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 in vitro study

INTRODUCTION

For decades, the orthodontic community has been concerned about the duration of orthodontic treatment. Long-term treatment not only increases the risk of side effects such as root resorption^[1] and dental caries^[2] but also reduces patient compliance.^[3] Researchers have documented various procedures to enhance the rate of tooth movement. Surgical methods^[4] include corticotomies, modified corticotomies (piezocision), osteotomies, micro-perforation, distraction osteogenesis, and others. Non-surgical methods include a variety of transmucosal interventions, low-level laser therapy, pharmacological measures, pulsed electromagnetic field (PEMF), extracorporeal shock wave therapy, and exogenous electric current application.^[5]

Periodontal defects,^[6] bony fractures,^[7] an increased rate of tooth movement,^[8] and a reduction in alveolar ridge resorption following extraction^[9] all benefit from electric stimulation (EStim). Two hypotheses have been proposed for alveolar bone remodeling, i.e., piezoelectricity (inorganic crystals, including those in bone and organic crystals, like collagen crystals in the periodontal ligament [PDL]) generated in alveolar bone bending and pressure tension in the PDL.

An electric field can cause a crystal to deform and produce a force, creating a reverse piezoelectric effect. Reverse piezoelectricity has no place in natural control systems. EStim’s pro-healing effect is attributed to its influence on the behavior and function of bone-forming stem cells, including migration,^[10] proliferation,^[11] differentiation,^[12] mineralization,^[13] extracellular matrix deposition, and attachment to scaffold materials.^[14]

When cells are exposed to an exogenous electric stimulus, a response known as electrocoupling is generated, which is caused by the high resistance of the plasma membrane. This high resistance prevents the penetration of electric stimuli, independent of the cytoplasmic conductive capacity.^[15] Asymmetric redistribution or diffusion of electrically charged cell membrane receptors in response to electric fields activates numerous downstream signaling cascades.^[16] The cell membrane depolarization is due to the direct activation of voltage-gated cytosolic calcium (Ca₂+) channels, leading to an increase in intracellular calcium ion concentration.^[17] Other mechanisms that play a role include mechanotransduction^[18] surface receptor redistribution, adenosine triphosphate synthesis stimulation^[19] activation of heat shock protein^[20] generation of reactive oxygen species^[21] and redistribution of lipid rafts.^[22]

For most applications, purpose-built chambers can be used to apply in vitro electrical stimulation (ES) to cells grown in 2-D or 3-D cultures. These chambers encompass a metallic electrode EStim chamber, a salt bridge EStim chamber, and microfluidic chip EStim chambers.^[23]

Direct, capacitive, or inductive coupling can apply EStim in vivo. Several in vitro studies^[12,24-26] have demonstrated that direct current (DC) and PEMFs enhance proliferation, differentiation factors such as osteocalcin, osteopontin, and Runt-related transcription factor 2, alkaline phosphatase activity, and calcium deposition in osteoblasts and mesenchymal stem cells. In their in vitro study on osteosarcoma cell lines, Fidan et al. found an increase in cell number following exposure to 0.5 kV/cm and 2.3 kV/cm DC electric fields; however, the authors did not consider the effect of current frequency and waveform.^[27]

Andrew and Bassett found that an electric current of 5–20 µA has been proven to aid osteogenesis in studies. The studies also reveal, however, that electric currents <5 µA do not promote bone formation, and that electric currents >20 µA cause cellular necrosis and bone death.^[28] France et al. have found it to be efficient in repair and bone strength.^[29] Davidovitch et al., in their animal study, applied 15 µA of DC. to the gingival tissues (near one maxillary canine) in a cat model.^[30] There was an intense staining of cyclic adenosine monophosphate and cyclic guanosine monophosphate evident, which suggested that ES enhances cellular enzymatic phosphorylation activities. However, none of the above studies have considered the waveform or frequency of the applied current.

Active bone cells produce bone markers such as tartrate-resistant acid phosphatase, osteoprotegerin (OPG), and receptor activator of nuclear factor kappa beta ligand (RANKL), which provide an estimate of the level of bone remodeling in serum or plasma. Therefore, we designed a study to understand the impact of applying exogenous electric current with a square waveform, varying in parameters such as pulsed frequency (seconds) and current-µA, on Saos2 cells, by estimating OPG and RANKL both qualitatively (histologically) and quantitatively enzyme-linked immunosorbent assay (ELISA). The clinical relevance of the study could pave the way for the development of adjunctive bioelectric stimulation therapies in orthodontics, accelerating tooth movement and reducing treatment duration.^[31] By identifying effective stimulation parameters, personalized protocols could be designed for different age groups or clinical conditions.^[32] The results of this study would provide a foundation for future in vivo and clinical trials, potentially extending to applications in bone regeneration, implantology, and periodontal therapy.^[33] Moreover, the use of bone markers like OPG and RANKL provides a basis for biomarker-based monitoring of bone remodeling, enabling more precise and adaptive treatment strategies in clinical practice.^[34]

