## 3 Results
### Impact of YB on Osteogenic Gene Expression  
Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALPL) and osteoclast (OC) gene expression was measured by real-time PCR, to assess the effect of two different doses of YB on ADSCs ability to differentiate towards osteogenic lineage. Our results showed that cells maintained in ODM had a higher expression of the osteogenic markers, compared to those cultured in BM (Figure 3).  
Analyzing, in depth, the effects of YB on cells cultured in basal conditions, we observed that Runx2 expression was downregulated at 7 days, with respect to control (\(p<0.05\)); on the other hand, after 14 days, Runx2 expression was similar in both the culturing conditions. The overcoming of Runx2 expression, with respect to the control, was reached after 21 days of treatment, with both of the YB concentrations (\(p<0.05\)) (Figure 3a).  
In the same basal conditions, ALPL gene expression was downregulated when cells were treated with YB at 50 \(\upmu\)g/mL, both after 14 and 21 days (\(p<0.05\)). Conversely, YB at 25 \(\upmu\)g/mL did not modify ALPL expression at all time points (\(p>0.05\)) (Figure 3b).  
The late osteogenic gene OC followed a different trend. At day 7, it was equally expressed by control and treated ADSCs; on the other hand, at day 14 and day 21, the treatment with YB at 25 \(\upmu\)g/mL significantly up-regulates OC expression (\(p<0.05\)) and the treatment with YB at 50 \(\upmu\)g/mL significantly down-regulated OC expression (\(p<0.05\)) (Figure 3c).  
The results were different for ADSCs cultured in ODM. The analysis of Runx2 gene expression showed that at day 7 and 21 there were no differences between the culturing conditions (\(p>0.05\)); at day 14, the 25 \(\upmu\)g/mL YB-treated cells expressed more Runx2, compared to the control (\(p<0.01\)) and the 50 \(\upmu\)g/mL YB-treated cells expressed less Runx2, compared to the control (\(p<0.01\)) (Figure 3d).  
In osteogenic committed cells, the treatment with the lower dose of YB promoted the up-regulation of ALPL gene at day 7 and 14 (\(p<0.01\)); at day 21, the ALPL expression was down-regulated (\(p<0.01\)) (Figure 3e).  
Figure 2: ADSCs viability measured by MTT assay. (**a**) Viability of control ADSCs and ADSCs treated with 25 \(\upmu\)g/mL or 50 \(\upmu\)g/mL of YB at 3, 7, 14 and 21 day of culturing in basal medium. (**b**) Viability of control ADSCs and ADSCs treated with 25 \(\upmu\)g/mL or 50 \(\upmu\)g/mL of YB at 3, 7, 14 and 21 day of culturing in osteogenic differentiation medium. Absorbance was measured at 570 nm. \({}^{*}\)\(p<0.05\); \({}^{**}\)\(p<0.01\); \({}^{***}\)\(p<0.001\) vs. CTRL.  
OC expression followed the same trend in all time points. Culturing conditions with YB at 25 \(\mu\)g/mL increased OC expression (\(p<0.05\)); YB at 50 \(\mu\)g/mL decreased OC expression, with respect to the control (\(p<0.01\)) (Figure 3f).  
Figure 3: Gene expression of osteogenic markers. (**–c**) Gene expression of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALPL) and osteoclastin (OC) in control ADSCs and ADSCs treated with 25 \(\mu\)g/mL or 50 \(\mu\)g/mL of YB at 3, 7, 14 and 21 day of culturing in basal medium. (**–f**) Gene expression of Runx2, ALPL and OC in control ADSCs and ADSCs treated with 25 \(\mu\)g/mL or 50 \(\mu\)g/mL of YB at 3, 7, 14 and 21 day of culturing in osteogenic differentiation medium. *\(p<0.05\); **\(p<0.01\) vs. CTRL.