## 4 Discussion  
Favorable effects of NTAPP on wound healing, immune responses, and stem cells involving increased expression of growth factors and cytokines have been reported in prior studies [19; 20; 21]. Similarly, this study demonstrated that NTAPP treatments of root dentin surfaces enhanced hDPSCs responses through cell adhesion, spreading and proliferation, which may benefit regeneration in endodontics. Pulp-dentin regeneration appears to be dependent on release of cell signaling molecules from reservoirs in root dentin [22; 23]. Their stimulation of stem cell attachment and spreading on dentin surfaces constitutes the initial phase of cellular function [15]. These bioactive factors may be released by dissolution, as EDTA pretreatments reportedly enhanced pulp stem cell migration, whereas water had no effects, and NaCl restrained migration [24]. Similarly, [PERSON] et al. (8) showed increased cell density on EDTA-conditioned dentin than on untreated dentin. However, EDTA had no significant effect on hDPSCs counts in this study, and only NTAPP significantly increased cell viability. EDTA's effects may have been hidden by the impact of NTAPP treatment. Pre-rinsing dentin with NaCl before EDTA was shown to reduce growth factor release, whereas chloroxhedine before EDTA increased their amounts [25]. Enhanced hDPSCs attachment on NTAPP-treated root dentin may have been due to improved hydrophilicity. NTAPP reportedly improved dentin wettability [26], and 30s of NTAPP brush treatment made human dentin super hydrophilic [27]. Their XPS analyses showed the oxygen/carbon ratio increased dramatically after NTAPP brush, suggesting that new oxygen-containing polar moieties had formed [27]. Similarly, [PERSON] et al. demonstrated improved human dentin wettability from NTAPP, which supported enhanced human osteoblast spreading [28]. Likely, NTAPP generates reactive spots or species through C-C and C-H bonds splitting from vigorous atom/ion bombardment of dentin surfaces, which increases their hydrophilicity. The resultant increased adsorption of cell adhesion proteins such as fibronectin enhances cellular attachment, spreading and proliferation. These indirect effects of NTAPP on the natural niche of stem cells could also be applied to synthetic biomaterials to promote cellular responses. For example, [PERSON] et al. reported that NTAPP changed nanoscale topography and elasticity of polymeric substrates, which enhanced human mesenchymal stem cell adhesion and spreading [29]. Figure 5: Live-cell imaging of hDPSCs attachment on root dentin. Holotomographic microscopy time series of NTAPP-treated (upper) and untreated controls (lower). Cells initiated attachment on NTAPP-treated dentin at 2 h, and on untreated controls at 4 to 6 h. hDPSCs on NTAPP-treated dentin had spread out over 4 to 6 h, and sprouted cellular processes into dental tubules at 8 h. Whereas, cells on untreated dentin were not spread until 6 to 8 h.  
Cell orientation, organization, morphology and function are strongly influenced by their underlying substrate [30], and the recruitment of odontblast precursors to dentin surfaces, and their extension of cellular processes into dental tubules is particularly important in regenerative endodontics. In this study, hDPSCs seen through SEM had attached and spread out in the direction of dental tubules exposed by EDTA and NTAPP treatments. They sprouted microvilli on dentin, with cellular extensions and filopodia often penetrating NTAPP-treated dental tubules. Additionally, hDPSCs observed in real-time by HTM live-cell imaging were attracted and attached to NTAPP-treated dentin surfaces faster than controls. Cells with enhanced attachment to NTAPP-treated dentin may have preferential expression of focal adhesion kinase (FAK) and faster progression through the cell cycle [3]. Enhanced subcellular activities were clearly visible in hDPSCs attached to NTAPP-treated surfaces.