MATERIAL AND METHODS

Group

An experiment was designed [Figure 1] to compare EStim Saos2 cells (Experimental) versus non-stimulated (Control) cell groups. The experimental group was divided into Group A with a 1s pulsed frequency and Group B with a 10s pulsed frequency. Each experimental group was further subdivided into five subgroups (A1–A5, B1–B5) based on the current levels subjected, i.e., 10, 15, 20, 25, and 30 µA. The control group did not receive any ES; however, the electrodes (0.17 × 0.25 inches) were placed similarly to those in the experimental groups. The cells in the control were subjected to the same environmental conditions and procedures as those for the experimental groups, except for the application of exogenous EStim.

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Figure 1:

(a and b) Experimental setup.

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RESULTS

Different pulse frequencies (1 s pulsed frequency and 10 s pulsed frequency), current levels (10–30 µA), and time duration (media collected at various intervals) individually had a significant influence on the expression of OPG [Table 1]. Interaction between pulse frequency and time, pulse frequency and current levels, and current level and time duration also showed a significant influence on the expression of OPG. The influence of interaction between pulse frequency, current level, and time on OPG expression was also found to be significant [Figure 2].

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Figure 2:

Qualitative analysis: Inverted cone microscopic view. (a) Control - normally dividing cells, no exogenous current. (b) 30 µA/10 s pulse frequency shows the cells are larger and are increased in number compared (e) to the 30 µA/1 s pulse frequency. (c) Rapidly dividing cells under the influence of exogenous electric stimulation. (d) 15 µA/10 s pulse frequency showing the cells are alive and spindle-shaped, but in comparison to (b) and (e), the exogenous current has not brought any significant changes. (e) 30 - µA/1 s pulse frequently shows a reduced number of cells as compared to (b) the 30 µA/10 s pulse frequency. The overall size of the cells is smaller compared to (b). No staining was used. The 6-well plate was directly taken under an inverted cone microscope to view the cells under 300X magnification.

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Similarly, different time intervals also had a significant influence on the expression of RANKL [Table 2]; however, different pulse frequencies and current levels did not have a significant influence on the expression of RANKL. The effect of interaction between pulse frequency and time, pulse frequency and current levels, and current level and time duration did not show a significant influence on the expression of RANKL. Finally, the effect of interaction between pulse frequency, current level, and time on RANKL expression was also non-significant.

OPG secretion peaked at 48 h (0.078 ng/mL, P < 0.001) [Table 3a] under a 1s pulsed frequency using a 30 µA current. A similar increase in OPG levels was observed at the 3 h time point with the application of 15 µA at the same frequency. Compared to the control group, OPG secretion consistently increased from 1 h to 72 h across the experimental timeline. Similarly, OPG expression reached its maximum at 24 h (0.090 ng/mL, P < 0.001) [Table 3b] with a 30 µA current under a 10 s pulsed frequency. Quantitative analysis revealed that overall OPG expression was higher at the 10 s pulsed frequency than at the 1 s frequency, with peak expression occurring 24 h earlier in the 10 s group. RANKL [Figure 3] expression at a 1s pulsed frequency [Table 3c] showed no consistent pattern of secretion across different current levels. In contrast, RANKL secretion was highest at 24 h (0.091 pg/mL, P < 0.001) [Table 3d] post-stimulation with a 30 µA current under the 10 s pulsed frequency, earlier than the peak observed under the same current at 1 s pulsed frequency.

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Figure 3:

Pictorial representation of Graph (group a and b). (a) The graph represents the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) under various direct current (DC) applications at 1 s pulsed frequency (SPF). The expression reaches a peak at 48 h, 0.077 pg/mL under a 30 µA current level, and the control lags. (b) The graph represents the expression of osteoprotegerin (OPG) under various DC applications at 1 SPF; the peak is reached again at 48 h, 0.078 ng/mL, with control lagging. (c and d) Both graphs showed that the expression of RANKL and OPG was maximum at 30 µA, as seen in (a) and (b); however, the expression increased at 10 s pulsed frequency, which was earlier at 24 h (RANKL – 0.091 pg/mL, OPG – 0.090 ng/mL). The X-axis in all line diagrams represents the time points of media extraction, Y axis in: RANKL line diagram represents – measurements in pg/ml OPG line diagram represents – measurements in ng/ml

