Background/Aims: The antimicrobial resistance of microorganisms in biofilms and the polymicrobial interactions in these biofilms that modulate resistance require novel strategies to evaluate the efficacy of caries-preventive compounds. The current study aimed to evaluate the effects of a caries-preventive agent in Streptococcus mutans and polymicrobial biofilms. Methods: We developed a novel high-throughput active attachment model. The model consisted of a custom-designed lid containing glass discs that fit on top of standard 24-well plates. Biofilms were formed using either S. mutans C180-2 or saliva. At the end of biofilm formation (up to 96 h) the biofilms were treated with amine fluoride (AmF) solutions. The viability of the biofilms was determined by CFU counts, and metabolic activity was measured via lactate production. Results: The effect of AmF on the viability of the polymicrobial biofilms was significantly less than that on the S. mutans biofilms, indicating a higher resistance in the complex biofilms. Both types of biofilms became more resistant to AmF with age. The higher resistance of the polymicrobial biofilms was not reflected in metabolic activity; in dose-response experiments AmF reduced lactate production in both types of biofilms to the same extent. Moreover, the age-induced increased resistance in the polymicrobial biofilms was less pronounced in terms of the inhibition of metabolic activity. Conclusions: This study clearly shows that when evaluating the efficacy of caries-preventive compounds it is essential to use appropriate polymicrobial biofilm models, and more importantly that efficacy needs to be judged based on the reduction of acid formation (i.e. cariogenic potential) as well as on bacterial viability.

Galla chinensis extract (GCE) interferes with de- and remineralization of dental enamel and the growth and metabolism in planktonic bacteria. However, no information is available on GCE effects on biofilms formed with saliva as inoculum. The aim of the current experiments was to investigate the effects of GCE at different stages of salivary microcosm biofilm formation. Biofilms formed on glass or enamel surfaces were treated with GCE solutions at different concentrations and at different time points. Effects were assessed by lactic acid formation and colony-forming unit (CFU) counts of the biofilms. The results showed that GCE treatments inhibited growth and acid metabolism of both nascent and mature microcosm biofilms. Pretreatment of the substratum with GCE solutions inhibited growth and lactic acid production of biofilms grown on enamel, but had little effects on biofilms formed on glass surfaces. A maximum GCE effect was found when biofilms, on either surface type, were treated after 8 h of formation with 40 h of subsequent growth. In medium with sucrose-fermenting biofilms, low concentrations of GCE (0.2 and 0.1 mg/ml) inhibited acid production without killing bacteria of the biofilm. Differences were found in GCE effects on biofilms formed with saliva from different donors, with reductions in acid formation and CFU values ranging between 0 and 78%. In conclusion, bioactive components in GCE reduce or inhibit both growth and lactic acid formation in biofilms.

Quantitative laser fluorescence has been reported as a useful method for the non-destructive in vitro and in vivo diagnosis of early enamel caries. A portable system for intraoral use has been developed with a new light source and filter system replacing the laser light to facilitate clinical application. This new device was validated with microradiographic and chemical analyses for assessment of mineral changes in enamel during lesion formation and remineralization in vitro and compared with the laser light equipment. A significant correlation was found between fluorescence changes and mineral loss: r = 0.79 (laser system) and r = 0.84 (portable lamp system). The correlation between the two fluorescence methods was r = 0.93. The portable fluorescence device seemed to be a promising new tool for reproducible and sensitive assessment of the severity of incipient enamel lesions.

Trimetaphosphate (TMP) effects on demineralized bovine enamel were studied after 15 days of pH cycling. Treatments included 30 wt% (weight percent) dilutions of 0, 500, 1,500 or 3,000 µg F/g aqueous NaF solutions with or without 3% TMP. Treated specimens were assessed by transverse microradiography. With the exception of the 3,000 µg F/g case, 3% TMP addition provided significant additional overall remineralization compared with F alone. Mineral content profiles differed significantly between corresponding F and F + TMP groups. Fluoride alone resulted in more remineralization in the original demineralized zone, whereas F + TMP caused less demineralization in the underlying, originally sound enamel.

Commensal oral biofilms, defined by the absence of pathology-related phenotypes, are ubiquitously present. In contrast to pathological biofilms commensal biofilms are rarely studied. Here, the effect of the initial inoculum and subsequent growth conditions on in vitro oral biofilms was studied. Biofilms were inoculated with saliva and grown anaerobically for up to 21 days in McBain medium with or without fetal calf serum (FCS) or sucrose. Pathology-related phenotypes were quantified and the community composition was determined. Biofilms inoculated with pooled saliva or individual inocula were similar. Denaturing gradient gel electrophoresis (DGGE) analysis allowed differentiation of biofilms grown with sucrose, but not with FCS. Lactate production by biofilms was significantly increased by sucrose and protease activity by FCS. McBain grown biofilms showed low activity for both phenotypes. Three clinically relevant in vitro biofilm models were developed and could be differentiated based on pathology-related phenotypes but not DGGE analysis. These models allow analysis of health-to-disease shifts and the effectiveness of prevention measures.

Caries prevention might benefit from the use of toothpastes containing over 1500 ppm F. With few clinical studies available, the aim of this pH-cycling study was to investigate the dose response between 0 and 5000 ppm F of de- and remineralization of advanced (> 150 microm) enamel lesions. Treatments included sodium and amine fluoride, and a fluoride-free control. Mineral uptake and loss were assessed from solution calcium changes and microradiographs. Treatments with 5000 ppm F both significantly enhanced remineralization and inhibited demineralization when compared with treatments with 1500 ppm F. Slight differences in favor of amine fluoride over sodium fluoride were observed. The ratio of de- over remineralization rates decreased from 13.8 to 2.1 in the range 0 to 5000 ppm F. As much as 71 (6)% of the remineralized mineral was calculated to be resistant to dissolution during subsequent demineralization periods. With 5000-ppm-F treatments, more demineralizing episodes per day (10 vs. 2 for placebo) would still be repaired by remineralization.

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This in vitro study aimed at investigating whether full remineralization would occur in white spot lesions when the surface porosity was increased by acid-etching. The effect of fluoride was also investigated. Enamel blocks with in vitro produced white spot lesions were used. Group A was exposed to a remineralizing solution only. In group B, the lesions were etched with 35% phosphoric acid for 30 s, then treated as in group A. Group C was treated as group A + daily treatment with a fluoride toothpaste slurry (1,000 ppm) for 5 min. Group D was treated as group B + the daily fluoride treatment of group C. The remineralization was measured weekly with Quantitative Light-induced Fluorescence during the experimental period. After 10 weeks of remineralization, mineral profiles were assessed with transverse microradiography. The enamel fluorescence was partly regained. There were significant differences in the lesion depth, mineral content at the surface layer, and integrated mineral loss between the groups. Addition of fluoride accelerated the remineralization only in the beginning; in later stages the process leveled out and even reached a plateau in all the groups. It was concluded that full remineralization was not achieved by etching, by the addition of fluoride, nor by the combination of both treatments in this in vitro study.