## 4 Discussion  
Human dental pulp stem cells (hDPSCs) are a kind of mesenchymal stem cells derived from the pulp of human tooth. Because hDPSCs demonstrate the capacity of self-renewal and multilineage differentiation, they have therapeutic potentials similar to those of bone marrow stem cells [36]. In addition, hDPSCs can be extracted from teeth recovered during routine dental procedures, making them a convenient source of stem cells for cell-based therapy. Furthermore, the multilineage differentiation of hDPSCs makes them an alternative strategy for treating various human diseases, rather than limiting to the treatment of dental-related problems [37]. Although the application of biochemical cues is the gold standard to induce cell differentiation, the promising promotion effects of physical stimulations, especially electrical stimulation (ES), have also been proven. For example, neural differentiation of PC12 cells in the presence of nerve growth factor (NGF) can be significantly enhanced by ES treatment [38]. [PERSON] et al. also demonstrated that ES improves osteogenic-related gene expression at specific time points with different gene expression patterns between bone marrow and adipose-derived MSCs [39]. These findings suggest that ES may regulate the physiology of the cell and the differentiation potential of stem cells. To date, the promotion effect of ES on the hDPSCs differentiation is rare. [PERSON] et al. have inserted electrodes in culture medium to stimulate hDPSCs by electrical current, and their results showed that this fluid-mediated ES treatment seems to improve cell proliferation, and the expression of OCN  
Figure 6: The normalized quantification of calcium deposition of hDPSCs under DCEF treatment at different stages of osteo-differentiation. To distinguish whether the calcium deposition results were affected by cell proliferation, the quantification results in Figure 5 were divided by cell numbers for normalization. Cells were lysed and the released LDH were evaluated to determine cell numbers. The normalized results were evaluated on (**A**) day 12, (**B**) day 14, and (**C**) day 21 after osteo-induction. (**D**) The results from all experimental groups were grouped to better understand the efficacy of ES. The negative and positive control groups were hDPSCs cultured on Ppy films using normal or osteogenic media, respectively. These two control groups were not treated by DCEF. (\(n=3\); *: \(p<0.05\), **: \(p<0.01\) compared with the positive control group) (N.D.: Non-detectable). is slightly enhanced [25]. However, whether ES may promote osteo-differentiation, especially the level of mineralization, is still unclear. In addition, the exact role of electrical signals in regulating the biosynthetic activity and homeostasis of osteogenesis remains elusive. In our previous study, conductive PPy films have been developed for ES to significantly improve the osteo-differentiation of rBMSCs [28]. These PPy films can be deposited onto various substrates, such as culture dishes, glass plates, and even metal devices. In addition, the electrical resistances of PPy films can be easily adjusted to meet specific requirements. These properties suggest that PPy-mediated ES treatment is a feasible approach to promote tissue regeneration [33]. Here, we demonstrated that hDPSCs could adhere on conductive PPy films with comparable proliferation rate to those on TCPS (Figure 2b). In addition, the lower resistances of PPy films resulted in the higher level of mineralization of hDPSCs (Figure 2c), which were in accordance with our previous finding of rBMSCs, suggesting that osteo-differentiation of hDPSCs can be improved by the conductivity of scaffolds [28]. Regarding the DCEF treatment, it can be either constant or in different waveforms, and the frequency of the electrical current may influence the biological effects [22]. Therefore, our previous study has treated rBMSCs using DCEF in different modes, including DCEF in constant and square waves in different frequency, offset, amplitude, and duty cycle [33].