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DISCUSSION

The bioelectrical cues found in natural living bone, such as piezoelectricity, pyroelectricity, and ferroelectricity, have been identified as an important factor in regulating metabolic activities, including growth, structural remodeling, and fracture healing.^[35,36] Researchers have suggested that the non-centrosymmetric collagen molecule is the primary reason behind bioelectricity in living bone.^[37-39] The collagen present in bone exhibits piezoelectric properties, generating a streaming potential. When subjected to stress, it leads to a decrease in hydraulic permeability and an increase in stiffness.^[40]

Bioelectric stimulation can be applied via exogenous sources and various techniques, including DC stimulation, alternating current stimulation, PEMF therapy, and others. The present study analyzes the effect of various parameters of direct electrical (amount of current--µA, (sec) pulsed frequency) stimulation of Saos2 cells on the in vitro secretion of RANKL and OPG. Soas2 cells are a widely used human osteosarcoma cell line in biomedical research. We chose Saos2 cells as they are widely used in research related to bone biology, including studies on osteoblast differentiation, bone mineralization, bone-related diseases, drug screening, and evaluating the effects of various factors on bone cell behavior.^[41,42] Despite being a valuable tool in bone-related research, it is important to note that Saos2 cells originate from a cancerous origin (osteosarcoma). Therefore, one should exercise caution when extrapolating findings from Soas2 cells to normal physiological conditions. In this study, we used six-well culture plates for the experimental setup, adhering Saos2 bone cells to the bottom of the polytetrafluoroethylene (PTFE) culture well and bathing them in bone cell-specific culture media. The DC source device required electrodes to transmit electric current to the media and cells. The choice of electrode material is a crucial factor that can significantly impact the effectiveness of ES in biological tissues. The electrodes generate and inject charges (electrons) through the anode’s oxidation process.^[43] The charge-injection mechanism is dependent on the characteristics of the electrode. In both in vitro and in vivo stimulation experiments, the electrode material should exhibit satisfactory biocompatibility, ensuring it does not induce irreversible faradaic corrosion reactions in the culture medium during ES.^[44] These materials include carbon (graphite rods), platinum (Pt), iridium (Ir), gold, titanium (Ti), tungsten (W), Pt-Ir alloys, Ti nitride, stainless steel (SS), indium tin oxide, and others.^[45] We used SS electrodes in this study, as it is readily available, bio-inert, have good mechanical strength, and possess a passive iron oxide layer that protects the surface. Cells behave in different ways when subjected to various external or internal stimuli. For instance, studies have reported that extracellular physical stimulation of osteoblasts inhibits osteoblast differentiation and cell proliferation. However, it has also been observed that sustained stimulation activates p38, a molecule involved in the mitogen-activated protein kinase cascade, thereby promoting osteoblast differentiation.^[46] ES can also start intracellular signaling through cell adhesion factors and increase the expression of the Src family kinases (Src) family^[47] in fibroblasts. This study observed dynamic changes in the expression of RANKL and OPG in controlled, electrically stimulated Saos2 cells (experimental groups) compared to the control group, which did not receive any ES. We also observed a significant increase in RANKL and OPG expression compared to the control group.

ES increases Ca2+ levels and activates cytoskeletal calmodulin. ES and mechanical strain trigger cellular responses that involve the modulation of calcium signaling pathways.^[48]. Katoh has demonstrated that cells increase staining with phosphotyrosine antibodies, tyrosinephosphorylated focal adhesion kinase, and tyrosinephosphorylated c-Scr, indicating that ES affects signal transduction-related proteins.^[49] Stephan et al. used continuous DC at 2.5–3.5 mV/m and 0.24–0.35 mV/m (60 kHz) for 3 days: 0.1 V improved metabolic rate, increased tissue inhibitor of metalloproteinases-1 (TIMP1) and OPG messenger ribonucleic acid.^[50] Dauben et al. used in vitro DC ES with 0.2 V or 1.4 V, 20 Hz, 45 min × 3/day over 3 days, on human osteoblasts–0.2 V increased procollagen type I expression, and 1.4 V enhanced osteocalcin transcription.^[51] Authors in the past have used different amounts of current/voltages in their in vivo experiments, but none of them have elucidated the reason for opting for such current levels.^[5] We therefore set up an in vitro study to investigate the various aspects of electric current and its impact on bone cells, utilizing ELISA to measure RANKL and OPG levels.

