Anti-biofilm and anti-metabolic effects of antimicrobial photodynamic therapy using chlorophyllin-phycocyanin mixture against Streptococcus mutans in experimental biofilm caries model on enamel slabs.

Abstract

Partial (selective) removal of dental caries is a suitable manner to treat deep carious lesions in vital teeth with asymptomatic pulps. Antimicrobial photodynamic therapy (aPDT) was proposed as a promising ancillary approach for reduction of the residual bacteria from the cavity. Therefore, the focus of this study was to investigate the influence of aPDT using diode laser (DL) plus PhotoActive+ (chlorophyllin-phycocyanin mixture [CHL-PC]) as photosensitizer (PS) on metabolic activity and the reduction in the number of living bacteria within the preformed biofilm caries model on enamel slabs of Streptococcus mutans. The lethal and sub-significant inhibitory (SSI) potential of aPDT using CHL-PC and 635 nm DL against experimental biofilm caries model on enamel slabs and metabolic activity of S. mutans was analyzed using crystal violet and XTT reduction assays, respectively. Intracellular ROS formation by DCFH-DA assay was measured in CHL-PC mediated aPDT treated bacterial samples. Tooth discoloration and cell cytotoxicity of CHL-PC were assessed in the CIEL*a*b* color space and neutral red assay, respectively. In this study aPDT at a maximum concentration level of CHL-PC (5000 μg/mL) with 3 min DL irradiation time (103.12 J/cm2) reduced the ex-vivo cariogenic biofilm of S. mutans by 36.93 % (P < 0.05). Although chlorhexidine (CHX) had an anti-biofilm effect about 1.7 fold compared to CHL-PC mediated aPDT, this difference was not significant (36.93 in comparison to 63.05 %; P > 0.05). CHL-PC mediated aPDT demonstrated a significant reduction in bacterial metabolic activity, with rates of 77 % at a SSI dose (using 156 μg/mL of CHL-PC and 3 min DL irradiation time with the energy density of 103.12 J/cm2). The treated bacterial cells exhibited significant (P < 0.05) increment in the ROS generation. The least color change (ΔE) was found using CHL-PC at a concentration of 156 μg/mL (ΔE = 2.74). CHL-PC in different concentrations showed no significant reduction in human gingival fibroblasts (HGFs) cell survival (P > 0.05). CHL-PC mediated aPDT not only reduces the number of living bacteria within the biofilms of S. mutans in an experimental biofilm caries model on enamel slabs but also its influences microbial virulence by reducing the metabolic activity of the S. mutants.