Ex-vivo effects of propolis quantum dots-nisin-nanoquercetin-mediated photodynamic therapy on Streptococcus mutans biofilms and white spot lesions.

BACKGROUND: White spot lesions (WSLs) remain one of the most critical adverse sequelae of fixed orthodontic treatment, despite materials and techniques advances in orthodontics. WSLs seem to be a multi-factorial interaction including increased microbial plaque due to intrabuccal appliances that limit the oral-cleansing mechanism and change in the oral microbiome during fixed appliance wear. The aim of this study was to investigate the synergistic effect of propolis quantum dots (PQD), nisin (Nis), and quercetin nanoparticles (nQCT)-mediated photodynamic therapy (PQD-Nis-nQCT-mediated aPDT) in the eradication of Streptococcus mutans biofilms and the remineralization of WSLs ex-vivo. MATERIALS AND METHODS: The cytotoxicity of PQD-Nis-nQCT composite on human gingival fibroblasts was evaluated using neutral red. Intracellular reactive oxygen species (ROS) generation following PQD-Nis-nQCT-mediated aPDT was measured. Enamel slabs were prepared and demineralized using a demineralization solution containing S. mutans. Demineralized enamel slabs were divided into 9 groups (n = 10) and treated in the following groups: 1) Artificial saliva (negative control), 2) 2% neutral sodium fluoride gel (NSF; positive control or treatment control, 3) PQD, 4) Nis, 5) nQCT, 6) Nis-nQCT, 7) PQD-Nis-nQCT 8) Blue laser irradiation (light), 9) PQD-Nis-nQCT with irradiation (PQD-Nis-nQCT-mediated aPDT). Then, the surface changes, microhardness, and surface topography of the demineralized slabs were examined following each treatment using DIAGNOdent Pen reading, digital hardness tester, and SEM, respectively. After the determination of minimum biofilm eradication concentration (MBEC) of PQD, Nis, and nQCT by microtiter plate assay, the synergistic antimicrobial effects of PQD and Nis-nQCT were determined via evaluation of fractional biofilm eradication concentration (FBEC) index. The anti-biofilm effects of each treatment on S. mutans were assessed using a colorimetric assay. The virulence­associated gtfB gene expression was assessed following PQD-Nis-nQCT-mediated aPDT by quantitative real­time PCR. RESULTS: PQD-Nis-nQCT at 2048 µg/mL had no significant cell cytotoxicity on human gingival fibroblasts compared to the control group (P > 0.05). A significantly increased (7.6 fold) in intracellular ROS was observed following PQD-Nis-nQCT-mediated aPDT (13.9 ± 1.41) when compared to the control (1.83 ± 0.13). Following each treatment, the microhardness of the demineralized enamel surface significantly increased except for the artificial saliva (negative) and blue laser irradiation groups. The highest change in microhardness improvement was detected in the PQD-Nis-nQCT-mediated aPDT group (P < 0.05). Also, DIAGNODent Pen reading revealed the highest significant improved change in the level of mineralization degree in the PQD-Nis-nQCT-mediated aPDT group. Nis and blue light irradiation groups, like the artificial saliva-treated demineralized enamel slabs (control group), did not lead to remineralization (P > 0.05). Also, the PQD-Nis-nQCT-mediated aPDT treatment results obtained from SEM revealed that remineralization of demineralized enamel slabs in that group has significantly improved compared to the others. Light-activated nQCT, PQD, Nis-nQCT, and PQD-Nis-nQCT composite significantly reduced pre-formed biofilms of S. mutans compared with unactivated forms of test materials. The relative expression level of the virulence gtfB gene was significantly decreased (7.53-fold) in the presence of PQD-Nis-nQCT-mediated aPDT (P < 0.05). CONCLUSION: PQD-Nis-nQCT-mediated aPDT can be used for the eradication of S. mutans biofilms and remineralization of WSLs. The found in vitro efficacy should be tested further through clinical studies.

Effect of nanomicelle curcumin-based photodynamic therapy on the dynamics of white spot lesions and virulence of Streptococcus mutans in patients undergoing fixed orthodontic treatment: A randomized double-blind clinical trial.

BACKGROUND/PURPOSE: The formation of white spot lesions (WSLs) around fixed orthodontic appliances is a major complication during treatment. The current double-blind, randomized clinical trial (RCT) study aims to investigate the varying effects of nanomicelle curcumin-based photodynamic therapy (NMCur-aPDT) on microbial count and virulence of Streptococcus mutans as well as the number and dynamics of WSLs. MATERIALS AND METHODS: Double-blind prospective RCT, comprised of 48 patients with fixed orthodontic appliances, were recruited for the current study. The patients were divided into four groups according to the type of the treatment (NMCur, LED, NMCur-aPDT or VITIS® anti-caries mouthwash), using block randomization. Antimicrobial and anti-virulence activities of the treatments against isolated S. mutans were assessed via colony counting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The visual inspection using the International Caries Detection and Assessment System (ICDAS II) score and laser fluorescence (LF) detection using a DIAGNOdent device were used for the detection and assessment of the dynamics of WSLs, respectively, on the labial surface in four areas (i.e., gingival, incisal, mesial, and distal) of the upper and lower anterior teeth at 30-, 60-, 90-, and 120-days follow-up after bonding of the lower and upper arches. RESULTS: The antimicrobial properties of NMCur, VITIS®, and NMCur-aPDT were time-dependent so the highest reduction in S. mutans population was observed following NMCur-aPDT (99.98%) on day 120 of the study. The gtfB gene expression levels in S. mutans isolates from the NMCur-aPDT group on days 60, 90, and 120 decreased by 2.07-, 2.32-, and 3.01-fold more than in S. mutans isolates from the VITIS® group, respectively (all P < 0.05), while NMCur and LED treatments could not significantly reduce gtfB gene expression up to 120 days of follow-up (P > 0.05). In patients who were treated with LED, an increase in the mean number of WSLs per patient (mean increase, 1.8; P < 0.05) was found, while in NMCur-aPDT and VITIS® groups, not only no increases were observed, but the mean number of WSLs per patient decreased (mean reductions, 0.5 and 0.9, respectively; not significant). LED treatment caused significant increases (P < 0.05) in the mean LF values at 90-and 120-days of follow-up in comparison with the baseline (mean increases, 5.1 and 6.5, respectively) while, in NMCur-aPDT, VITIS®, and NMCur groups 11.8-, 7.1-, and 4.4-reductions in the mean LF values were observed, respectively (all, P < 0.05). CONCLUSIONS: The antimicrobial and anti-virulence activities of NMCur-aPDT against S. mutans were higher than the other treatment groups. In patients who were treated with NMCur-aPDT, the mean number and LF values of WSLs per patient were significantly lower than the other groups in 90-and 120-days of follow-up.