# Effects of Black Jade on Osteogenic Differentiation of Adipose Derived Stem Cells under Benzopyrene
## 3 Results and Discussion
In sharp contrast, without the extract, the fibroblast cells showed downregulated anti-apoptotic markers besides increased apoptotic markers under benzo(a)pyrene (Figure 3a, Supplementary Materials S1). Unlike the cellular viability, which was attenuated about 8.3 times more than the control under benzo(a)pyrene, the extract enhanced cellular viability about 2.9 times more than the exposure to benzo(a)pyrene (Figure 3b). Based on reports from the World Health Organization (WHO), fine particles could induce several diseases including lung cancer, cardiovascular diseases, respiratory diseases, skin inflammation, and allergy [21]. Notably, skin is directly affected by atmospheric fine particles leading to dermatological problems, including dermatitis and dermal irritations [22]. In addition, fibroblast cells under stress upregulate the release of proinflammatory cytokines and secrete detrimental signals to various peripheral cells types including immune cells, adipocytes, and stem cells [22]. Overall, the black jade extract protects dermal cells against oxidative stress induced by benzo(a)pyrene. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Composition** & **Control * (\%)** & **Heated Black jade (\%)** \\ \hline SiO\({}_{2}\) & 41.30 \(\pm\) 0.1 & 41.80 \(\pm\) 0.2 \(\lx@math@degree\) \\ MgO & 34.20 \(\pm\) 0.45 & 34.80 \(\pm\) 0.1 \(\lx@math@degree\) \\ Total Fe & 6.78 \(\pm\) 0.1 & 6.62 \(\pm\) 0.2 \(\lx@math@degree\) \\ Al\({}_{2}\)O\({}_{3}\) & 2.81 \(\pm\) 0.2 & 1.32 \(\pm\) 0.2 \(\lx@math@degree\) \\ CaO & 1.57 \(\pm\) 0.3 & 1.68 \(\pm\) 0.1 \(\lx@math@degree\) \\ FeO & 0.59 \(\pm\) 0.02 & 0.98 \(\pm\) 0.01 \(\lx@math@degree\) \\ K\({}_{2}\)O & 0.42 \(\pm\) 0.02 & 0.62 \(\pm\) 0.02 \(\lx@math@degree\) \\ Na\({}_{2}\)O & 0.38 \(\pm\) 0.01 & 0.74 \(\pm\) 0.01 \(\lx@math@degree\) \\ TiO\({}_{2}\) & 0.16 \(\pm\) 0.02 & 0.025 \(\pm\) 0.0002 \(\lx@math@degree\) \\ Cr & 0.07 \(\pm\) 0.01 & \textless{}0.005 \(\pm\) 0.0001 \(\lx@math@degree\) \\ Mn & 0.08 \(\pm\) 0.001 & \textless{}0.005 \(\pm\) 0.0002 \(\lx@math@degree\) \\ \hline \hline \end{tabular}  
* The control is normal black jade without heating.
* Significant difference (\(p<0.05\), \(n=3\)), independent \(t\) test.  
\end{table} 
Table 2: Comparison of compositions and contents between the black jade and the heated black jade. Figure 4, Supplementary Materials S1 and S2 show the expression of osteogenic genes in ASCs exposed to conditioned media (the supernatants of fibroblast cells exposed to  
Figure 3: Expression levels for anti-apoptotic and apoptotic markers in fibroblast cells exposed to black jade extract. (**a**) The bar graphs indicating relative fold changes for the markers. (**b**) Histograms for the cellular viability under benzo(a)pyrene (BP) or the black jade extract (Ext). BP+Ext represents that fibroblast cells were exposed to BP after treatment using Ext. Con; control, (\(p<0.05\), \(n=3\)). Figure 2: Diameters of particles in the grinded black jade. Distributions of particle size in one and two cycles of heating and grinding. (**a**) Red dotted lines indicate the peaks for the particle size of the greatest portion in the black jade particles. (**b**) Comparison of peak sizes between first and second cycles (\(p<0.05\), \(n=3\)). several conditions: control, black jade extract and benzo(a)pyrene). Unlike ASCs in the medium conditioned with benzo(a)pyrene (BPS) that showed downregulated osteogenic genes, the medium conditioned with black jade extract (ES) showed activated expression of pre-osteobatic genes. Under BPS, all markers were downregulated and notably, BMP6 and COL1A1 were strongly decreased in ASCs (Figure 4). However, all markers in ASCs were strongly upregulated under BPS+ESm (Figure 4). Comparing between BPS and BPS+ES conditioned media, the levels of Runx2 and Dlx5 were 3.89 and 2.40 times higher, respectively, in the latter (BPS+ES) (Figure 4). Interestingly, although the conditioned medium (BPS) suppressed osteoblastic differentiation of ASCs, BPS+ES and ES conditioned media showed the opposite effect (Figure 5a,b).