Avaliação da citotoxicidade da quimioterapia fotodinâmica antimicrobiana sobre culturas de células pulpares in vitro

Abstract

Photodynamic antimicrobial chemotherapy (PACT) has been used successfully to eradicate cariogenic microorganisms, but there is a lack of knowledge regarding PACT effects on dental pulp tissue. To evaluate the cytotoxicity of PACT with methylene blue (MB) associated to white light or diode laser on primary pulp cell cultures, 1.5 x 105 cells/well were seeded in 96-well plates for 48 h, incubated with MB (12.5, 25 e 50 µg/ml) and irradiated with a white light source (500-800 nm; 400 mW/cm2) or red laser (660 nm; 1000 mW/cm2) for 1 minute. Cell metabolism was evaluated by the MTT (methyltetrazolium), NR (neutral red) and ALP (alkaline phosphatase activity) assays. The type of cell death was evaluated by annexin-V and propidium iodide assay by flow cytometry. Analysis of variance (ANOVA) followed by Tukey´s test were performed to compare all groups. Only MB at 12.5 µg/ml exposed to red laser caused slightly cytotoxic effects on pulp cells. There was a statistically significant decrease of cell metabolism for all MB concentrations irradiated by white light and at concentrations of 25 and 50 µg/ml irradiated by laser light. After 24 h, toxic effects were even more severe characterizing damage without cell recovery. Cell detachment and morphological alterations could also be observed under light microscope. After exposure to white light there was a predominance of necrotic cell death, whereas when laser did the exposure there was a predominance of apoptotic cell death. MB at 12.5 and 25 µg/ml exposed to red laser seems to have the potential to be applied in vivo. However, news studies should be performed to further optimization of light dosimetry of PACT directly applied on pulp cells.