Optimizing Alternating Current Electrical Stimulation Parameters to Enhance Osteoblasts Differentiation

Abstract

Electrical stimulation (ES) has emerged as a promising technique in the field of bioengineering and biomedicine, particularly inbone regeneration and cell differentiation. ES using alternating current (AC) is based on the periodic reversal of current direc-tion, which generates oscillating electric fields. The application of an electric field has effects on cell growth and differentiation,as well as on morphology and migration. This study aimed to explore the effect of applying AC electrostimulation within theproliferation, differentiation, and morphology process of osteoblastic cells. The electrical stimulation signals were daily appliedfor 3 h during 14 days. Different frequencies were tested (1 Hz, 10 Hz, 100 Hz, and 1 kHz), with amplitudes of 125, 250, 500, 750,1000, and 1500 mV/mm. Cell viability was estimated using the AlamarBlue, and MC3T3-E1 differentiation levels were eval-uated through alkaline phosphatase (ALP) activity. RUNX2, OSX, ALP, OPG, and RANKL gene expression was assessed byRT-PCR. Morphological analysis was performed through cell transfection followed by immunofluorescence. Statistical analysiswas conducted by SPSS.23 and graphs generated through Graph-pad. Viability and ALP activity were optimal at 10 Hz. Oncethe frequency was defined, RUNX2, OSX, ALP, OPG, and RANKL gene expression revealed an increase in the differentiationand osteogenic activity levels at 10 Hz and 500–750 mV/mm. As well as, morphological studies showed an increase in the area,pseudopodia length, and numbers at 500 mV 10 Hz conditions. The optimal ES condition to differentiate MC3T3-E1 cells is 10 Hz500–750 mV/mm. Electrostimulation has emerged as a promising technique in the field of bioengineering and biomedicine, par-ticularly in bone regeneration and cell early maturation.