## 3 Results
### Influence of Conditioned Osteoblastic Supernatants on Pre-Osteoclasts
#### 3.3.3 Released Mediators  
The release of TRAP5b, BMP-2, cystatin C, IFN-\(\beta\), IL-1\(\beta\), -4, -6, -10, -17A, -23, OPN, TGF-\(\beta\)1, and TNF-\(\alpha\) were analyzed for the pre-osteoclasts stimulated with ESO and NSO, and the control\({}_{\text{invc}}\). The concentration of the control\({}_{\text{invc}}\) was subtracted from the concentration of the cultivated pre-osteoclasts. The data for IFN-\(\beta\) are not shown as no proteins could be detected.  
The different stimulation conditions did not significantly influence the concentration of IL-1\(\beta\), OPN, cystatin C, TRAP5b, TGF-\(\beta\)1, and IL-17A (Figure S3). For TNF-\(\alpha\) and IL-6 lower amounts of proteins were detected after the stimulation with pre-osteoclasts compared to the incubated medium without any cells (control\({}_{\text{invc}}\)). As the protein concentrations of the control\({}_{\text{invc}}\) were subtracted from the protein concentrations of the sample, negative concentrations are shown (Figure 6c).  
The concentration of IL-4 was downregulated by 7 d, 14 d, and 21 d ESO media compared to NSO. This downregulation was significant for 7 d (\(p\) = 0.0008) and for 14 d (\(p\) = 0.0087). A similar trend was observed for IL-10, IL-23, and BMP-2. The IL-10 concentration was downregulated with 7 d and 14 d ESO media and significantly downregulated for 21 d compared to the NSO (\(p\) = 0.0002). For IL-23, differences between the ESO and the control were the most pronounced after 7 d (\(p\) = 0.008) and slighter at 14 d (\(p\) = 0.051) and 21 (\(p\) = 0.08). The concentration of BMP-2 was mainly changed through indirect stimulation with the 21 d medium. BMP-2 was reduced with the ESO supernatant (\(p\) = 0.059) compared to the NSO (Figure 6b).  
For TNF-\(\alpha\), the lowest protein amount was detected for the cultivation with 7 d ESO supernatant compared to the NSO (\(p\) < 0.0001). With 14 d and 21 d, no changes between the ESO and NSO were observed. IL-6 was mainly regulated by the 7 d supernatant; stimulation with ESO led to a significant upregulation of IL-6 compared to NSO (\(p\) = 0.0036). Media generated after 14 d and 21 d stimulation did not affect the IL-6 release (Figure 6c).  
Figure 6: Gene expression and protein data of pre-osteoclasts after 48 h incubation with pooled supernatant of osteoblast (ESO) stimulated over 7, 14, and 21 d and without electric stimulation (NSO). (**a**) Gene expression data were related to the NSO using the 2\({}^{-\Delta\Delta\text{Ct}}\) method. Boxplots are used to present the distribution of the total results with significant changes [n = 8]. Significant differences were determined using the two-way ANOVA with Tukey. # indicates significant differences between the ESO and NSO: # \(p<0.05\). * indicates significant differences between stimulation time points: * \(p<0.05\). (**b**, **c**) Protein concentration related to the total protein concentration of the supernatant. The respective protein concentration from the control\({}_{\text{invc}}\) was subtracted. Boxplots are used to present the distribution of the total results [n \(\geq 6\)]. Significant differences were determined using the two-way ANOVA with Tukey. * indicates significant differences: * \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\), *** \(p<0.0001\). (**b**) Proteins that were produced by the pre-osteoclasts (**c**) proteins that were consumed by the pre-osteoclasts.