## 3 Results
### Cell Differentiation  
The results of the hMSC differentiation experiments following exposure to the NTAPPJs are shown in Figure 2. The expression of PPARG, COL II, and OPN indicated the adipogenesis, chondrogenesis, and osteogenesis, respectively.  
Generally, for the expression of all genes tested in this study, the air-flowing NTAPPJ resulted in better differentiation than did the nitrogen-flowing NTAPPJ (Figure 3a-c).  
In terms of adipogenesis, expression of PPARG was increased as the duration of exposure to the NTAPPJ increased through the direct method (Figure 3a). However, there was no significant effect when the NTAPPJ exposure was carried out using the media-treatment method (Figure 3a).  
In terms of chondrogenesis and osteogenesis, the expression of COL II and OPN following exposure to the NTAPPJ, either by the direct or media-treatment method, showed significantly higher levels compared to those of the control for the longer duration of exposure (Figure 3b,c).  
The results were confirmed using the fluorescent staining of OPN, as it showed the most dramatic increase of gene expression in RT-PCR analysis following the NTAPPJ exposure, compared to PPARG or COL II. The results were in concordance with RT-PCR analysis, as brightly colored green staining was evident for hMSCs directly exposed to 4 min of the air-flowing NTAPPJ by the direct and media-treatment methods (Figure 4b,c) and hMSCs exposed to 4 min of the nitrogen-flowing NTAPPJ by the direct method (Figure 4d), when compared to the numbers of cells present in the culture (Figure 4f). There was no evidence of any fluorescence for the control samples (Figure 4a), and hMSCs  
Figure 3: Relative gene expression in the human mesenchymal stem cells (hMSCs) after 14 days of cell culture following different durations of plasma exposure by the NTAPPJ: (**a**) PPARG, (**b**) COL II, and (**c**) OPN. The results are expressed as the relative fold increase compared to that of the control, with GAPDH as the housekeeping gene. Each test group was coded as follows: AD, exposed to the air-flowing NTAPPJ by the direct method; AM, exposed to the air-flowing NTAPPJ by the media-treatment method; ND, exposed to the nitrogen-flowing NTAPPJ by the direct method; and NM, exposed to the nitrogen-flowing NTAPPJ by the media-treatment method. The number after each code indicates the duration of the exposure to the NTAPPJ in minutes (* significant at \(p<0.05\)).  
exposed to 4 min of the nitrogen-flowing NTAPPJ by the media-treatment method showed a brighter color than control samples but a weaker signal than other test samples (Figure 4e).  
Finally, the formation of mineralized tissues was examined by considering calcium deposition as indicated by the alizarin red staining (Figure 5). The results were similar to the trends reported in the other results: the hMSCs directly exposed to 4 min of the air-flowing NTAPPJ by the direct and media-treatment methods showed significantly higher levels of optical density (\(p<0.05\)) as did the hMSCs exposed to 4 min of the nitrogen-flowing NTAPPJ by the direct method.  
Figure 4: Immunofluorescence images of osteepontin following staining with anti-osteepontin antibody and the goat anti-mouse IgG-FITC stain (green) of hMSCs of: (**a**) control, (**b**) direct exposure with the air-flowing NTAPPJ for 4 min, (**c**) media-treatment exposure with the air-flowing NTAPPJ for 4 min, (**d**) direct exposure with the nitrogen-flowing NTAPPJ for 4 min, and (**e**) media-treatment exposure with the nitrogen-flowing NTAPPJ for 4 min. (**f**) Brightfield image of control. Scale bar is 100 \(\upmu\)m.