## 3 Results  
### Osteogenic Potential of Human Dental Pulp Stem Cells on Conductive PPy Films  
For treatment of skeletal defects, osteo-conductive materials are critical to promote bone healing [34]. To determine whether PPy is a suitable substrate for cell culture in vitro, hDPSCs were seeded on PPy films. The morphology of the hDPSCs on PPy was maintained as spindle-like, which was similar to that of cells grown on TCPS (Figure 2A). The results of lactose dehydrogenase (LDH) assay revealed that there was nearly no difference in the proliferation rates of hDPSCs cultured on TCPS and PPy, suggesting the good biocompatibility of PPy (Figure 2B). To investigate the effects of conductivity of cell substrate on osteogenesis, differentiation concentrations of pyrrole were used to prepare PPy films. Four-point probe analysis was applied to measure sheet resistances of PPy films, and the results indicate that electrical resistances decreased with increasing pyrrole concentrations (Table 2), suggesting that the conductivity of PPy films can be easily manipulated. These PPy films were then applied as substrates to examine their effects on osteo-differentiation of hDPSCs. These seeded cells were induced by osteogenic medium, and alizarin red S staining was applied on day 14 to determine the level of calcium deposition (Figure 2C). The results showed that cells grown on PPy films with lower electrical resistances demonstrated higher levels of mineralization. \begin{table}
\begin{tabular}{c c} \hline \hline
**Gene** & **Primers** \\ \hline GAPDH & 5′-CTCTGCTCCTCCTTTGAC \\  & 3′-ACGACAAATCCGTTGACTC \\ \hline BMP1 & 5′-ACCCTGGGCAGCTACAAGT \\  & 3′-TGAGAATCCGCCCAAG \\ \hline BMP2 & 5′-CAGACCACCGGTTGGAGA \\  & 3′-CCCACTCGTTTTCGGTAGTTTCT \\ \hline BMP3 & 5′-CCCAAGTCCTTTGACTGTAC \\  & 3′-TCTGATGGTACCATGATTTGA \\ \hline BMP4 & 5′-CTTTACCGGCTTCAGGTCTG \\  & 3′-TGGGATGTTTCCGATGTTC \\ \hline BMP5 & 5′-AACCGAATAATCCCAGCTC \\  & 3′-TTTTGTCTCACTTTGTTAAAATCC \\ \hline BMP6 & 5′-ACATGCTATGACTTTTGA \\  & 3′-ACTCTTGTGGTGCCTGGA \\ \hline BMP7 & 5′-ACCACTGGGGTCAAATCC \\  & 3′-  
Analysis of Gene Expression of the BMP Family and BMP Receptors in hDPSCs under Electrical Stimulation  
Because PPy films were not only suitable but also beneficial to osteogenesis, these conductive substrates were further applied to investigate the potential of ES on facilitating hDPSCs differentiation. Bone morphogenetic proteins ( BMPs) are well-known signal proteins in osteogenesis [35]; thus, the gene expression profiles of the BMP family and BMP receptors were evaluated in this study. After seeding hDPSCs on PPy films for one day, these cells were osteo-induced by replacing the culture \begin{table}
\begin{tabular}{c c} \hline \hline
**Solutions for PPy Preparation** & **Sheet Resistances of PPy Films (kf)/Square)** \\ \hline
0.1 M pyrrole & 25.72 \(\pm\) 1.52 \\
0.3 M pyrrole & 10.58 \(\pm\) 0.65 \\
0.5 M pyrrole & 7.83 \(\pm\) 0.47 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The sheet resistances of PPy films prepared by different concentrations of pyrrole. Figure 2: **(A)** Phase contrast images of human dental pulp stem cells (hDPSCs) on TCPS (left) and PPy films (right). The hDPSCs were cultivated on TCPS or PPy for 3 days. The cells on both materials exhibited almost the same typical fibroblast-like morphology with comparable confluency, indicating that cell adhesion and extension were similar on these two surfaces (scale bar = 100 \(\upmu\)m). **(B)** The LDH assays were applied to quantify the amounts of hDPSCs on TCPS or PPy films. All cell numbers were compared to those in day 1. The results showed comparable cell viability and proliferation between two surfaces, suggesting the good biocompatibility of PPy films (\(n=3\)). **(C)** Alizarin red S staining was performed to evaluate the level of mineralization. The hDPSCs were seeded on PPy films prepared by pyrrole solutions in concentrations of 0.1, 0.3, and 0.5 M, respectively.