In vitro effect of curcumin-mediated antimicrobial photodynamic therapy on fibroblasts: viability and cell signaling for apoptosis.

Although it was demonstrated that curcumin-mediated antimicrobial photodynamic therapy (aPDT) is effective for reducing the viability of microbial cells and the vitality of oral biofilms, the cytotoxicity of this therapeutic approach for host cells has not been yet elucidated. Hence, the aim of this study was to evaluate the cytotoxicity and apoptotic effects of curcumin-mediated aPDT on mouse fibroblasts. Cells were treated with 0.6 or 6 µmol.L-1 curcumin combined with 0.075 or 7.5 J.cm-2 LED at 455 nm. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while quantitative reverse transcriptase-PCR (qRT-PCR) was used to assess the expression of Bax, Bad, Bcl-2, VDAC-1, cytochrome C, and Fas-L genes for apoptosis. 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 qRT-PCR (P < 0.05). The effect of 0.6 µmol.L-1 curcumin plus 0.075 J.cm-2 LED (minimum parameter) did not differ statistically from control group; however, the combination of 0.6 µmol.L-1 curcumin plus 7.5 J.cm-2 LED reduced viable cells in 34%, while the combinations of 6 µmol.L-1 curcumin plus 0.075 and 7.5 J.cm-2 LED reduced viable cells in 47% and 99%, respectively. aPDT increased significantly the relative expression of Bax/Bcl-2, cytochrome C, VDAC-1, and Fas-L genes, without influence on the ratio Bad/Bcl-2. Therefore, curcumin-mediated aPDT activated Bcl-2 apoptosis signaling pathways in mouse fibroblasts regarding present conditions, reducing the viability of cells with the increase of curcumin concentrations and light energies.

Could a chelant improve the effect of curcumin-mediated photodynamic antimicrobial chemotherapy against dental intact biofilms?

To our knowledge, there is still no evidence in relation to the combination of curcumin with chelants to improve the effects of antimicrobial photodynamic therapy (aPDT) on complex dental caries biofilms. Therefore, the aim of this study was to evaluate the antimicrobial effect of curcumin-ethylenediaminetetraacetic acid (EDTA)-mediated aPDT on the vitality of intact biofilms of dentin caries microcosms. Biofilms were grown on glass slabs in McBain medium plus 1% sucrose in microaerophily at 37 °C for 5 days. Then, biofilms were treated with associations of 600 µmol L-1 curcumin combined or not with 1% EDTA and 37.5 or 75 J cm-2 LED (455 nm). The vitality was determined by a confocal laser scanning microscopy (CLSM) after staining biofilms with a mixture of 2.5 g L-1 fluorescein diacetate and 0.25 g L-1 ethidium bromide. Statistical analysis was conducted by Kruskal-Wallis and post hoc Dunn's test (P < 0.05). Three treatments were able to reduce the vitality of overall biofilms: curcumin + 75 J cm-2 LED, curcumin-EDTA + 37.5 J cm-2 LED, and curcumin-EDTA + 75 J cm-2 LED. Also, the vitality of inner layers of biofilms was significantly reduced only after the combination of aPDT with EDTA. Therefore, the association of curcumin and EDTA improved the antimicrobial effect of aPDT on dentin caries microcosms, considering the application of lower light densities and deeper layers of biofilms.

Could chlorhexidine be an adequate positive control for antimicrobial photodynamic therapy in- in vitro studies?

BACKGROUND: Chlorhexidine digluconate (CHX) is commonly applied as positive control of new antimicrobials, because it is considered the gold-standard for chemical plaque control. The aim of this study was to compare the effect of treatments with curcumin-mediated aPDT and CHX in relation to the viability of specific microorganism groups in two distinct times (immediately and 24 h later). METHODS: Dentin caries microcosms were grown on bovine dentin discs (37 °C, anaerobiosis) for 3 days in the Active Attachment Amsterdam Biofilm Model. The biofilms were treated with 300 µM curcumin and 75 J.cm-² LED, or 0.06% and 0.12% CHX. Then, total microorganisms, total streptococci, mutans streptococci, and total lactobacilli counts were determined. The statistical analysis was conducted by Kruskal-Wallis and post-hoc Dunn's tests (P < 0.05). RESULTS: Curcumin-mediated aPDT (C + L+), 0.06% and 0.12% CHX reduced mutans streptococci counts (0.19, 0.10 and 0.07 log10 respectively) in the immediate analysis. After 24 h, it was observed a re-growth of microorganisms treated by curcumin-mediated aPDT, whereas both CHX concentrations demonstrated a decrease of the viable microorganisms. CONCLUSION: This study confirmed the substantive effect of CHX and the immediate effect of aPDT. The use of a neutralizer solution was important to block the substantivity of CHX and permit its fair comparison with aPDT, allowing its use as a positive control in further studies.