## 4 Discussion  
The present study indicated that the amelogenin exon 5-encoded peptide (SP) promoted cell proliferation and osteogenic differentiation of human DPSCs. Moreover, we found that SP activated the MAPK pathway. EMD and another enamel proteins promote the cell viability of dental pulp cells [20]. In our previous study, we found that SP could also promote PDL cell proliferation [21]. In this study, we found that a concentration of 100 ng/mL SP most enhanced DPSC proliferation; this was similar to our previous reported study [14]. Although, the cell proliferation at 1000 ng/mL SP was decreased compared with 100 ng/mL SP, the cell proliferation was increased compared with 0 ng/mL SP. Therefore, the concentration of 1000 ng/mL was not toxic. From these results, we used this concentration to further investigate the effects of 100 ng/mL SP on osteogenic differentiation. ALP and OCN are regarded as markers of the osteogenesis phenotype [22; 23]. In this study, we found that SP promoted the activity of ALP and the production of OCN. This result is similar to the effect of EMD and SP in human MSCs [13] and PDL stem cells [14]. Thus, we concluded that SP can also induce differentiation of DPSCs. Runx2, ON, and COL1A1 are essential factors required during the early stages of osteogenic differentiation [24; 25; 26]. In particular, COL1A1 has a critical role in the formation of new hard tissue [27]. Previous work has shown that amelogenin and EMD can promote the expression of all three genes in human osteoblasts and human dental pulp cells [28], as well as in human MSCs [29]. Similarly, SP can promote expression of ON in human PDL stem cells [13], and it promoted the expression of type I collagen in our previous in vivo study [11]. In this study, the SP enhanced the expression of Runx2, ON, and COL1A1 in DPSCs, suggesting that the SP enhances osteogenic differentiation in DPSCs during early osteogenic differentiation. Previous work has shown that EMD promotes the mineralization of dental pulp cells [28], and that SP could also increase the mineralization of PDL stem cell [14]. Here, we examined qualitatively and quantitatively changes in calcified nodule formation induced by SP. We found that SP induced greater nodule formation and calcium deposition at 7 and 14 days compared with cultures that lacked SP. Furthermore, SP promoted an increased Ca/P ratio at 7 days of culture compared with control conditions. The theoretical Ca/P ratio of 1:67 is indicative of stoichiometrically pure hydroxyapatite [30]. The Ca/P ratio at day 7 is significantly different, but day 14 is no different. These results suggest that SP induces high-quality calcified nodule formation on dental pulp at an early stage (day 7). However, the effect is limited, meaning the late stage (day14) is no different. We consider that forming calcifications at an early stage in protecting the dental pulp is very important in endodontic treatments. Therefore, we believe that SP induces the dentin bridge at an early stage, and could contribute to the successes of endodontic treatments. The MAPK signal is an essential regulator of cell viability and osteogenic differentiation in dental tissue cells [31; 32; 33]; however, the mechanism by which the amelogenin exon 5 peptide exerts its effects remains unclear. Figure 5 shows that SP activated the phosphorylation of MAPK components in human DPSCs. EMD and another amelogenin peptides have the ability to regulate cellular functions through MAPK signaling. Our findings support the role of the SP in regulating cellular function through MAPK signaling in a manner similar to that of EMD and other enamel proteins. Amelogenin has various biological functions, such as cell proliferation and differentiation. As well known, amelogenin can activate physiological actions through various signaling pathways. In our previous study, SP promotes the cell proliferation via the ERK pathway in human MSCs [13]. Therefore, we believe that SP promotes the cell proliferation via MAPK, such as ERK signaling.