Fig 1.
Structure of cranberry-derived flavonoids for combination therapy.
Myricetin, one of the most active cranberry flavonols, is characterized by the presence of an unsaturated double bond between C2 and C3 and three hydroxyl-groups in the B ring. Proanthocyanidins (PACs) in cranberry are predominantly found in oligomeric forms (up to 13 monomeric units) with at least one A-type double interflavan linkage [epicatechin-(4β→8, 2β→O→7)-epicatechin] between the lower or lowest two units of the oligomer. The degree-of-polymerization (DP) is variable depending on the number of monomeric units; DP4 (tetramer) and DP9 (nonamer) are among the most abundant and bioactive cranberry PAC.
Fig 2.
Experimental design for topical treatment regimen.
Mixed-species cariogenic biofilm model and treatment regimen of selected combination of cranberry flavonoids (CranFlav). CranFlav or vehicle was topically applied twice daily with 5 min exposure.
Fig 3.
Three-dimensional (3D) architecture, EPS content and microbiological composition of mixed-species biofilms treated with CranFlav.
(A) Representative 3D rendered images of mixed-species biofilms following topical treatments. Selected areas in panel (A) show detailed views of merged confocal images of bacterial cells (green) and EPS (red). (B) COMSTAT analysis of bacterial cells and EPS distribution across biofilm thickness (from the disc surface to the fluid phase interface). (C) Quantitative analysis of confocal images using COMSTAT software for determination of total, bacteria and EPS biomass (as well as EPS/bacteria ratio). The asterisks indicate that total biovolume, EPS biovolume and EPS/bacteria ratio of biofilm treated with CranFlav are significantly different from vehicle-control. A pairwise comparison between vehicle-control and CranFlav was conducted using a t-test. Values are significantly different from each other at *p<0.01 or **p<0.001. (D) Biochemical quantification of water-soluble EPS and insoluble EPS via colorimetric (phenol-sulfuric) assay (n = 6). A pairwise comparison between vehicle and CranFlav was conducted using a t-test. Values are significantly different from each other at **p<0.001. (E) Dynamics of microbial populations changes of mixed-species biofilms as determined by viable cell (colony forming units; CFU) counting (n = 6). A pairwise comparison between S. oralis and S. mutans was conducted. Values are significantly different from each other at **p<0.001.
Fig 4.
Mechanical stability and in situ pH within intact mixed-species biofilms treated with CranFlav.
(A) The amount of remained biofilm dry-weight before and after application of shear stress was measured to determine the mechanical stability and attachment strength of CranFlav (or vehicle)-treated biofilms on sHA surface. We also determined biofilm removal at 0.184 and 0.449 N/m2 (vs. no shear, 0 N/m2) (n = 8). (B) In situ biofilm pH values were determined every 2 μm from the sHA surface, and averaged based on pH measurements across the biofilm interface (n = 3). The molar concentration of hydrogen ion (H+) at the biofilm/sHA interface (10–15 μm from the sHA surface) was estimated via the equation: [H+] = 10-in situ pH. Values are significantly different from each other at *p<0.01 or **p<0.001.