Endodontic antimicrobial photodynamic therapy: safety assessment in mammalian cell cultures

Abstract

Objectives: The purpose of this study was to assess the in vitro synergistic effect of methylene blue (MB) and red light on human gingival fibroblasts and osteoblasts with parameters similar to those that may be applied in a clinical setting for endodontic disinfection.

Materials and methods: Both cell types were sensitized with 50 microg/mL MB followed by exposure to red light at 665 nm for 5 minutes with an irradiance of 10, 20, and 40 mW/cm(2). After photodynamic therapy (PDT), cell viability and mitochondrial activity were evaluated by the neutral red and MTT assay, respectively. The assessment of PDT-induced apoptosis was investigated by western blot analysis using cleaved poly(ADP-ribose) polymerase-specific antibodies.

Results: Light at 20 and 40 mW/cm(2) with MB had modest effects at 24 hours on osteoblasts in both assays, whereas sodium hypochlorite completely eliminated cells. Western blot analysis revealed no signs of apoptosis in either cell type.

Conclusion: The data suggest that there is a safe therapeutic window whereby PDT can inactivate endodontic pathogens without affecting host cell viability.

Figures

Figure 1

Exposure of the root canal system of a tooth specimen to red light with a power density of 100 mW/cm2 (A). The power of light emitted by the specimen was found to be 0.012 W (12 mW) which corresponded with a power density of 4 mW/cm2 (B). The maximum power density obtained was approximately 10 mW/cm2.

Figure 2

Growth of MB-sensitized gingival fibroblasts as determined by the neutral red assay (A) and MTT assay (B) following their exposure to red light. Bars represent the means of optical density (± standard error). L-MB- (no light/no drug); L-MB+ (MB-treated cells but not irradiated with light); L10+MB-, L20+MB-, and L40+MB- (MB-untreated cells but irradiated with light of 10, 20 and 40 mW/cm2); L10+MB+, L20+MB+, and L40+MB+ (MB-treated cells and irradiated with light of 10, 20 and 40 mW/cm2); NaOCl (cells treated with sodium hypochlorite).

Figure 3

Growth of MB-sensitized osteoblasts as determined by the neutral red assay (A) and MTT assay (B) following their exposure to red light. Bars represent the means of optical density (± standard error). The groups are the same as in Fig. 2.

Figure 4

Osteoblasts (A) and fibroblasts (B) were treated with MB only (lane MB+), light only (lane L+) or light and MB (lane L+MB+). Control cultures were left untreated (lane −) or treated with 200 μM staurosporine (lane +). Twenty-four hours following treatment, cultures were stopped and analyzed by Western Blot using cleaved PARP- and β-Actin-specific antibodies. Neither treatment resulted in PARP cleavage.