Cytotoxic effect and apoptosis pathways activated by methylene blue-mediated photodynamic therapy in fibroblasts.

Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment of oral infections as a minimal intervention clinical approach. Its antimicrobial efficacy was demonstrated in several studies; however, there is a lack of evidence on its cytotoxic effect on mouse fibroblasts (NIH/3T3). The aim of this study was to evaluate the cytotoxicity and apoptotic pathways of methylene blue-mediated aPDT on mouse fibroblasts. Cells were treated with 0.1 or 1.0 mg.L-1 methylene blue (MB), and 0.075 or 7.5 J.cm-² LED at 630 nm. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while cDNA expression for Bax, Bad, Bcl-2, VDAC-1, cytochrome C and Fas-L was assessed by qRT-PCR (1, 3, 6 and 24 h). The differences between groups were detected by Kruskal-Wallis and post-hoc Dunn's tests for MTT and CV assays, and by ANOVA and post-hoc Tukey test for qPCR (P < 0.05). The combination of 1.0 mg.L-1 MB and 7.5 J.cm-² LED significantly reduced the cellular viability, whereas MB and LED alone were innocuous to fibroblasts. MB-mediated aPDT increased the expression of cytochrome C and Fas-L after 3 h, and Bax/Bcl-2, Bad/Bcl-2, and VDAC-1 after 6 h from treatment. Based on these results, MB-mediated aPDT induced cytotoxicity on mouse fibroblasts, with consequent activation of Bcl-2 apoptosis signaling pathways. Further studies are needed to determine the adequate parameters of aPDT to inactivate microorganisms without damaging fibroblasts.

Curcumin-mediated antimicrobial photodynamic therapy reduces the viability and vitality of infected dentin caries microcosms.

BACKGROUND: To our knowledge, there is a lack of evidence on the effect of Antimicrobial Photodynamic Therapy (aPDT) by the application of curcumin against complex biofilms of dental caries lesions. This study aimed to evaluate the viability, vitality, and acid metabolism of infected dentin caries microcosms treated with curcumin-mediated aPDT. METHODS: After microcosm biofilms growing anaerobically on bovine dentin disks immersed in McBain medium with 1% sucrose at 37 °C for 5 days, the biofilms were treated by the association of DMSO water solution or 600 µmol L-1 curcumin with 0, 37.5 or 75 J cm-2 blue LED (455 nm). Then, the colony-forming units (CFU) counts of total microorganisms, total streptococci, mutans streptococci, and total lactobacilli were determined by plating. The lactic acid concentration was analyzed by enzymatic spectrophotometry method, while the vitality of intact biofilms was evaluated by confocal laser scanning microscope (CLSM). Statistical analysis was performed by Kruskal Wallis and post-hoc Dunn's tests (P < 0.05). RESULTS: Curcumin alone did not affect the viability of microorganisms and the vitality of intact biofilms. However, 75 J cm-2 LED alone decreased the total microorganisms and total lactobacilli counts. The combination of curcumin and LED reduced significantly the counts of all microorganism groups and the vitality of intact biofilms. Differences were not observed between the lactic acid concentrations of distinct groups. CONCLUSIONS: Therefore, curcumin-mediated aPDT was effective in reducing the viability and the vitality of infected dentin caries microcosms, without interfering in their acidogenicity.