## 4 Discussion  
In this study, the effects of an NTAPPJ with varying levels of supply gas and exposure time on the differentiation of hMSCs were considered in terms of three possible differentiation states of hMSCss: adipogenesis, chondrogenesis, and osteogenesis. The experiments were carried out while cells were suspended in medium, rather than fully attached and cultured, in order to accurately assess the attachment of the hMSCs [12; 13]. However, despite the similar conditions, the same NTAPPJ treatments, and the previous results in which the mouse osteoblasts showed an increased number of cells attached after the NTAPPJ exposure [13], here the results with hMSCs indicated minimal effects of the NTAPPJ exposure. This finding may be due to differences in the animal cell lines used in the previous study and the human cells used in this study. Animal cell lines exhibit heteroloid chromosome patterns, whereas human cells are diploid in nature, and these two types of cells are known to react differently to surrounding chemicals [19]. Cell attachment is important in cellular activity as it may subsequently affect both the cell proliferation and differentiation [23]. The results for the numbers of cells attached following the NTAPPJ exposure (Figure 1) indicated that there were no significant differences in the numbers of cells attached, except for those cells exposed to 4 min of the NTAPPJ by the direct method, which showed a slight but significant increase in number (Figure 1a). The results concur with the results of experiments under similar conditions that considered exposures of no longer than 4 min [7; 8; 13]. Additionally, the morphology of cells appeared similar between the NTAPPJ-exposed and control cells (Figure 2). Previous studies investigated the effects of non-thermal plasma on the attachment and proliferation of cells [13; 14; 15]. In a previous study that applied helium-based NTAPPJ to adipose-tissue-derived stem cells, more cells proliferated with the NTAPPJ exposure and their stem-like properties were unaffected [16]. Here, different gas sources were used for the NTAPPJ and the focus was on markers of stemness--namely, CD44 and CD105. In another study in which the effect of the helium-gas-supplied NTAPPJ exposure was considered on human mesenchymal stem cells isolated from periodontal ligaments (hPDL-MSCs), it was evident that the hPDL-MSCs possessed all the typical MSC properties, including the capacity for multilineage differentiation [17]. Hence, this study is unique because, to the best of our knowledge, it is the first to investigate the effects of non-thermal atmospheric pressure plasma on the differentiation of human mesenchymal stem cells using air or nitrogen as the source with the results based on the genetic markers related to adipogenesis, chondrogenesis, and osteogenesis. In the physiological state, the differentiation of stem cells is known to be affected by the interactions between cells and the extracellular matrix (ECM), which results in cell-signaling cascades [2]. Therefore, the extracellular environment in tissue engineering is important for the control of cell functions, where the biomimetic chemical functionalities surrounding the cells have been shown to affect the differentiation of stem cells [24]. The application of the NTAPPJ to the hMSCs by both the direct and media-treatment methods resulted in changes in the chemicals in the medium by the interactions between electrons, various ions, and radicals from the plasma with the proteins and ions in the medium. Such chemical changes may have resulted in chemical changes to the extracellular environment, as well as in cellular differentiation. This explains the increased differentiation of stem cells in both the directly treated and media-treated groups for chondrogenesis and osteogenesis (Figure 3b,c). In fact, it is already known that chemicals such as OH' or COO' result in increased chondrogenesis [25; 26].