## 4 Discussion
Although these systematic examinations are helpful for optimization, we only applied 0.33 V/cm of continuous DCEF in this study because the goal of this study is to determine whether ES treatment may promote osteo-differentiation, and this constant electric field has been proven to stably improve osteo-differentiation of rBMSCs [28; 33]. The DCEF treatment of hDPSCs not only enhanced osteogenic capacity but also promoted mineralization. In addition, only ES performed before day 6 resulted in increasing calcium deposition and mineralization. Therefore, we conclude that ES mainly triggers pathways in the early stages of osteo-differentiation. Gene regulation plays an important role in osteogenesis. It has been shown that mesenchymal stem cells and osteo-progenitor cells can be differentiated into osteoblasts by certain key cytokines and functional proteins, including proteins in the BMP family, Runx2, and certain ECM proteins [40; 41]. Bone morphogenesis proteins ( BMPs) belong to the transforming growth factor-\(\beta\) (TGF-\(\beta\)) superfamily. Because BMPs comprise a group of proteins participating in bone formation [42], they are important in adult tissue homeostasis [43]. BMPs may initiate Smad-dependent or non-canonical pathways via binding to type I and type II heterotetrameric receptors [44]. According to a previous sequence alignment analysis, BMP2/4 and BMP5/6/7 are two groups of structurally related proteins; however, BMP1 and BMP3 are more distantly related [42]. BMP1 exhibits no sequence similarity to BMP2/4 or BMP5/6/7 because BMP1 is a metalloprotease that participates in collagen maturation and is therefore independent of BMP-mediated pathways [45]. In our study model, there was a 3.9-fold up-regulation in the expression of BMP3 on day 0. Although BMP3 has been shown to be a negative regulator of osteogenesis [46], it also has been reported that BMP3 expression in the perichondrium of chick limbs may regulate cartilage cell proliferation to ensure proper ossification [47]. Therefore, we speculate that the up-regulated expression of BMP3 by ES may play a role in modulating the levels of other BMP signaling, thereby enhancing mineralization. However, further experiments are needed to confirm this hypothesis. The expression levels of BMP1 and BMP6 in our study model fluctuated in both control and ES-treated groups during the experimental time period, indicating that these BMPs may not be involved in ES-stimulated osteogenesis. BMP7 was reported to participate in eye and kidney development [48], but its expression is undetectable in hDPSCs. BMP2 has been studied extensively in osteogenesis [49], and numerous evidence indicates that BMP2 plays a crucial role in osteogenesis via its modulation of RUNX2 expression, especially in the early stages of osteogenesis [50]. In our study model, the expression of BMP2 was up-regulated 7.7-fold by ES on day 1. It was a significant change because no other BMPs exhibited such a profound up-regulation by ES in the early stages of osteogenesis. Therefore, we deduce that the ES-induced promotion of osteogenesis may be directly modulated via BMP2. There are two types of BMP receptors, i.e., type I and type II, and these receptors participate in BMP-mediated signal transduction [51]. When these serine/threonine kinase receptors are triggered by a ligand, they form a heterotetrameric complex in which the type II receptor transphosphorylates the type I receptor, and the signal conducts though Smads to the nucleus [52]. In hDPSCs, the expression of BMPR1-A was undetectable with qPCR; therefore, we assume that BMPR1-B and BMPR2 are expressed in hDPSCs as heterotetramers to accept BMP protein-ligands. In this study, we comprehensively investigated mRNA of BMPs and their receptors through qPCR analysis because BMPs are the most well-known growth factors to initiate osteo-differentiation. However, these qPCR results did not represent the corresponding protein expression levels.