Electrical Stimulation Promotes Osteoconduction via Calcium-Mediated β-Catenin Site-Specific Phosphorylation

Objectives: Electrical stimulation (ES) has been known to promote osteoconduction by modulating membrane potential. However, the mechanism linking ES to the cellular response remains unclear. In this study, we aim to elucidate this connection and propose a definitive mechanism underlying ES-induced osteoconduction.
Methods: ES was applied to human bone marrow stem cells (hBMSCs) at 60 V and 18 Hz for 8 hours daily. Alizarin Red S and Bulk RNA-seq were performed on both ES and non-ES groups to evaluate the potency of osteoconduction and identify differentially expressed genes. Gene Ontology analysis was applied to validate in vitro osteoconduction and related signaling pathways. Real-time live imaging using the intracellular calcium biosensor was performed to examine calcium oscillation. In addition, live imaging and immunocytochemical approaches were utilized with β-catenin overexpression models to elucidate the mechanism of ES-induced osteoconduction.
Results: ES induced osteoconduction in hBMSCs, as evidenced by increased calcium deposition detected by Alizarin Red S staining. RNA-seq analysis revealed significant changes in transcriptome upon ES stimulation, highlighting the potential involvement of calcium signaling and WNT-related pathways in mediating the osteoconduction process. Real-time live cell imaging validated that ES triggers calcium oscillations via voltage-gated calcium channels (VGCCs). Specifically, we identified that the intranuclear accumulation of β-catenin is facilitated by site-specific phosphorylation at the S552 and S675 residues. Furthermore, this site-specific phosphorylation of β-catenin is directly mediated by calcium-dependent kinases, particularly CaMKII and PKC. This calcium-mediated site-specific phosphorylation of β-catenin represents a previously unrecognized mechanism underlying ES-mediated osteoconduction.
Conclusions: This study provides mechanistic insight into ES-induced osteoconduction. Our findings suggest that this phenomenon is mediated by calcium-directed stabilization of β-catenin via site-specific phosphorylation. These insights deepen our understanding of how bioelectrical stimulation influences osteoconduction, improving therapeutic strategies for bone regeneration.