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

Abstract

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. 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. Thevirulence‑associated gtfB gene expression was assessed following PQD-Nis-nQCT-mediated aPDT byquantitative real‑time PCR. PQD-Nis-nQCT at 2048µg/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). 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.