A qualitative and quantitative analysis of various parameters of electric current and its effect on biomarkers (receptor activator of nuclear factor kappa-B ligand, osteoprotegerin): A <i>in vitro</i> study

Objectives: The objectives of the study are to examine the qualitative and quantitative effects of exogenous electric current (square waveform with varying pulsed frequencies and current intensities) on sarcoma osteogenic-2 (Saos-2) cells, focusing on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL). Material and Methods: The experimental group was divided into two groups: Group A with a 1s pulsed frequency and Group B with a 10s pulsed frequency. Each experimental group (i.e., Group A and Group B) was further subdivided into five subgroups (A1–A5, B1–B5) based on the current levels applied (10–30 µA). Saos2 cells seeded in 6-well plates were subjected to initial compression forces using glass coverslips for 1 h. Reverse piezoelectricity was applied continuously for 24 h a day over 3 days. A total of 2 µL of media was collected at specific time points of 1, 3, 5, 7, 12, 24, 48, and 72 h, and analyzed using an enzyme-linked immunosorbent assay for OPG and RANKL concentrations. The cells underwent histological examination for qualitative analysis after 72 h. All experiments were performed in triplicate. The data obtained were analyzed statistically. Results: Maximum OPG (0.090 ng/mL, P &lt; 0.001) and RANKL (0.091 pg/mL, P &lt; 0.001) expression occurred at 30 µA and 24 h in Group B; in Group A, the peak was delayed to 48 h with decreased expression levels (OPG: 0.078 ng/mL, P &lt; 0.001; RANKL: 0.077pg/mL, P &lt; 0.001). OPG levels showed a progressive increase at earlier time points. OPG levels rose early and were influenced by the timing of media collection, intensity, and frequency. RANKL was influenced only by the time points ( P &lt; 0.001). Phase contrast microscopy revealed an enlarged size of Saos-2 cells at 30 µA with a 10s frequency. Conclusion: The findings of this study show that bioelectric stimulation can directly modulate both OPG and RANKL. There are unique frequencies that may be applied to bone cells to regulate, alter, and optimize the expression of OPG and RANKL. These findings suggest that bone cells, and possibly other cells, possess biochemical pathways that respond reliably to specific frequencies of electrical stimulation.