Methylene blue mediated photobiomodulation on human osteoblast cells.

Abstract

Photobiomodulation (PBM) and photodynamic therapy (PDT) are two major methods, which use light in medicine and dentistry. PBM uses low-level laser light to induce cell proliferation and activity. In contrast, PDT use laser light combined with a photosensitizer (PS) to cause cell death. Due to similar, not fully understood mechanisms and biphasic response of light, unexpected and complex outcomes may be observed. In the present study, the effect of 635nm laser light, with power density 50mW/cm2, at three different energy densities (0.5, 1, and 2J/cm2 which last 10, 20, and 40s, respectively) mediated by methylene blue (MB) on the human osteoblast cell line (ATCC-CRL-11372, Rockville, MD, USA) was investigated. Cell viability (MTT assay and acridine orange/propidium iodide staining) and proliferation (Alamar Blue assay) were assessed at 24, 48, and 72h post irradiation. Alkaline phosphatase (ALP) activity, mineralization (Alizarin Red staining) and gene expressions (RT-PCR analysis) were analyzed at 7th and 14th days after treatment. Five groups were formed as the control group (no MB, no irradiation), MB (only 0.05μM MB), MB+0.5J/cm2, MB+1J/cm2, and MB+2J/cm2. Cell viability was decreased at 72h (ANOVA; p<0.05) for MB+0.5J/cm2, MB+1J/cm2, and MB+2J/cm2 groups. Although proliferation does not seem to be effected by MB-mediated laser application, osteo-anabolic activity is altered. ALP activity was significantly increased at day 7 (ANOVA; p<0.05) for MB-combined laser groups; on the other hand, mineralization was significantly decreased (ANOVA; p<0.05) in all treatment groups. Alkaline phosphatase and collagen-I expressions were upregulated in MB+2J/cm2 group at 7th and 14th days, respectively. These results may contribute to the low-dose PDT researches and understanding PBM effects on osteoblast behavior but further studies are needed since inappropriate conditions may lead to undesirable results for both therapies.