Determinants of Microscale pH in In Situ-Grown Dental Biofilms.

Dental biofilm pH is the most important determinant of virulence for the development of caries lesions. Confocal microscopy-based pH ratiometry allows monitoring biofilm pH with high spatial resolution. Experiments performed on simplified biofilm models under static conditions identified steep pH gradients as well as localized acidogenic foci that promote enamel demineralization. The present work used pH ratiometry to perform a comprehensive analysis of the effect of whole saliva flow on the microscale pH in complex, in situ-grown 48-h and 96-h biofilms (n = 54) from 9 healthy participants. pH was monitored in 12 areas at the biofilm bottom and top, and saliva flow with film thicknesses corresponding to those in the oral cavity was provided by an additively manufactured microfluidic flow cell. Biofilm pH was correlated to the bacterial composition, as determined by 16S rRNA gene sequencing. Biofilm acidogenicity varied considerably between participants and individual biofilms but also between different areas inside one biofilm, with pH gradients of up to 2 units. pH drops were more pronounced in 96-h than in 48-h biofilms (P = 0.0121) and virtually unaffected by unstimulated saliva flow (0.8 mm/min). Stimulated flow (8 mm/min) raised average biofilm pH to near-neutral values but it did not equilibrate vertical and horizontal pH gradients in the biofilms. pH was significantly lower at the biofilm base than at the top (P < 0.0001) and lower downstream than upstream (P = 0.0046), due to an accumulation of acids along the flow path. pH drops were positively correlated with biofilm thickness and negatively with the thickness of the saliva film covering the biofilm. Bacterial community composition was significantly different between biofilms with strong and weak pH responses but not their species richness. The present experimental study demonstrates that stimulated saliva flow, saliva film thickness, biofilm age, biofilm thickness, and bacterial composition are important modulators of microscale pH in dental biofilms.

Effect of photodynamic therapy in combination with various irrigation protocols on an endodontic multispecies biofilm ex vivo.

AIM: To analyse the antibacterial effect of photodynamic therapy (PDT) in combination with various irrigation protocols on a multispecies biofilm in root canals ex vivo. METHODOLOGY: A total of 160 extracted human single-rooted teeth were divided into four groups (n = 40). In group G1, root canals were instrumented up to size 60 (control group), whereas in G2 to G4 canals were enlarged up to size 40. All root canals were inoculated with a multispecies biofilm (Enterococcus faecalis, Streptococcus oralis, Prevotella intermedia) for 5 days. In G2 to G4, instrumentation up to size 60 was performed using 0.9% sodium chloride (NaCl) (G2), 1% sodium hypochlorite (NaOCl) (G3), 1% NaOCl and a final irrigation with 2% chlorhexidine (CHX) (G4), respectively. In all groups half of the specimens received adjunctive PDT using phenothiazine chloride as photosensitizer and a diode laser (wavelength 660 nm). Counts of colony-forming units (CFUs) in each group were analysed separately for planktonic and dentine-adherent bacteria immediately after therapy (T1) and after 5 days of further incubation (T2). Descriptive statistics and two-way analysis of variance were carried out to analyse reduction of planktonic bacteria and nonparametric tests were used to analyse dentine-adherent bacteria. RESULTS: CFU reduction in planktonic bacteria was significantly affected by the irrigation protocol at T1 and T2 (P < 0.0001), but PDT significantly reduced CFUs only at T2 (P = 0.01; anova). Irrigation using NaOCl, CHX and adjunctive PDT significantly reduced CFUs at T2 (P < 0.0001; Tukey HSD) compared to the control group. In 85.6% of all samples the same categories of CFU counts in both planktonic and dentine-adherent bacteria were detected at T1 and T2. CONCLUSIONS: Adjunctive photodynamic therapy in combination with an irrigation protocol including NaOCl and CHX was an effective method for reduction of bacterial biofilm inside the root canals of extracted teeth.

Osteopontin adsorption to Gram-positive cells reduces adhesion forces and attachment to surfaces under flow.

The bovine milk protein osteopontin (OPN) may be an efficient means to prevent bacterial adhesion to dental tissues and control biofilm formation. This study sought to determine to what extent OPN impacts adhesion forces and surface attachment of different bacterial strains involved in dental caries or medical device-related infections. It further investigated if OPN's effect on adhesion is caused by blocking the accessibility of glycoconjugates on bacterial surfaces. Bacterial adhesion was determined in a shear-controlled flow cell system in the presence of different concentrations of OPN, and interaction forces of single bacteria were quantified using single-cell force spectroscopy before and after OPN exposure. Moreover, the study investigated OPN's effect on the accessibility of cell surface glycoconjugates through fluorescence lectin-binding analysis. OPN strongly affected bacterial adhesion in a dose-dependent manner for all investigated species (Actinomyces naeslundii, Actinomyces viscosus, Lactobacillus paracasei subsp. paracasei, Staphylococcus epidermidis, Streptococcus mitis, and Streptococcus oralis). Likewise, adhesion forces decreased after OPN treatment. No effect of OPN on the lectin-accessibility to glycoconjugates was found. OPN reduces the adhesion and adhesion force/energy of a variety of bacteria and has a potential therapeutic use for biofilm control. OPN acts upon bacterial adhesion without blocking cell surface glycoconjugates.

Infiltration of natural caries lesions in relation to their activity status and acid pretreatment in vitro.

This study aimed at testing how active and inactive enamel caries lesions differ by their degree of resin infiltration, and whether the choice of acid pretreatment plays a crucial role. Four examiners assessed 104 human molars and premolars with noncavitated enamel lesions and classified them as 'active' or 'inactive' using the Nyvad criteria. Forty-five teeth were included in this study after independent unanimous lesion activity assessment. Lesions were cut perpendicularly into 2 halves. Each half lesion was pretreated with either 15% hydrochloric acid or 35% phosphoric acid. The lesions were infiltrated after staining with rhodamine isothiocyanate. Thin sections of 100 µm were prepared and the specimens were bleached with 30% hydrogen peroxide. The specimens were then counterstained with sodium fluorescein, subjected to confocal laser scanning microscopy and analyzed quantitatively. Outcome parameters were maximum and average infiltration depths as well as relative penetration depths and areas. In active lesions no significant difference of percentage maximum penetration depth and percentage average penetration depth between lesions pretreated with hydrochloric or phosphoric acid could be observed. In inactive lesions, however, phosphoric acid pretreatment resulted in significantly lower penetration compared to hydrochloric acid pretreatment. Surface conditioning with hydrochloric acid led to similar infiltration results in active and inactive lesions. Moreover, inactive lesions showed greater variability in all assessed infiltration parameters than did active lesions. In conclusion, caries lesion activity and acid pretreatment both influenced the infiltration. The use of phosphoric acid to increase permeability of the surface layer of active lesions should be further explored.