Stimulatory Effects of Low-energy Laser Irradiation on the Initial Proliferation of Rat Calvarial Osteoblasts

Objective: Low-power laser irradiation accelerates healing, and is thought to accelerate bone formation after fracture. However, the mechanism of laser healing is not clear. To clarify the bone healing effect of laser irradiation, we investigated the effects of laser irradiation on rat osteoblasts in vitro. Methods: Osteoblasts from 3-day-old Wistar rat calvaria were irradiated using a low-power Gallium-Aluminium-Arsenide (Ga-Al-As) diode laser under various conditions. Bone formation, osteoblast differentiation, and cell proliferation were evaluated with von Kossa staining, alkaline phosphatase (ALP) staining, 5-bromo-2'-deoxyuridine (BrdU) uptake, and fluorescence activated cell sorter (FACS) analysis. Results: The greatest bone formation with laser irradiation was observed the first week after seeding and with an irradiation energy of 3.75 J/cm2. Laser irradiation induced an increase in ALP activity 7 days after seeding, and an increased number of cells beginning 4 days afterwards. By contrast, in the first two days after laser irradiation, the number of cells in the irradiation group was significantly lower than in the control group. The BrdU uptake indicated lower proliferation 12 and 24 hours after irradiation as compared with the control. Furthermore, FACS data demonstrated a higher proportion of cells in the S+G2 phase of the cell cycle 12 hours after irradiation as compared with the control. Conclusion: These results demonstrate that low-power laser irradiation initially depresses the cell cycle of osteoblastic cells, and then stimulates cell proliferation, resulting in increased bone formation.