## 4 Discussion  
BiodentineTM is a capping material that is designed to stimulate the odontogenesis and dentinogenesis processes in vital pulp therapy. Consequently, such material is required to be biocompatible, with high physiochemical standards, and most importantly, to have the ability to stimulate the formation of dentin and odontoblastic differentiation of dental pulp cells [31]. Hence, the biocompatibility of BD with cellular processes, such as proliferation and differentiation, is mandatory. Many previous studies have examined the biocompatibility of BD with different types of dental stem cells, such as DPSCs [22], SHED [30], SCAP [32] and PDLSCs [29], in addition to evaluating its role in the differentiation processes, such as dentinogenesis, osteogenesis and odontogenesis, for different types of stem cells [26; 27; 28; 33]. However, no previous study has evaluated the signaling pathways that are activated through the dentinogenic differentiation process. Therefore, this study was performed to evaluate the impact of BD on the molecular pathways and signaling networks that are activated through odonto/osteogenic differentiation of hDPSCs at both gene and protein levels. At gene level, our data show that BD has stimulated the expression level of BMP genes, which are highly involved in teeth development, growth and formation [27; 28; 29]. The BMP proteins (2, 4, 5, 6 and 7) are products of genes that are involved in odonto/osteogenic differentiation and ossification, as they are known for their high odonto/osteogenic capacity. These genes play essential roles in odontogenic and osteogenic differentiation signaling pathways by increasing the expression of odonto/osteoblastic differentiation markers, such as osterix (SP7), osteopontin (SPP1), ALPL and osteoclastin (BGLAP), RUNX2, the calcium mineralization potential, and collagen forming genes, such as CoL2A [34; 35; 36]. The BD-treated group has shown a downregulation of the expression levels of BMP4 and 6. This downregulation could be explained by the fact that both BMP4 and 6 are expressed at early stages of the odontogenesis and osteogenesis pathways [37]. In addition, the odonto/osteogenic potency of each BMP might depend on the stage of differentiation of the treated cells. Thus, BD-treated cells might be at different stages of differentiation [38]. For the BMP3 gene, the BD-treated group showed a moderate, insignificant increase in its expression level, compared to other groups, which is reflected in their potency to differentiate into odonto/osteoblasts, as overexpression of BMP3 opposes odont and osteoblast differentiation by activating activin receptor (AcvrIIB)-Smad2/3 signaling [39]. Moreover, our results are consistent with the previously published data regarding the enhancement of expression levels of SP7, SPP1 and COL2A, in addition to the downregulation that was observed in the expression levels of RUNX2 and ALP [32; 35]. Both RUNX2 and ALP markers play essential roles in the differentiation pathway, as RUNX2 promotes and regulates odontblast and osteoblast formation, and ALP is responsible for their development [36; 40; 41; 42]. Additionally, the expression level of ALP is used to monitor the maturation stage of MSCs toward osteogenesis and tooth mineralization; therefore, it is considered an indicator for the commitment of cells to continue odonto/osteo-lineage differentiation [40]. Thus, secreted ALP is accumulated in the media, and this accumulation is a sign of extended mineralization [43; 44]. Our results are also in agreement with the previously published data on RUNX2 and ALPL being expressed in early days of differentiation, then declining rapidly in expression with time in a remarkable manner [32; 40; 41; 42]. The downregulation in the expression level of these two genes is consistent with what other groups have reported [37; 40], except for the results from [PERSON] et al., where DPSCs were cultured in media containing BD in addition to other mineralizing factors [26].