Effects of micro-electrical stimulation on osteoblasts were examined using osteogenic MC 3T3-E1 cells which have retained the capacity to differentiate into osteoblasts and form mineralized tissue in in vitro.
The cells in 0.3% type I collagen gel at 2×106 cells/ml were cultured in α-MEM supplemented with 10% fetal bovine serum and 5mM β-glycerophosphate at 37°C in a fully humidified atmosphere of 5% CO2. Two platinum wire electrodes (0.3mm in diameter) were inserted into collagen gel at 10mm-intervals. The cells were stimulated by 1, 5, 10 or 20μA of constant direct currents via the electrodes for 6 days. At 5μA of current, their alkaline phosphatase (ALP) activities were maximum. Thus, they were stimulated by 5μA of current for a short or long term. ALP activity and 45Ca incorporation were significantly increased in long-term stimulated cultures. These results showed that long-term stimulation provided an optimal environment for MC 3T3-E1 cells to differentiate into osteoblasts. Next, the cells were stimulated by 5μA of current for a long term. ALP activity and 45Ca incorporation significantly increased in the stimulated cultures compared to the controls. Histological observations showed that the cells surrounding the cathode apparently differentiated into osteoblasts and synthesized abundant fibrous matrix. Moreover, many matrix vesicles containing needlelike crystals were observed on cathode at 9 days. On day 12, the stimulated cultures were positively stained with von Kossa.
These results suggest that micro-electrical stimulation directly affects MC3T3-E1 cells and induces osteoblastic differentiation and calcification.