## 3 Results
### Osteogenic Potential of Human Dental Pulp Stem Cells on Conductive PPy Films
The photos were taken 14 days after osteo-induction, which indicated that PPy films prepared in higher concentrations of pyrroles resulted in the better mineralization (scale bar = 500 \(\upmu\)m). medium with osteogenic medium (day 0) and ES was immediately performed once for 4 h. The mRNAs were extracted from the hDPSCs with or without ES treatment on different days for qPCR analysis. Gene expressions were investigated from day 0 to day 6 because genes affected in early stage of differentiation may participate in the ES-driven signaling pathways. The qPCR results demonstrated increasing expression levels of BMP2, BMP3, BMP4, BMP5, and BMPR1B in the ES group (Figure 3). Among these up-regulated genes, the differences were significant for BMP2 on day 0 and 1, BMP3 on day 0 and day 4, BMP4 on day 0 and 1, BMP5 on day 0 and 2, and BMPR1B on day 0. It is worth noting that the expression of BMP2 on day 1 exhibited 7.7-fold increase, BMP3 on day 0 exhibited 3.9-fold increase, BMP4 on day 1 exhibited 2-fold increase, BMP5 on day 2 exhibited a nearly 2-fold increase, and BMPR1B on day 0 exhibited 2.2-fold increase by ES, compared to those of the control group. These significant changes suggested that these genes may be directly influenced by ES. The expression levels of BMP1, BMP6, and BMPR2 did not demonstrate significant difference between two groups. The gene expressions of BMP7 and BMPR1-A were also evaluated; however, the expressions of these two genes were undetectable in hDPSCs. Electrical Stimulation Enhanced the Calcium Deposition of hDPSCs on PPy Films Under Osteogenesis Induction  
The qPCR results indicated that some of BMPs were up-regulated by ES treatment in the early osteogenesis stage. It is essential to evaluate whether these up-regulated BMPs indeed promoted  
Figure 3: Gene expressions of bone morphogenesis proteins ( BMPs) and BMP receptors family in osteogenesis-induced hDPSCs under ES. To determine the ES effects on gene expressions during osteo-differentiation, hDPSCs were seeded to PPy films for 1 day and then were induced by osteogenic medium. In the same time, one-time DCEF treatment was performed for 4 h to stimulate cells immediately after medium replacement (day 0). The mRNAs were harvested from differentiated hDPSCs on different days, and the transcriptional levels of BMP family were determined using quantitative real-time PCR (qPCR). All relative results were compared to those from undifferentiated hDPSCs, and the red and black circles represent the relative gene expression levels of hDPSCs with or without DCEF treatment, respectively. Each value is the average \(\pm\) SD of three independent experiments (\(n=3\); *: \(p<0.05\), **: \(p<0.01\)). osteo-differentiation and eventually improved bone matrix formation. In addition, the optimal ES treating time is still undetermined. To investigate the temporal effects of ES on osteogenesis, hDPSCs were stimulated by DCEF once for 4 h at different time points after the induction with osteogenic medium. Mineralized matrix was stained by alizarin red S (Figure 4), and the deposited calcium was analyzed using Ca-OCPC complex method on day 12, day 14, and day 21 after ostee-induction (Figure 5). The alizarin red S results demonstrated that DCEF highly improved mineralization (Figure 4). For the day 12 results, hDPSCs treated with DCEF on day 0, 2, and 4 all exhibited great enhancement in calcium deposition compared to that of the control group (no ES) (Figure 4A). However, the promotion effects were reduced when the ES was performed after day 6 or later. The results evaluated on day 14 demonstrated a similar trend (Figure 4B). These results indicate that the ES seemed to work mainly on the early stage of osteogenesis. However, when the alizarin red S staining was performed on day 21, there was almost no difference among groups (Figure 4C). In addition to qualitative alizarin red S staining, Ca-OCPC complex method was also applied to measure the deposited calcium in ECM to quantitatively evaluate the level of mineralization (Figure 5).