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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.

Exposure to extremely low frequency pulsed electromagnetic fields (ELF-PEMF) is supposed to simulate local EMF generated during mechanical stimulation of bone and may therefore be used to improve bone regeneration. This study aimed at optimizing the exposure strategy and investigating the underlying mechanisms of a 16 Hz ELF-PEMF, previously reported to boost osteoblast function. Comparing influences of daily continuous (30 min every 24 h) and intermittent (10 min every 8 h) exposure to the 16 Hz ELF-PEMF on osteoprogenitor cells revealed that the intermittent exposure strategy enhanced the 16 Hz ELF-PEMF effects regarding cell numbers and osteogenic function. Gene expression of piezo 1 and related Ca 2+ influx were significantly increased in SCP-1 cells with the daily intermittent exposure. Pharmacological inhibition of piezo 1 with Dooku 1 largely abolished the positive effect of the 16 Hz ELF-PEMF exposure on osteogenic maturation of SCP-1 cells. In summary, the intermittent exposure strategy enhanced the positive effects of 16 Hz continuous ELF-PEMF exposure in terms of cell viability and osteogenesis. This effect was shown to be mediated by an increased expression of piezo 1 and related Ca 2+ influx. Thus, the intermittent exposure strategy is a promising way to further optimize the therapeutic effects of the 16 Hz ELF-PEMF regarding fracture healing or osteoporosis.

The effects of extremely low-frequency electromagnetic field (ELF-MF) exposure on living systems have been widely studied at the fundamental level and also claimed as beneficial for the treatment of diseases for over 50 years. However, the underlying mechanisms and cellular targets of ELF-MF exposure remain poorly understood and the field has been plagued with controversy stemming from an endemic lack of reproducibility of published findings. To address this problem, we here demonstrate a technically simple and reproducible EMF exposure protocol to achieve a standardized experimental approach which can be readily adopted in any lab. As an assay system, we chose a commercially available inflammatory model human cell line; its response to magnetic fields involves changes in gene expression which can be monitored by a simple colorimetric reporter gene assay. The cells were seeded and cultured in microplates and inserted into a custom-built, semi-automated incubation and exposure system which accurately controls the incubation (temperature, humidity, CO2) and magnetic-field exposure conditions. A specific alternating magnetic field (<1.0% spatial variance) including far-field reduction provided defined exposure conditions at the position of each well of the microplate. To avoid artifacts, all environmental and magnetic-field exposure parameters were logged in real time throughout the duration of the experiment. Under these extensively controlled conditions, the effect of the magnetic field on the cell cultures as assayed by the standardized operating procedure was highly reproducible between experiments. As we could fully define the characteristics (frequency, intensity, duration) of the pulsed magnetic field signals at the position of the sample well, we were, for the first time, able to accurately determine the effect of changing single ELF-MF parameters such as signal shape, frequency, intensity and duty cycle on the biological response. One signal in particular (10 Hz, 50% duty cycle, rectangular, bipolar, 39.6μT) provided a significant reduction in cytokine reporter gene expression by 37% in our model cell culture line. In sum, the accuracy, environmental control and data-logging capacity of the semi-automated exposure system should greatly facilitate research into fundamental cellular response mechanisms and achieve the consistency necessary to bring ELF-MF/PEMF research results into the scientific mainstream.

Delayed fracture healing and fracture non-unions impose an enormous burden on individuals and society. Successful healing requires tight communication between immune cells and bone cells. Macrophages can be found in all healing phases. Due to their high plasticity and long life span, they represent good target cells for modulation. In the past, extremely low frequency pulsed electromagnet fields (ELF-PEMFs) have been shown to exert cell-specific effects depending on the field conditions. Thus, the aim was to identify the specific ELF-PEMFs able to modulate macrophage activity to indirectly promote mesenchymal stem/stromal cell (SCP-1 cells) function. After a blinded screening of 22 different ELF-PEMF, two fields (termed A and B) were further characterized as they diversely affected macrophage function. These two fields have similar fundamental frequencies (51.8 Hz and 52.3 Hz) but are emitted in different groups of pulses or rather send-pause intervals. Macrophages exposed to field A showed a pro-inflammatory function, represented by increased levels of phospho-Stat1 and CD86, the accumulation of ROS, and increased secretion of pro-inflammatory cytokines. In contrast, macrophages exposed to field B showed anti-inflammatory and pro-healing functions, represented by increased levels of Arginase I, increased secretion of anti-inflammatory cytokines, and growth factors are known to induce healing processes. The conditioned medium from macrophages exposed to both ELF-PEMFs favored the migration of SCP-1 cells, but the effect was stronger for field B. Furthermore, the conditioned medium from macrophages exposed to field B, but not to field A, stimulated the expression of extracellular matrix genes in SCP-1 cells, i.e., COL1A1 , FN1 , and BGN . In summary, our data show that specific ELF-PEMFs may affect immune cell function. Thus, knowing the specific ELF-PEMFs conditions and the underlying mechanisms bears great potential as an adjuvant treatment to modulate immune responses during pathologies, e.g., fracture healing.

