## 4 Discussion  
NTAPP is a partially ionized gas containing electrically charged particles and radicals at atmospheric pressure [18]. Previous studies reported that NTAPP exhibits various physiological effects beneficial for applications in regenerative medicine. It activates the proliferation of various mesoderm-derived adult stem cells, including human adipose tissue-derived stem cells, bone marrow-derived (BM)-MSCs, and hematopoietic stem cells, in vitro without affecting their stem-like properties [15,19]. In the present study, we investigated the effectiveness of NTAPP in activating the proliferation of cells from the deciduous dental pulp for potential use in regenerative dentistry. Our results showed that the cell proliferative capacity of cells treated 3 times with NTAPP for 20 s each time was approximately 1.2-fold higher than that of non-treated cells. In this study, the increased rate of cell proliferation was detected by MTS which is an indirect indicator. To confirm that proliferation (and not only metabolic activity) was increased by the NTAPP treatment, work should be carried out to directly compare cell numbers between the control group and the group treated with the optimum NTAPP protocol in future study. The optimal NTAPP conditions vary depending on plasma generator devices, the gas type used for plasma generation, and cell type. For instance, it has been reported that the proliferative potential of MSCs and hematopoietic stem cells is increased when treated with helium-based NTAPP 10 times for 50 s each time, in contrast to that of human synovial cells when treated with argon-based NTAPP for 60 s [19,20]. Here, the proliferative potential of hDDPFs decreased when treated >3 times with NTAPP for >20 s each time. Thus, the appropriate treatment conditions for each cell line need to be established before any clinical application. In plasma medicine, NTAPP acts directly on cells and indirectly through the plasma-treated medium. For instance, NTAPP-activated media have anti-cancer effects on various human cancer cells [16], and He-based low-temperature atmospheric plasma jet-activated media have anti-bacterial properties [17,21]. Here, we found that cells cultured in NTAPP-treated medium had a higher proliferative capacity than those cultured in non-treated medium. Therefore, NTAPP enhanced cell proliferation both directly and indirectly by modifying the culture medium. Many oral peripacil tissues contain large amounts of water. For instance, the dental pulp is a soft gelatinous tissue and water comprises 75-80% of its volume [22]. Therefore, it is essential to consider the effects of NTAPP on the cells and the surrounding fluid. Our findings showed the effectiveness of NTAPP in modifying the cell culture media, possibly expanding the applications of NTAPP using dental pulp. Figure 5: Effect of non-thermal atmospheric pressure plasma (NTAPP) on the expression of stem cell-specific markers (CD44, CD105, and CD146) in human deciduous dental pulp fibroblast-like cells (hDDPFs). \(\Delta\)MFI (%), change in median fluorescence intensity. Previous studies reported that _Oct4_, _Sox2_, and _Nanog_ play essential roles in maintaining stem cell pluripotency and promoting cell proliferation in various adult stem cells [23; 24; 25; 26]. To determine whether NTAPP treatment (3 times, 20 s each) could promote the proliferation of hDDPFs without affecting pluripotency, we examined the expression of _Oct4_, _Sox2_, and _Nanog_. We also studied the expression of _Sox9_, a marker of pluripotency of undifferentiated neural crest cells, and _ALP_, a marker of pluripotency of undifferentiated osteoblasts since hDDPFs are known to differentiate into chondrocytes and osteoblasts [27; 28; 29]. Our results showed that mRNA expression of _Oct3/4_, _Sox2_ and _Nanog_ was upregulated in NTAPP-treated cells. A study on multipotent myoblasts has reported that the expression of _Sox9_ increases after 3 to 5 days of incubation with material trioxide aggregate (MTA), which promotes hard tissue formation [30].