Pulsed-electromagnetic-field induced osteoblast differentiation requires activation of genes downstream of adenosine receptors A2A and A3

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.

Pulsed electromagnetic field (PEMF) transiently stimulates the rate of mineralization in a 3-dimensional ring culture model of osteogenesis

Pulsed Electromagnetic Field (PEMF) has shown efficacy in bone repair and yet the optimum characteristics of this modality and its molecular mechanism remain unclear. To determine the effects of timing of PEMF treatment, we present a novel three-dimensional culture model of osteogenesis that demonstrates strong de novo generation of collagen and mineral matrix and exhibits stimulation by PEMF in multiple stages over 62 days of culture. Mouse postnatal day 2 calvarial pre-osteoblasts were cast within and around Teflon rings by polymerization of fibrinogen and cultured suspended without contact with tissue culture plastic. Ring constructs were exposed to PEMF for 4h/day for the entire culture (Daily), or just during Day1-Day10, Day11-Day 27, or Day28-Day63 and cultured without PEMF for the preceding or remaining days, and compared to no-PEMF controls. PEMF was conducted as HF Physio, 40.85 kHz frequency with a 67 ms burst period and an amplitude of 1.19 mT. Osteogenesis was kinetically monitored by repeated fluorescence measurements of continuously present Alizarin Red S (ARS) and periodically confirmed by micro-CT. PEMF treatment induced early-onset and statistically significant transient stimulation (~4-fold) of the mineralization rate when PEMF was applied Daily, or during D1-D10 and D11-D27. Stimulation was apparent but not significant between D28-D63 by ARS but was significant at D63 by micro-CT. PEMF also shifted the micro-CT density profiles to higher densities in each PEMF treatment group. Ring culture generated tissue with a mineral:matrix ratio of 2.0 by thermogravimetric analysis (80% of the calvaria control), and the deposited crystal structure was 50% hydroxyapatite by X-ray diffraction (63% of the calvaria and femur controls), independent of PEMF. These results were consistent with backscatter, secondary electron, and elemental analysis by scanning electron microscopy. Thus, in a defined, strong osteogenic environment, PEMF applied at different times was capable of further stimulation of osteogenesis with the potential to enhance bone repair.

Letter to the Editor: Strawberries: Facts, Truth, Misinformation, Publication Bias and the Importance of Negative Results.

We commonly make the assumption that information provided in instructions and/or publications is correct and based on proven and unbiased knowledge. In a recent study about strawberry seed germination, which will be reported concurrently with this letter, seed supplier’s instructions (based on accepted wisdom among growers) were actually tested, and the results of that part of the study brought to mind the insidious nature of publication bias. This testing was done because the seed planting in question was a crucial component of a scientific study I recently completed on the effects of electromagnetic fields on seed germination in which more than 7,000 seeds were planted, carefully and individually, and their germination rates were studied closely. Briefly, the recent study to which I will make reference describes the interaction between three different treatments prior to planting the seeds: pre-freezing, pre-soaking, and the application of PEMF. It is widely held that the first two, especially pre-freezing, is essential for strawberry seed germination. These procedures are also often reported in the methods section as a side note in scientific reports, so I took them as a given. Our goal was to determine the extent to which the application of PEMF interacted with either or both of the widely accepted pre-treatments. But when the data came in, I did not see evidence of any positive effects on germination resulting from either pre-treatment when studied separately or in combination. In fact, both pre-treatments appeared to have a slight negative effect on the germination rate, and neither under any circumstances interacted with PEMF treatment in a positive way at any level of practical importance. These negative results will be submitted for publication in JoSaM, in keeping with our policy to fight against publication bias.

Pulsed Electromagnetic Field Inhibits Synovitis via Enhancing the Efferocytosis of Macrophages

Synovitis plays an important role in the pathogenesis of arthritis, which is closely related to the joint swell and pain of patients. The purpose of this study was to investigate the anti-inflammatory effects of pulsed electromagnetic fields (PEMF) on synovitis and its underlying mechanisms. Destabilization of the medial meniscus (DMM) model and air pouch inflammation model were established to induce synovitis in C57BL/6 mice. The mice were then treated by PEMF (pulse waveform, 1.5 mT, 75 Hz, 10% duty cycle). The synovitis scores as well as the levels of IL-1β and TNF-α suggested that PEMF reduced the severity of synovitis in vivo. Moreover, the proportion of neutrophils in the synovial-like layer was decreased, while the proportion of macrophages increased after PEMF treatment. In addition, the phagocytosis of apoptotic neutrophils by macrophages (efferocytosis) was enhanced by PEMF. Furthermore, the data from western blot assay showed that the phosphorylation of P38 was inhibited by PEMF. In conclusion, our current data show that PEMF noninvasively exhibits the anti-inflammatory effect on synovitis via upregulation of the efferocytosis in macrophages, which may be involved in the phosphorylation of P38.