Cigarette smoking (CS) is one of the main factors related to avoidable diseases and death across the world. Cigarette smoke consists of numerous toxic compounds that contribute to the development of osteoporosis and fracture nonunion. Exposure to pulsed electromagnetic fields (PEMF) was proven to be a safe and effective therapy to support bone fracture healing. The aims of this study were to investigate if extremely low frequency (ELF-) PEMFs may be beneficial to treat CS-related bone disease, and which effect the duration of the exposure has. In this study, immortalized human mesenchymal stem cells (SCP-1 cells) impaired by 5% cigarette smoke extract (CSE) were exposed to ELF-PEMFs (16 Hz) with daily exposure ranging from 7 min to 90 min. Cell viability, adhesion, and spreading were evaluated by Sulforhodamine B, Calcein-AM staining, and Phalloidin-TRITC/Hoechst 33342 staining. A migration assay kit was used to determine cell migration. Changes in TGF-β signaling were evaluated with an adenoviral Smad2/3 reporter assay, RT-PCR, and Western blot. The structure and distribution of primary cilia were analyzed with immunofluorescent staining. Our data indicate that 30 min daily exposure to a specific ELF-PEMF most effectively promoted cell viability, enhanced cell adhesion and spreading, accelerated migration, and protected TGF-β signaling from CSE-induced harm. In summary, the current results provide evidence that ELF-PEMF can be used to support early bone healing in patients who smoke., Competing Interests: The authors declare no conflict of interest. Sachtleben GmbH provided the ELF-PEMF devices (Somagen®) and the background on the physical parameters, but were not involved in the study design or the data evaluation.

The finding that alterations in electrical potential play an important role in the mechanical stimulation of the bone provoked hype that noninvasive extremely low frequency pulsed electromagnetic fields (ELF-PEMF) can be used to support healing of bone and osteochondral defects. This resulted in the development of many ELF-PEMF devices for clinical use. Due to the resulting diversity of the ELF-PEMF characteristics regarding treatment regimen, and reported results, exposure to ELF-PEMFs is generally not among the guidelines to treat bone and osteochondral defects. Notwithstanding, here we show that there is strong evidence for ELF-PEMF treatment. We give a short, confined overview of in vitro studies investigating effects of ELF-PEMF treatment on bone cells, highlighting likely mechanisms. Subsequently, we summarize prospective and blinded studies, investigating the effect of ELF-PEMF treatment on acute bone fractures and bone fracture non-unions, osteotomies, spinal fusion, osteoporosis, and osteoarthritis. Although these studies favor the use of ELF-PEMF treatment, they likewise demonstrate the need for more defined and better controlled/monitored treatment modalities. However, to establish indication-oriented treatment regimen, profound knowledge of the underlying mechanisms in the sense of cellular pathways/events triggered is required, highlighting the need for more systematic studies to unravel optimal treatment conditions., Competing Interests: The authors declare no conflict of interest.

Extremely low-frequency pulsed electromagnetic field (ELF-PEMF) therapy is proposed to support bone healing after injuries and surgical procedures, being of special interest for elderly patients. This study aimed at investigating the effect of a specific ELF-PEMF, recently identified to support osteoblast function in vitro, on bone healing after high tibial osteotomy (HTO). Patients who underwent HTO were randomized to ELF-PEMF or placebo treatment, both applied by optically identical external devices 7 min per day for 30 days following surgery. Osseous consolidation was evaluated by post-surgical X-rays (7 and 14 weeks). Serum markers were quantified by ELISA. Data were compared by a two-sided t-test (α = 0.05). Device readouts showed excellent therapy compliance. Baseline parameters, including age, sex, body mass index, wedge height and blood cell count, were comparable between both groups. X-rays revealed faster osseous consolidation for ELF-PEMF compared to placebo treatment, which was significant in patients ≥50 years (∆ mean = 0.68%/week; p = 0.003). Findings are supported by post-surgically increased bone-specific alkaline phosphatase serum levels following ELF-PEMF, compared to placebo (∆ mean = 2.2 µg/L; p = 0.029) treatment. Adverse device effects were not reported. ELF-PEMF treatment showed a tendency to accelerate osseous consolidation after HTO. This effect was stronger and more significant for patients ≥50 years. This ELF-PEMF treatment might represent a promising adjunct to conventional therapy supporting osseous consolidation in elderly patients.