RANKL is a transmembrane protein available in two forms: Membrane-bound and secreted. The membrane-bound form can be converted into the secreted form through proteolytic cleavage or alternative splicing.^[52] Various factors promote osteoclast formation and activity and stimulate the expression of RANKL in osteoblasts or stromal cells. The expression of RANK and RANKL increases bone resorption through osteoclastic differentiation, which starts bone softening and leads to tooth movement (Ikebuchi et al., 2018).^[53] In our study, comparing RANKL expression, the available 10s pulsed frequency group showed that a current level of 30-µA increased RANKL expression, which peaked at 24 h and then decreased below the control level by 72 h. The 1-s frequency interval group experienced a 48 h delay in peak expression, whereas the 10s frequency interval group experienced it at 24 h. We observed significant alteration in expression within the 1s pulsed frequency group after 12 and 24 h. When we compared different current intensities, we found that 30 µA significantly increased RANKL expression compared to all other current levels. Similarly, within the 10s frequency interval group, significant differences were observed between the 10 µA and 30 µA current levels. Therefore, we could comment that a current level of 30 µA at 10s pulsed frequency showed the most pronounced effects in enhancing RANKL expression.

Osteoblasts express OPG in various tissues, including the heart, kidney, liver, spleen, and bone marrow.^[54] Bone softening is prevented by inhibiting osteoclastogenesis and promoting bone hardening through enhanced OPG expression (Baud’huin et al., 2013).^[55] The same factors that induce RANKL expression in osteoblasts also regulate OPG expression, similar to RANKL. OPG expression was highest under the influence of a 30 µA current within a 1-s time interval. Compared to the 10 s time interval, OPG showed an increasing trend from 3 h to 24 h and decreased at 72 h with 30-µA current. This experiment has consistently demonstrated that OPG expression peaked with a 30-µA current and a 10-s time interval. Pair-wise comparisons of OPG expression within the 1-s pulse frequency (5–72 h) showed significant statistical differences between the control group and the 30-µA current group. These effects are primarily due to changes in cellular activity and the microenvironment. Changes in oxygen concentration and pH near the negative electrode directly affect cellular activity. An alkaline environment, induced by ES, promotes the release of calcium, which in turn promotes osteogenesis. These mechanisms contribute to the observed changes in cellular behavior and bone-related activity.^[56] Kuehne et al.^[57] advocated that ES at 100 mV, 60 kHz significantly increased OPG expression in MG-63 cells cultured on biphasic calcium phosphate scaffolds, enhancing osteogenic differentiation. In contrast, 1 V showed no added benefit and slightly reduced cell viability. The RANKL/OPG ratio was favorably altered, indicating a potential role in regulating bone remodeling.^[57] Under physiological conditions, the interaction between RANKL (produced by osteoblasts) and RANK (present on the surface of osteoclast precursors) plays a crucial role in osteoclastogenesis regulation. When RANKL binds to RANK, it recruits the adaptor protein TNF receptor-associated factor 6 (TRAF6), which initiates a signaling cascade leading to the activation of nuclear factor kappa-B (NF-κB) and its subsequent translocation to the nucleus. Turning on NFκB increases the production of c-Fos, a transcription factor that collaborates with (nuclear factor of activated T cells [NFATc1], cytoplasmic 1). The interaction between c-Fos and NFATc1 is essential for the transcription of genes involved in osteoclast differentiation and function. Together, they trigger osteoclastogenic gene expression, promoting osteoclast formation and activity. However, OPG acts as a regulatory factor by binding to RANKL and inhibiting its interaction with RANK. By inhibiting this interaction, OPG interferes with the initiation of the osteoclastogenesis process. As a result, OPG serves as a counterbalance to RANKL, maintaining bone remodeling homeostasis by controlling osteoclast activation and differentiation.^[58] An increase in the ratio of RANKL to OPG favors osteoclastogenesis, promoting osteoclast formation and activity. The RANKL/OPG ratio has been widely recognized as a significant determinant of bone mass.^[59]

The findings of this study show that bioelectric stimulation can directly modulate both OPG and RANKL. OPG expression was on the rise at intervals of 1, 5, 12, and 24 h, and OPG expression increased with an increase in the current levels. As the current levels increased from 10 µA to 30-µA, we observed an increasing trend in the amount of RANKL content at 1, 5, and 24 h in groups with a 10 s time interval. These outcomes cannot be considered just a universal onset of cellular protein expression; instead, there are unique electrical frequencies that may be applied to bone cells to regulate, alter, and optimize the expression of OPG and RANKL.

CONCLUSION

Our results show that square-wave EStim modulates OPG and RANKL expression in Saos-2 cells, with peak expression dependent on pulse frequency (earlier at 24 h for the 10s interval, and delayed for the 1-s interval), time duration, and current levels. These findings suggest that specific EStim parameters can fine-tune osteogenic signaling, supporting their therapeutic potential. Future studies may include validating findings in primary human cells, extending stimulation protocols, and conducting in vivo studies to assess long-term biological effects.