Effects of Smads by Laser-Irradiation on Calcification Ability of pulp

Objectives: Recently, the clinical use of lasers has increased, and lasers are now employed frequently even for endodontic treatment. Ga-Al-As laser is used for coagulation of deep tissue in the body and to promote hard tissue formation. Against this background, we also showed that calcification of human dental pulp (HDP) cells are stimulated by laser irradiation, and that free radicals generated by laser irradiation are triggers for the promotion of HDP cell mineralization. However, issues such as cell-propagated signaling immediately after laser irradiation and hard tissue-related mRNA expression have received little attention. We investigated the effects of Ga-Al-As laser irradiation on the calcification ability of HDP cells, and on Smads and BMPs production as one mechanism for the transmission of laser photochemical energy to cells.

Methods: The cells were cultured in alpha-MEM containing 10% FCS, and laser irradiation at 1.0W for 500s. After laser irradiation (Day 0), the cells were incubated with culture medium supplemented with mineral-inducing factors comprising 50micro-g/ml ascorbic acid and 10mM beta-glycerol-phosphate. Expression of mRNAs of smads, BMPs, ALPase, OCN, we used RT-PCR analysis. Production of BMPs were measured using ELISA technique. ALPase activity was measured using the method described by Ohshima et al. Calcified nodule formation was determined by Alizarin red S staining, and the amount of calcium was then measured.

Results: Calcified nodule formation and production of calcium were increased by in the laser irradiation. ALPase activity was higher after laser irradiation. Expression of mRNAs of Smad1, Smad7, BMPs, ALPase, and OC were greater with laser irradiation. However, Smad6 mRNA expression in HDP cells was inhibited by laser irradiation. Production of BMP-2 and BMP-4 in conditioned medium was also higher after laser irradiation.

Conclusion: These results suggested that Smads and BMPs play important roles in calcification upon laser irradiation of HDP cells.