Enhancement of Proliferation and VEGF Secretion in Osteoblast by Biphasic u current Stimulation
It has been known that electrical stimulation and pulsed electromagnetic stimulation promote osteogenesis and healing of bone fracture. In this study, we showed that u level charge balanced biphasic current stimulation could promote the proliferation of osteoblasts and increase the secretion of the VEGF protein significantly. We designed a current stimulator IC and a specific culture system so that biphasic u current pulses might stimulate the osteoblast being on the conductive Au metal plate fabricated by thermal evaporation on silicon wafer with a thickness of 3,000A. Each culture system is composed of an upper (Channel Electrode) and lower(Reference Electrode) plate connected with a ucurrent stimulator and Teflon(r) well with a dimension of 30mm X 30mm. Rat calvarias osteoblasts inoculated at a density of 2,500 cells/cm2 were exposed to electrical stimulation of 1.5 uA/cm2 at 1,000 Hz during 6 hours and 24 hours respectively compared to control without stimulation. After 1 and 2 days with stimulation, the increased cells were counted for measuring proliferation. The number of osteoblasts increased from 33,800 11,292 (mean SEM) cells to 44,300 9,287, which represented a 31% increase in proliferation of osteoblasts. VEGF induction At day 4 after exposure to stimulation, mRNA expression was investigated for differentiation promoting cytokines such as BMP 2, BMP 4, TGF b1, IGF 1, 2 and angiogenesis promoting vascular endothelial growth factor(VEGF) The amount of secreted VEGF was measured using an ELISA kit. The VEGF protein was significantly increased in both stimulated group compared to control. In conclusion, this study showed that BEC contributes to enhanced osteogenesis by increasing cell proliferation and by induction of angiogenesis, which are important for initiation of bone formation. Continuous stimulation by BEC was more effective than interrupted exposure. The results in this study indicate that BEC can act as a potential osteogenic inducer. To evaluate this potential effect in bone formation, further studies are needed in vivo.