspensions of Streptococcus mutans ATCC 25175 or Streptococcus sobrinus ATCC 33478 for initiating single-species biofilm formation for either 4 or 24 h. The relative amount of adherent, viable cells was determined using a Resazurin and a MTT assay. Metabolic activity was assessed by quantifying lactate production with a modification of the commercial Clinpro Cario L-Pop kit. Results. Both assays identified similar S. sobrinus biofilm formation on the two substrata; for S. mutans, the MTT test showed significantly fewer streptococci on the glass-ionomer cement than on the ceramic. Concerning metabolic activity, for S. sobrinus, significantly higher lactate production was observed for biofilms formed on the glass-ionomer cement in comparison to the ceramic, whereas similar values were identified for S. mutans. Conclusions. Within the limitations of the study, the results suggest that the pure amount of adherent streptococci does not a priori indicate the metabolic activity of the cariogenic bacteria organized in the respective biofilm. Thus, comparisons between the relative amount of adherent streptococci and their metabolic activity may allow for an improved understanding of the effect of dental material surfaces on the formation and metabolic activity of streptococcal biofilms.

Bioceramic Materials Show Reduced Pathological Biofilm Formation

The aim of the present work was to assess the surface ability of three bioceramic materials (A: alumina BIOLOX®forte; B: Si3N4; C: alumina matrix composite BIOLOX®delta) to inhibit bacterial biofilm formation. For this purpose, ceramic disks at standardized roughness (Ra = 0,25 μm) were used as test materials while commercial polystyrene was considered as control. Two biofilm-producing bacterial strains (S. epidermidis ATCC14990, Escherichia coli ATCC25922) were used for experiments. The viable biomass was assessed by the metabolic MTT assay after 24h incubation. Morphological data regarding biofilms structure were obtained by scanning electron microscopy. In general, results revealed that all bioceramics materials were significantly less colonized compared to polystyrene. The degree of biofilm formation onto bioceramics ranged between about 30 to 60% less than the polystyrene control. Moreover, some differences were noticed by comparing the three bioceramics inhibition ratio: bioceramic A showed significanlty less S. epidermidis biofilm formation (p<0.005) compared to B and C that showed similar performance. Conversely, no difference were noted for E. coli biofilm amount for A, B and C. In conclusion, the tested materials showed capability to reduce biofilm formation to a different extent depending on the tested bacterial strains.

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.