## 4 Discussion
### Activation of Pre-Osteoclasts  
Osteoclasts are the major antagonists for osteoblasts, and their interplay is essential for bone turnover and remodeling [36]. On the one hand, excessive activity of osteoclasts can lead to diseases such as osteoporosis, periprosthetic osteolysis, or bone tumors; on the other hand, reduced activity can lead to osteopterosis [49]. Therefore, studying osteocl cell behavior under electric stimulation is important to understand bone remodeling processes. Before the pre-osteoclast cells were incubated with the specific osteoblast supernatants, they were first stimulated with RANKL and M-CSF. Both are essential factors that induce and promote osteoclastogenesis in vitro [50]. The subsequent change of cell culture conditions with the osteoblast media was intended to clarify whether the factors secreted by osteoblasts can maintain osteoclastogenesis.  
As for the PBMCs, most of the changes were observed with the 7 d medium. _MMP9_ and _CA2_ were upregulated in pre-osteoclasts cultured with the ESO supernatant comparedto the NSO. _MMP9_ plays an important role in bone resorption processes and remodeling by degrading the organic matrix and releasing the extracellular matrix proteins [51]. [PERSON] et al. reported an upregulation of _MMP9_ during the improved healing process of an amputated rat limb through electric stimulation [52]. This highlights the importance of _MMP9_ and its role as a marker for osteoclastness through electric stimulation. _CA2_ is important for bone resorption as it regulates the pH through H+ production, leading to an acidification of the extracellular environment [53]. The simultaneous upregulation of _MMP9_ and _CA2_ indicates an increased induction of osteoclast activity through the cultivation with 7 d ESO supernatants. This increase in activity can be related, as explained before, to the increased TNF-\(\alpha\) concentration in the ESO medium compared to the NSO medium. Further, the medium of the NSO contained a higher concentration of the osteocl antagonists IFN-\(\beta\) and IL-10. This could have led to the higher activity of the pre-osteoclasts which were cultured with the ESO medium compared to the cells cultured with the NSO medium. In addition, a higher IL-6 protein release and _MCP1_ expression were observed. IL-6 is known to trigger the expression of _MCP-1_ in cells, and the resulting higher _MCP-1_ expression may, in turn, further increase osteoclastness [54; 55; 56], resulting in the enhanced recruitment of monocytic cells through the 7 d ESO supernatant. It can be postulated that the stimulation with electric fields alters the osteoblastic secretion of proteins, which in turn influence the differentiation and activity of osteocl cells and therefore affect remodeling processes. This fact might be more marked at the earlier stimulation time points than the later ones, as the induction and the maintenance of osteoclastness were more prominent with 7 d ESO medium.  
It was not possible to generate multinuclear osteoclasts through the pre-stimulation or through the cultivation with the ESO or NSO media. To improve the outcome in the generation of osteoclasts, the usage of mineralized surfaces or scaffolds can be implemented. Through the cultivation on apatite-containing materials, osteoclastness is enhanced and more robust sealing zones are formed compared to the cultivation on plastic [57]. Further, resorption pits, as results from osteoclast activity, can be used to make assumptions about the resorption activity [57; 58; 59].