Translational Insights into Extremely Low Frequency Pulsed Electromagnetic Fields (ELF-PEMFs) for Bone Regeneration after Trauma and Orthopedic Surgery

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.

Pulsed Electromagnetic Field Therapy Improves Osseous Consolidation after High Tibial Osteotomy in Elderly Patients—A Randomized, Placebo-Controlled, Double-Blind Trial

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.

Global Responses of Il-1β-Primed 3D Tendon Constructs to Treatment with Pulsed Electromagnetic Fields

Tendinopathy is accompanied by a cascade of inflammatory events promoting tendon degeneration. Among various cytokines, interleukin-1β plays a central role in driving catabolic processes, ultimately resulting in the activation of matrix metalloproteinases and a diminished collagen synthesis, both of which promote tendon extracellular matrix degradation. Pulsed electromagnetic field (PEMF) therapy is often used for pain management, osteoarthritis, and delayed wound healing. In vitro PEMF treatment of tendon-derived cells was shown to modulate pro-inflammatory cytokines, potentially limiting their catabolic effects. However, our understanding of the underlying cellular and molecular mechanisms remains limited. We therefore investigated the transcriptome-wide responses of Il-1β-primed rat Achilles tendon cell-derived 3D tendon-like constructs to high-energy PEMF treatment. RNASeq analysis and gene ontology assignment revealed various biological processes to be affected by PEMF, including extracellular matrix remodeling and negative regulation of apoptosis. Further, we show that members of the cytoprotective Il-6/gp130 family and the Il-1β decoy receptor Il1r2 are positively regulated upon PEMF exposure. In conclusion, our results provide fundamental mechanistic insight into the cellular and molecular mode of action of PEMF on tendon cells and can help to optimize treatment protocols for the non-invasive therapy of tendinopathies.

Pulsed electromagnetic field stimulation may improve fusion rates in cervical arthrodesis in high-risk populations

Objectives Pulsed electromagnetic field (PEMF) stimulation was evaluated after anterior cervical discectomy and fusion (ACDF) procedures in a randomized, controlled clinical study performed for United States Food and Drug Administration (FDA) approval. PEMF significantly increased fusion rates at six months, but 12-month fusion outcomes for subjects at elevated risk for pseudoarthrosis were not thoroughly reported. The objective of the current study was to evaluate the effect of PEMF treatment on subjects at increased risk for pseudoarthrosis after ACDF procedures. Methods Two evaluations were performed that compared fusion rates between PEMF stimulation and a historical control (160 subjects) from the FDA investigational device exemption (IDE) study: a post hoc (PH) analysis of high-risk subjects from the FDA study (PH PEMF); and a multicentre, open-label (OL) study consisting of 274 subjects treated with PEMF (OL PEMF). Fisher’s exact test and multivariate logistic regression was used to compare fusion rates between PEMF-treated subjects and historical controls. Results In separate comparisons of PH PEMF and OL PEMF groups to the historical control group, PEMF treatment significantly (p < 0.05, Fisher’s exact test) increased the fusion rate at six and 12 months for certain high-risk subjects who had at least one clinical risk factor of being elderly, a nicotine user, osteoporotic, or diabetic; and for those with at least one clinical risk factor and who received at least a two- or three-level arthrodesis. Conclusion Adjunctive PEMF treatment can be recommended for patients who are at high risk for pseudoarthrosis. Cite this article: D. Coric, D. E. Bullard, V. V. Patel, J. T. Ryaby, B. L. Atkinson, D. He, R. D. Guyer. Pulsed electromagnetic field stimulation may improve fusion rates in cervical arthrodesis in high-risk populations. Bone Joint Res 2018;7:124–130. DOI: 10.1302/2046-3758.72.BJR-2017-0221.R1.

Novel protective effects of pulsed electromagnetic field ischemia/reperfusion injury rats

Pulsed electromagnetic field (PEMF) treatment protected ischaemia/reperfusion (I/R) injury from apoptosis via B-cell lymphoma 2 (Bcl-2), Bax and nitric oxide (NO) releasing.