Optimal Low-Energy Laser Irradiation Causes Temporal G₂/M Arrest on Rat Calvarial Osteoblasts

Low-energy laser irradiation (LELI) accelerates wound healing and is thought to accelerate bone formation. However, the mechanism of laser healing is not clear. To clarify the biological mechanism of LELI healing, we investigated the effects of LELI on rat osteoblasts in vitro. Osteoblastic cells from 3-day-old Wistar rat calvaria were irradiated using a low-energy gallium-aluminum-arsenide (Ga-Al-As) diode laser. Bone formation, osteoblast differentiation, and cell proliferation were evaluated by von Kossa staining, reverse-transcription polymerase chain reaction, alkaline phosphatase (ALP) staining, 5-bromo-2′-deoxyuridine (BrdU) uptake, and fluorescence-activated cell sorter (FACS) analysis. At 21 days after LELI, the greatest bone formation was observed with irradiation energy of 3.75 J/cm² and the first week after seeding. LELI (3.75 J/cm²) induced an increased number of cells at day 3. LELI-stimulated differentiation in osteoblastic cells was demonstrated by the increases of Runx2 expression and ALP-positive colonies. By contrast, at 1 day after laser irradiation, the number of cells in the irradiation group was significantly lower than that in the control group. BrdU uptake indicated lower proliferation 12 and 24 hours after irradiation compared with the control. Furthermore, FACS data demonstrated a higher proportion of cells in the G₂/M phase of the cell cycle 12 hours after irradiation compared with the control. G₂/M arrest was confirmed by the appearance of G₂/M arrest marker 14-3-3-σ or phospho-p53. These results demonstrate that LELI induces not only acceleration of bone formation but also initial G₂/M arrest, which may cause wound healing like tissue repair.