Résumé
Objective To explore in vitro the best time window for using sinusoidal electromagnetic fields to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods BMSCs were isolated and cultured from 4-week-old Sprague-Dawley rats (male and female,80-120g).The BMSCs (from passage 3) were exposed 0,1,4 or 8h/d for 7d,14,or 28d,respectively,to 15Hz sinusoidal electromagnetic fields with a maximum amplitude of lmT.Those exposed 0h/d served as the control.The relative expressions of runt related gene-2 (RUNX2),bone sialoprotein (BSP) and osteopontin (OPN) were determined using real-time,quantitative reverse transcription-polymerase chain reactions (RT-PCRs).The level of RUNX2 protein was determined by Western blotting after 14d.Alizarin red staining was used to compare calcium distribution in each group.Results Obvious promotion of differentiation to osteoblasts was observed after 7 days of exposure to the15 Hz sinusoidal electromagnetic fields,most obviously manifested by an outstanding increase of the early osteogenic index RUNX2 in those exposed 4h/d.After 14 days of intervention,the 1h/d exposure showed to be most effective,especially in inducing the changes of the late osteogenic index OPN.The trends of changes in RUNX2 protein were similar in all groups.After stimulating 1h/d for 14 and 28days,calcium deposition increased to the greatest extent.Conclusions Exposure to sinusoidal electromagnetic fields induces osteogenic differentiation to osteoblasts in rat BMSCs in vitro.There is an apparent window effect.The best results are observed with more days of exposure and shorter exposure time (1h) every day.
Résumé
Objective To investigate the effects of 620nm noncoherent red light emitted from light-emitting diode (LED) on proliferation and osteogenic differentiation of rat osteoblasts.Methods Osteoblasts were isolated from SD rat and cultured in vitro.The LED was placed at 2cm above the cell layer.Cells were divided into four groups and each group was irradiated for 0s, 150s, 300s and 600s at energy dose of 0, 1, 2, and 4J/cm2, respectively.Cells were irradiated once every day.Cellular proliferation activities were evaluated by using cell counting kit-8 ( CCK-8 ) at 2nd and 4th d.The alkaline phosphatase (ALP) ratio activity was evaluated by alkaline phosphatase activity kit at the 9th d, and expressions of osteoblast master genes ( Collα1, Bglap and Runx2) were monitored as indicators of osteoblast differentiation by reverse transcription-polymerase chain reaction (RT-PCR) technique.Results In the group irradiated with 1 and 2 J/cm2 proliferation activities of osteoblasts enhanced significantly compared with the control group at the 4th d ( P < 0.05 ).In the group irradiated with 4 J/cm2 ALP ratio activity elevated significantly compared with the control group at the 9th d ( P <0.05 ).In the group of red light irradiation expressions of Bglap and Runx2 enhanced significantly.Conclusion The 620nm nonconherent red light emitted from LED can promote proliferation of osteoblasts and enhance osteogenic differentiation significantly.