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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.