## 4 Discussion
### Differentiation of PBMCs  
Monocytes as well as macrophages play an important role in inflammation, homeostasis, tissue development, and injury repair [38]. They have the potential to differentiate into osteoclasts. Therefore, understanding their differentiation potential is important for further information on bone remodeling processes [39]. This study focused on the impact of indirect stimulation with ESO on the induction of PBMC differentiation into osteoclasts. It was possible to initiate osteocl differentiation of PBMCs without the external differentiation factors M-CSF and RANKL [28] by using supernatants from stimulated osteoblasts. A higher number of osteoclast-like cells were seen with 7 d and 14 d ESO media, and an increase in _ACP5_ gene expression could be observed. Further, a significant upregulation in _CTSK_ gene expression could be observed with the 7 d medium, a gene that is an important marker for the activity of osteocl cells [40]. As no major changes in the _RANK_ gene expression were noticeable, it can be assumed that RANKL was not the key factor for the induced osteoclastogenesis. Studies have shown that other mediators such as TNF-\(\alpha\), TGF-\(\beta\), and IL-33 have the potential to enhance osteoclast differentiation in the absence of the RANK-RANKL pathway [41; 42; 43]. In this study, TNF-\(\alpha\) was doubled in the ESO supernatants; thus, the differentiation through TNF-\(\alpha\) can be assumed [41; 44; 45]. Further, [PERSON] et al. revealed the inhibition of RANKL-induced osteoclastogenesis in directly electrically stimulated macrophages, which would underline the assumption of a RANKL-independent differentiation observed in this study [20]. Another argument supporting the hypothesis is the IFN-\(\beta\) concentration, as IFN-\(\beta\) is an anti-osteoclastogenic cytokine [46]. The highest amount of released IFN-\(\beta\) was detectable in PBMCs cultivated with the 7 d NSO medium. Comparing the protein concentration of ESO and NSO proves that, especially after seven days, the presence of osteocl antagonists such as IFN-\(\beta\) and IL-10 were present in the NSO medium, but not in the ESO medium. IL-10, like IFN-\(\beta\), is known for its possible role in inhibiting osteoclast formation [47]. Vice versa, the induction of _ACP5_ and _CTSK_ mRNA transcripts in PBMCs cultivated with the 7 d ESO medium correlates with the absence of IFN-\(\beta\) and IL-10 proteins. Although the anti-osteoclast mediators IL-10 and IFN-\(\beta\) were detectable in the ESO medium after 14 days, the induction of the osteocl differentiation and activity markers was still demonstrated. However, besides the presence of higher IL-6 protein concentrations, the clearly enhanced concentration of the TNF-\(\alpha\) protein in particular seemed to be more inductive for osteoclastogenesis. Although IFN-\(\beta\) and IL-10 were no longer detectable in the 21 d ESO medium, the composition of the medium did not affect osteoclastogenesis. This could be due to the presence of other mediators in the medium, which we did not consider in our setup. However, in our previous work, we could show that, especially on day 21, the highest mineralization of osteoblasts was detectable following stimulation with alternating electric fields [30]. Since osteoclastogenesis is particularly inhibited by enhanced calcium ion levels [48] the accumulation of calcium in the 21 d ESO medium might have a greater inhibitory effect on osteoclastogenesis than the ESO media of the earlier time points.