Extremely low-frequency pulsed electromagnetic field (ELF-PEMF) devices have been used in the clinic for the treatment of bone disorders over the past 30 years. However, the underlying mechanism of which ELF-PEMFs exert an effect on tissues at a cellular level is not well understood. Hence, in this study, we explored the potential of different ELF-PEMF signals in modulating human adipose-derived mesenchymal stromal cells' (hAMSC) osteogenic capability. The cell proliferation rate was assessed using carboxyfluorescein succinimidyl ester (CFSE) method. The osteogenesis potential of cells was determined by alkaline phosphatase (ALP) activity, Alizarin-Red S staining, and RT-qPCR. Finally, the intracellular signaling pathway of a selected ELF-PEMF signal was examined using the PathScan Intracellular Signaling Array. Among the tested ELF-PEMF signals, program 20 (26 Hz) showed activation of the Akt and MAPK/ERK signaling cascade and significant upregulations of collagen I, alkaline phosphatase, and osteocalcin when compared to nonstimulated cells. This study demonstrates the potential of certain ELF-PEMF signal parameters to induce osteogenic differentiation of hAMSC and provides important clues in terms of the molecular mechanisms for the stimulation of osteogenic effects by ELF-PEMF on hAMSC.

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.)

Human adipose-derived mesenchymal stem cells (Ad-MSCs) have been proposed as suitable option for cell-based therapies to support bone regeneration. In the bone environment, Ad-MSCs will receive stimuli from resident cells that may favor their osteogenic differentiation. There is recent evidence that this process can be further improved by extremely low frequency pulsed electromagnetic fields (ELF-PEMFs). Thus, the project aimed at (i) investigating whether co-culture conditions of human osteoblasts (OBs) and Ad-MSCs have an impact on their proliferation and osteogenic differentiation; (ii) whether this effect can be further improved by repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz); (iii) and the effect of these ELF-PEMFs on human osteoclasts (OCs). Osteogenic differentiation was improved by co-culturing OBs and Ad-MSCs when compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1 had best effects on total protein content, alkaline phosphatase (AP) activity, and matrix mineralization. Osteogenic differentiation was further improved by both ELF-PEMFs investigated. Interestingly, only repetitive exposure to 26 Hz ELF-PEMF increased Trap5B activity in OCs. Considering this result, a treatment with gradually increasing frequency might be of interest, as the lower frequency (16 Hz) could enhance bone formation, while the higher frequency (26 Hz) could enhance bone remodeling., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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.

For many years electromagnetic fields (EMFs) have been used clinically with various settings as an exogenous stimulation method to promote fracture healing. However, underlying mechanisms of action and EMF parameters responsible for certain effects remain unclear. Our aim was to investigate the influence of defined EMFs on human osteoblasts' and osteoclasts' viability and function. Primary human osteoblasts and osteoclasts were treated 3 times weekly for 21 days during their maturation process using the Somagen® device (Sachtleben GmbH, Hamburg, Germany), generating defined extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs). Certain ELF-PEMF treatment significantly increased the total protein content (up to 66%), mitochondrial activity (up to 91.1%) and alkaline phosphatase (AP) activity (up to 129.9%) of human osteoblasts during the entire differentiation process. Furthermore, ELF-PEMF treatment enhanced formation of mineralized matrix (up to 276%). Interestingly, ELF-PEMF dependent induction of AP activity and matrix mineralization was strongly donor dependent - only osteoblasts with a poor initial osteoblast function responded to the ELF-PEMF treatment. As a possible regulatory mechanism, activation of the ERK1/2 signaling pathway was identified. Maturation of osteoclasts from human monocytes was not affected by the ELF-PEMF treatment. In summary the results indicate that a specific ELF-PEMF treatment with the Somagen® device improves viability and maturation of osteoblasts, while osteoclast viability and maturation was not affected. Hence, ELF-PEMF might represent an interesting adjunct to conventional therapy supporting bone formation during fracture healing or even for the treatment of osteoporosis.

Background: The promotion of the healing process following musculoskeletal injuries comprises growth factor signalling, migration, proliferation and apoptosis of cells. If these processes could be modulated, the healing of tendon tissue may be markedly enhanced. Here, we report the use of the Somagen™ device, which is certified for medical use according to European laws. It generates low-frequency pulsed electromagnetic fields that trigger effects of a nature that are yet to be determined., Methods: A 1.5-cm wide, linear scrape was introduced into patellar tendon fibroblast cultures (N = 5 donors). Treatment was carried out every second day. The regimen was applied three times in total with 30 minutes comprising pulsed electromagnetic field packages with two fundamental frequencies (10 minutes of 33 Hz, 20 minutes of 7.8 Hz). Control cells remained untreated. All samples were analyzed for gap closure time, proliferation and apoptosis one week after induction of the scrape wound., Results: The mean time for bridging the gap in the nontreated cells was 5.05 ± 0.33 days, and in treated cells, it took 3.35 ± 0.38 days (P <0.001). For cell cultures with scrape wounds, a mean value for BrdU incorporation of OD = 0.70 ± 0.16 was found. Whereas low-frequency pulsed electromagnetic fields treated samples showed OD = 1.58 ± 0.24 (P <0.001). However, the percentage of apoptotic cells did not differ between the two groups., Conclusions: Our data demonstrate that low-frequency pulsed electromagnetic fields emitted by the Somagen™ device influences the in vitro wound healing of patellar tendon fibroblasts and, therefore, possibly increases wound healing potential.