## 4 Discussion  
MSCs differentiation is a fundamental step toward bone repair. The Yunnan Baiyao (YB), a Chinese herbal medicine, is well-known for its beneficial effects, especially on bone tissue healing. YB has been reported to have several biological effects, such as hemostatic properties; effective hemostasis after bone trauma is an important step to achieve good healing. Currently, surgeons have the challenge to choose a hemostatic material to ensure an efficient and immediate reduction of hemorrhagic phenomena in the surgical site. A good hemostasis followed by a strong anti-inflammatory effect ensures the proper biological environment to emphasize the osteogenic properties, here investigated, of YB. In our article, we decided to investigate the effects of YB on MSCs osteogenic differentiation, so as to better understand its potential applications in bone regeneration. Our MTT assay highlighted that YB administered at doses higher than 50 \(\upmu\)g/mL, severely affects the cell viability. These results are in accordance with the study of [PERSON] et al., although they worked on other cell types [14]. Starting from this information, we established 50 \(\upmu\)g/mL as the highest dose of YB to be safely administered. We analyzed the gene expression of three osteogenic markers: Runx2, ALPL, and OC. Runx2 is an early osteogenic marker: It is considered essential for the osteogenic differentiation, because it promotes the expression of osteoblastic genes, such as osteocalcin [20]. Moreover, it was demonstrated that Runx2 improves the ADSCs differentiation towards osteoblasts, by increasing the ALP expression in addition to OC expression as well [21]. ALP is also considered an early marker of osteogenic differentiation and it is assumed that its function is to promote matrix mineralization [22]. Several studies demonstrated that ALP gene expression increases in the middle of the osteoinduction process, and then quickly decrease at the end of the osteoinduction process, followed by an intense deposition of bony matrix [23; 24; 25]. OC is a major protein of the bone extracellular matrix and it is expressed exclusively by mature osteoblasts. OC plays a pivotal role in the production of hydroxyapatite crystals [26; 27]. Furthermore, it is considered an indicator of osteoblast differentiation. After a careful analysis of the gene expression carried out from our study, the substantial increase in Runx2, ALPL and OC expression observed in ODM cultured ADSCs indicates that ADSCs were differentiating into osteoblasts; the addition of 25 \(\upmu\)g/mL of YB enhanced the overall expression of these markers, triggering osteogenesis. On the  
Figure 5: Alizarin red S (ARS) stainingassay. (**a**) ARS assay in control ADSCs and ADSCs treated with 25 \(\upmu\)g/mL or 50 \(\upmu\)g/mL of YB at 7 and 21 day of culturing in basal medium. Magnification 20X. (**b**) Quantification of ARS assay in control ADSCs and ADSCs treated with 25 \(\upmu\)g/mL or 50 \(\upmu\)g/mL of YB at 7 and 21 day of culturing in basal medium. (**c**) ARS assay in control ADSCs and ADSCs treated with 25 \(\upmu\)g/mL or 50 \(\upmu\)g/mL of YB at 7 and 21 day of culturing in osteogenic differentiation medium. Magnification 20X. (**d**) Quantification of ARS assay in control ADSCs and ADSCs treated with 25 \(\upmu\)g/mL or 50 \(\upmu\)g/mL of YB at 7 and 21 day of culturing in osteogenic differentiation medium. \({}^{*}p<0.05\); \({}^{**}p<0.01\); \({}^{***}p<0.001\) vs. CTRL. contrary, the treatment with 50 \(\upmu\)g/mL of YB reduced the MSCs differentiation, decreasing the expression of all the osteogenic genes at each time point. Recent studies highlighted a reduced expression of Runx-2 in MSCs from type 2 diabetes patients, correlated to a decreased ability of MSCs to naively differentiate into osteoblasts [9; 28; 29]. Furthermore, new bone formation in diabetic patients is typically reduced by an impairment of osteoblast activity, associated with a reduction of OC production, while the activity of the osteoclasts remains unchanged [30; 31].