Cellular-extracellular biofilm components on resin composites after treatment with mouthwashes

Objectives: To compare the 3-dimensional structures of selected cellular and extracellular components of Streptococcus mutans biofilms grown on resin-composites after treatment with six mouthwashes.  Methods: Specimens of Point 4 (Kerr Corp.) and TPH3 (Dentsply/Caulk) resin-composites were sequentially wet-polished using 180-1200grit SiC-disks, and 1.00µm and 0.50µm polycrystalline-diamond suspensions (final dimensions: d=6mm, h=3mm), and sterilized (ethylene oxide gas). Streptococcus mutans (strain UA159) biofilms were grown microaerophilically on specimens (batch culture, 0.5X Tryptone-yeast extract medium with 10mM sucrose, 37°C, 100rpm; media replenished 24h). After 48h of bacterial growth, treatment group specimens (n=5/group) were immersed for 60s in Biotene (BIO) or Listerine Total Care (LTO), or for 30s in Crest Pro-Health (CPH), Listerine Antiseptic (LIS), Listerine Advanced (LTC) or Plax (PLX) on a shaker (150rpm), then washed (5x; sterile, ultrapure water; 150rpm). Untreated specimens immersed in water for 60s or 30s constituted control groups C60 (n=10) and C30 (n=20), respectively. Biofilms were stained [Concanavalin A-Alexa Fluor 647 conjugate (AF; extracellular polymeric-substances/EPS), Sypro Red (SR; proteins), Syto 9 (SY; nucleic-acids); Invitrogen], then imaged using a confocal laser scanning microscope (CLSM; Leica TCS SP2-MP) at three comparable locations/specimen. Biofilm structural parameters calculated using ISA3D software were: biovolume (BV; μm³), fractal dimension (FD), and mean biofilm thickness (MT; μm). Separate mixed models statistical analyses compared mean values of structural parameters between mouthwashes and corresponding control groups (α=0.05).  Results: Mouthwashes that differed significantly from controls (p<0.05) have highlighted p-values in the table below. Data for CPH and LTC are not shown because there were no differences from the C30 group (p>0.05).

Conclusions: CLSM scans of 48-hour S. mutans biofilms grown on two resin-composites demonstrated significant differences in the abilities of the mouthwashes tested to remove selected cellular/extracellular biofilm components. BIO removed more extracellular polymeric-substances, proteins and nucleic-acids in biofilms grown on both resin-composites than the other mouthwashes.