Engineered Salivary Peptides Reduce Enamel Demineralization in Cariogenic Streptococcus mutans Biofilm

Objectives: Dental biofilm, the necessary factor responsible for caries, is formed on dental surfaces covered by a layer of selective salivary proteins and peptides named acquired enamel pellicle (AEP). The AEP may modulate enamel demineralization provoked by bacteria biofilm sugar fermentation. It was previously determined that the AEP engineered peptides DR9-DR9 and DR9-RR14 were able to reduce enamel demineralization and displayed antimicrobial activity, respectively, but under experimental conditions not mimicking caries development induced by biofilm and sucrose exposure, the most cariogenic dietary sugar. Here we tested the carious-protective effect of our engineered peptides when cariogenic Streptococcus mutans biofilms were grown for 144h on enamel slabs previously treated with statherin, histatin 3, DR9, DR9-DR9, DR9-RR14, RR14, fluoride (positive control), or NaCl (negative control), to form the AEP.
Methods: Biofilms were exposed to sucrose for 8h/day and let rest overnight in a culture medium without sucrose. The pH of the culture medium was determined 2x/day, before and after exposing the biofilms to sucrose, as an indicator of bacterial acidogenicity. The exchanged culture medium was collected daily to determine its calcium concentration (mM), as an indicator of enamel demineralization. In an additional experiment, biofilms were treated 2x/day with the peptides before and after exposure to sucrose. At 144h, biofilms were collected to determine the bacterial viability (CFU/biofilm) and biofilm mass (mg protein/biofilm). Intracellular and extracellular bacterial proteins were extracted and identified by mass spectrometry.
Results: We found that DR9-DR9 and DR9-RR14 significantly reduced enamel demineralization (p<0.0001). This effect was observed when the peptides were adsorbed onto the enamel surface forming the AEP or when the biofilms were additionally treated 2x/day with the engineered peptides. However, the peptides did not have a significant effect (p>0.05) on biofilm biomass nor modulate the expression of the intracellular and extracellular bacterial proteins involved in biofilm cariogenicity.
Conclusions: The findings suggest that the engineered DR9-DR9 and DR9-RR14 peptides may modulate caries development mainly by a physicochemical mechanism.