# Effects of Black Jade on Osteogenic Differentiation of Adipose Derived Stem Cells under Benzopyrene
## 3 Results and Discussion
Osteoblastic differentiation markers osteeponin and osteocalcin were upregulated in BPS+ES, along with the highest cell counts at day 10 (Figure 5a,b). At day 10, osteeponin and osteocalcin levels in the BPS+ES conditioned medium were 1.84 and 2.63 times higher, respectively, than those in the BPS conditioned medium (Figure 5c). The conditioned media of activated fibroblast cells enhanced the osteogenic differentiation of the ASCs. With BPS, osteogenic differentiation was attenuated significantly in ASCs (Figure 4). However, after exposure to ES, ASCs activated their osteogenic differentiation under benzopyrene (Figures 4 and 5). Based on several reports, Notch signaling controls osteogenic differentiation of ASCs as well as osteogenic development [23, 24, 25]. Although Notch protein suppresses osteogenic differentiation from mesenchymal stem cells by downregulating Wnt/\(\beta\)-catenin and Runx2, Notch activates mineralization during osteogenic development [25]. Bioactive components from black jade in the media-affected Notch-related osteogenic pathways reduced the suppression of osteogenic differentiation in dermal cells, and activated the differentiation besides the protection of dermal cells against fine dust. Figure 4: Expression of osteogenic genes in adipose-derived stem cells (ASCs) exposed to conditioned media. Bands indicating cDNA levels for the osteogenic markers. Histograms indicating relative fold changes for the corresponding intensity levels. BPS+Ext represents that ASCs were exposed to the conditioned medium after treatment using black jade extract (Ext). The conditioned medium (BPS) contained the supernatant from the fibroblast cells exposed to benzo(a)pyrene. The conditioned medium (ES) contained the supernatant from the fibroblast cells exposed to the extract. Con; Control, (\(p<0.05\), \(n=3\)). BPS: benzo(a)pyrene inducing supernatants; ES: extract supernatants. Unlike BPS inhibited osteoblast differentiation, ES protected and activated osteoblastic differentiation of ASCs (Figure 6a,b and Figure 7). Cell counts for osteocytes were about 3.9 times higher than those in BPS at day 10 (Figure 6b). Moreover, osteoblastic differentiation was gradually increased during the 10 days in BPS+ES (Figure 6c). Despite being under BPS, ASCs exposed to ES strongly activated osteogenic differentiation (Figure 7). Although sclerostin secreted by osteoclasts inhibits osteoblastic maturation and bone formation, the protein is an important marker to differentiate between the osteoblast and osteocyte during early osteogenesis development [26]. The upregulation of sclerostin has been reported in osteocytes surrounding bone, osteoclasts under stresses, developing bone and bone marrow-derived osteoclast precursors, to decrease the formation of osteoclasts [26]. These  
Figure 5: Protective effect of black jade extract on osteoblastic differentiation in adipose-derived stem cells (ASCs) under conditioned mediums. These results are from ASCs under conditioned mediums (BPS, BPS+EX). (**a**) Histograms indicating cell counts and expression levels for the osteoponin and osteocalcin detected using flow cytometry. (**b**) Relative fold changes for cell counts in the corresponding graphs. (**c**) Relative fold changes between BPS and BPS+ES. After exposure to ES, ASCs were exposed to BPS. The conditioned medium (BPS) contained the supernatant from the fibroblast cells exposed to benzo(a)pyrene. The conditioned medium (ES) contained the supernatant from the fibroblast cells exposed to the extract. Con; Control, (\(p<0.05\), \(n=3\)). results suggest that the extract has the potential to modulate the formation of osteoclasts during bone development. Figure 6: Protective effect on osteoblast differentiation by black jade extract in adipose-derived stem cells (ASCs) under conditioned mediums. These results are from ASCs under conditioned mediums (BPS, BPS+EX). (**a**) Graphs indicating sclerosin cell counts detected using flow cytometry. (**b**) Relative fold changes for sclerostin cell counts.