Enhancement of Differentiation and Mineralisation of Osteoblast-like Cells by Degenerate Electrical Waveform in an In Vitro Electrical Stimulation Model Compared to Capacitive Coupling

Electrical stimulation (ES) is effective in enhancing bone healing, however the best electrical waveform, mode of application and mechanisms remains unclear. We recently reported the in vitro differential healing response of a novel electrical waveform called degenerate sine wave (DW) compared to other forms of ES. This study further explores this original observation on osteoblast cells. Here, we electrically stimulated SaOS-2 osteoblast-like cells with DW in an in vitro ES chamber (referred to as ‘DW stimulation’) and compared the intracellular effects to capacitive coupling (CC) stimulation. ES lasted for 4 h, followed by an incubation period of 20 h and subsequent ES for 4 additional hours. Cytotoxicity, proliferation, differentiation and mineralisation of the osteoblast-like cells were evaluated to determine the cell maturation process. DW significantly enhanced the differentiation of cells when compared to CC stimulation with increased alkaline phosphatase and collagen I gene expression by quantitative real time- polymerase chain reaction analysis (p<0.01). Moreover, DW significantly increased the mineralisation of cells compared to CC stimulation. Furthermore the transcription of osteocalcin, osteonectin, osteopontin and bone sialoprotein (p<0.05) was also up regulated by DW. However, ES did not augment the proliferation of cells. Translational analysis by immunocytochemistry and Western blotting showed increased collagen I, osteocalcin and osteonectin expression after DW than CC stimulation. In summary, we have demonstrated for the first time that DW stimulation in an in vitro ES chamber has a significant effect on maturation of osteoblast-like cells compared to CC stimulation of the same magnitude.