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.

To assess the cytotoxicity of an experimental hybrid-glass-based infiltrant and its effect on biofilm attachment, growth and metabolic activity, and to compare it to the resin-based infiltrant Icon. Cytotoxicity of hybrid-glass-based material (EXP) and resin-based infiltrant Icon (Icon) was tested in direct contact tests on freshly cured (direct_mat) and on materials kept for 24 h in cell culture medium (direct_exmat), and extract test with materials 24-h extracts (extract). Cell viability of L929 mouse fibroblast cell line was measured with MTT assay, according to ISO10993-5:2009. Biofilm attachment (5 h), growth (24 h and 48 h) and lactic-acid production (24 h and 48 h) on glass-disk specimens coated with EXP or Icon, or uncoated (control), were assessed using a microcosm biofilm model and Amsterdam Active Attachment system. At indicated time points, biofilms were harvested, plated, and CFU counts were determined, while lactic-acid production was measured colorimetrically. Cell viability reduction by EXP was below 30%-threshold in direct contact tests, while in extract test an increased cell viability was observed. Icon reduced cell viability substantially in all three tests. Significantly less bacteria attached to the surface of EXP after 5 h compared to Icon and control. Biofilm growth was significantly lower on EXP than on Icon and control after 24 h, but this difference was smaller and statistically insignificant after 48 h. There was no difference in lactic-acid production among groups. Novel hybrid-glass-based infiltrant seems to have a better biocompatibility and accumulates on its surface less bacteria than resin-based infiltrant, which makes it an attractive resin-free alternative.

Microbiome modulation, aiming to restore a health-compatible microbiota, is a novel strategy to treat periodontitis. This study evaluated the modulation effects of antimicrobial peptide LL-31 and its D-enantiomer (D-LL-31) on saliva-derived microcosm biofilms, spiked with or without Porphyromonas gingivalis. To this end, one-day-old biofilms were incubated for 24 h with biofilm medium alone, or medium containing 40 µM LL-31 or D-LL-31, after which biofilms were grown for 5 days. Biofilms were assessed at 1 day and 5 days after intervention for the total viable cell counts, dipeptidyl peptidase IV (DPP4) activity, P. gingivalis amount (by qPCR) and microbial composition (by sequencing). The results showed that D-LL-31, not LL-31, significantly reduced the total viable cell counts, the P. gingivalis amount, and the DPP4 activity of the biofilms spiked with P. gingivalis, but only at 1 day after intervention. In the biofilms spiked with P. gingivalis, D-LL-31 tended to reduce the α-diversity and the compositional shift of the biofilms in time as compared to the control and LL-31 groups. In conclusion, D-LL-31 showed a better performance than LL-31 in biofilm modulation. The biofilm modulation function of the peptides could be impaired when the biofilms were in a severely dysbiotic state.

This study compared the effect of topically applied fluoride products on dentine lesions in an in vitro experiment. Demineralized bovine dentine specimens were treated once with either SDF solution (35,400 ppm F), NaF varnish (22,600 ppm F), TiF4 solution (9,200 ppm F), SnF2 gel (1,000 ppm F), no treatment (control), or preserved as baseline lesions. After the application and subsequent removal of the fluoride products, the specimens were subjected to pH-cycling. Calcium loss and uptake in the de- and remineralization buffers were assessed daily. Fluoride release into the buffers was analyzed on days 1, 2, 3, 5, 8, and 13. After the pH-cycling period, mineral distribution throughout the lesion depth was analyzed using transversal microradiography (TMR). X-ray energy-dispersive spectroscopy (EDS) examined the deposition of silver, titanium, and tin after application of SDF, TiF4, and SnF2, respectively. Overall, calcium loss and uptake analysis in the de- and remineralization buffers revealed that the SDF product was the most effective in inhibiting lesion progression, followed by the TiF4, NaF, and SnF2 products. Fluoride analysis disclosed a steep reduction of the amount of fluoride released into de- and remineralization buffers with time. The fluoride effects on de- and remineralization continued beyond the days that fluoride was released into the buffers. TMR analysis showed significant remineralization in the outer zone of the dentine lesions for all fluoride products, with SDF giving hypermineralization in this zone. In the inner zone, lesions developed in all fluoride groups, with the smallest in the SDF group. EDS showed silver and titanium deposition in depth up to 85 μm and 8 μm, respectively, while no tin deposition was observed. The silver in the dentine lesions did not contribute significantly to the density of the TMR profiles in the SDF group. In conclusion, all topical fluoride products protected the dentine lesions against lesion progression, but at different degrees. SDF showed a superior effect in protection against further demineralization and enhancement of remineralization. This was probably attributed to its fluoride concentration that was the highest among the fluoride products.