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Fracture-healing is a highly complex and timely orchestrated process. Non-healing fractures are still a major clinical problem and treatment remains difficult. A 16 Hz extremely low-frequency pulsed electromagnetic field (ELF-PEMF) was identified as non-invasive adjunct therapy supporting bone-healing by inducing reactive oxygen species (ROS) and Ca 2+ -influx. However, ROS and Ca 2+ -influx may stimulate neutrophils, the first cells arriving at the wounded site, to excessively form neutrophil extracellular traps (NETs), which negatively affects the healing process. Thus, this study aimed to evaluate the effect of this 16 Hz ELF-PEMF on NET formation. Neutrophils were isolated from healthy volunteers and exposed to different NET-stimuli and the 16 Hz ELF-PEMF. NETs were quantified using Sytox Green Assay and immunofluorescence, Ca 2+ -influx and ROS with fluorescence probes. In contrast to mesenchymal cells, ELF-PEMF exposure did not induce ROS and Ca 2+ -influx in neutrophils. ELF-PEMF exposure did not result in basal or enhanced PMA-induced NET formation but did reduce the amount of DNA released. Similarly, NET formation induced by LPS and H 2 O 2 was reduced through exposure to ELF-PEMF. As ELF-PEMF exposure did not induce NET release or negatively affect neutrophils, the ELF-PEMF exposure can be started immediately after fracture treatment.

Experiments on cell cultures exposed to extremely low frequency (ELF, 3-300 Hz) magnetic fields are often subject to multiple sources of uncertainty associated with specific electric and magnetic field exposure conditions. Here we systemically quantify these uncertainties based on exposure conditions described in a group of bioelectromagnetic experimental reports for a representative sampling of the existing literature. The resulting uncertainties, stemming from insufficient, ambiguous, or erroneous description, design, implementation, or validation of the experimental methods and systems, were often substantial enough to potentially make any successful reproduction of the original experimental conditions difficult or impossible. Without making any assumption about the true biological relevance of ELF electric and magnetic fields, these findings suggest another contributing factor which may add to the overall variability and irreproducibility traditionally associated with experimental results of in vitro exposures to low-level ELF magnetic fields. Bioelectromagnetics. 39:231-243, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Recently, we identified a specific extremely low-frequency pulsed electromagnetic field (ELF-PEMF) that supports human osteoblast (hOBs) function in an ERK1/2-dependent manner, suggesting reactive oxygen species (ROS) being key regulators in this process. Thus, this study aimed at investigating how ELF-PEMF exposure can modulate hOBs function via ROS. Our results show that single exposure to ELF-PEMF induced ROS production in hOBs, without reducing intracellular glutathione. Repetitive exposure (>3) to ELF-PEMF however reduced ROS-levels, suggesting alterations in the cells antioxidative stress response. The main ROS induced by ELF-PEMF were •O 2 - and H 2 O 2 , therefore expression/activity of antioxidative enzymes related to these ROS were further investigated. ELF-PEMF exposure induced expression of GPX3, SOD2, CAT and GSR on mRNA, protein and enzyme activity level. Scavenging •O 2 - and H 2 O 2 diminished the ELF-PEMF effect on hOBs function (AP activity and mineralization). Challenging the hOBs with low amounts of H 2 O 2 on the other hand improved hOBs function. In summary, our data show that ELF-PEMF treatment favors differentiation of hOBs by producing non-toxic amounts of ROS, which induces antioxidative defense mechanisms in these cells. Thus, ELF-PEMF treatment might represent an interesting adjunct to conventional therapy supporting bone formation under oxidative stress conditions, e.g. during fracture healing.