## 3 Results
### Odonto/Osteogenic Differentiation of hDPSCs
#### 3.2.1 PCR Arrays for Odonto/Osteogenic Signaling Pathway  
At the gene level (Figure 3), it was found that the investigated 84 genes play major roles in the odontogenesis and osteogenesis pathway, and alteration in the expression level of any of these genes apart from the normal expression level would impact the overall outcome of the pathway. While comparing the effect of the BiodentineTM group with the positive control group, we here report similar expression patterns of the odontogenesis- and osteogenesis-related genes in both groups in terms of fold regulation and p values as compared to day 1 of treatment (Figure 3). As shown in the scatter plots of Figure 3B-D, each treatment group was compared to day 1 of treatment, showing the number of genesthat were upregulated, downregulated and unchanged genes. The results of the PCR array were divided into three major signaling pathways of odontogenesis differentiation and ossification, extracellular matrix-related gene clusters during odonto/osteogenic differentiation, and Ca\({}^{2+}\) signaling. Odontogenic Differentiation and OSsification  
The following BMP proteins are genes that are involved in odonto/osteogenic differentiation and ossification, as they are known for their high odonto/osteogenic capacity: 2, 4, 5, 6 and 7. Interestingly, the BiodentineTM-treated group and both control groups showed a significant upregulation in the expression level of BMP2, -3, -5 and -7, whereas only the BD-treated group showed a significant downregulation of BMP4 and -6 (\(p<0.05\)). Meanwhile, BMP3 was upregulated in the BD-treated group in a moderate amount, compared to the other treated groups. As a consequence, our results show that the expression of other odonto/osteogenic markers. SP7, SPP1 and COL2A, were upregulated significantly on all groups, resulting in accelerating the process of matrix mineralization. Additionally, after 21 days of treatment, the BiodentineTM-treated group showed a significant downregulation of ALP and RUNX2 expression levels (\(p<0.05\)) (Table 1). Figure 2: Flow cytometric analysis of mesenchymal stem cells (MSC) surface markers expression of DPSCs derived from pulp tissue. DPSC histograms show that cells are positive for MSCs surface markers (**A**) CD90, (**B**) CD105, (**C**) CD73, and (**D**) CD44, and (**E**) have negative expression of hematopoietic stem cells surface markers (negative cocktail), including CD45, CD34, CD19, CD14 and HLA-DR.  
Figure 3: _Cont._  
Overall, none of the three treatment groups showed a significant change in the expression pattern of TGF \(\beta\) genes. Except for TGF\(\beta\)2 gene, as BD-treated cells showed a remarkable decrease in the expression level of TGF\(\beta\)2, which stimulates their state in osteogenesis. Apparently, BD stimulates the expression of these genes in a range that enables the treated hDPSCs to differentiate into odontoblasts and osteoblasts. Figure 3: Evaluation of human odonto/osteogenesis pathway by quantitative PCR array. (**A**) Heat map of gene expression levels in human odonto/osteogenesis pathway in hDPSCs treated with Biodentine, osteogenic differentiation media (positive control), and uninduced cells, cultured in their own growth media (negative control), compared to day 1 of treatment. Scatter plots of odonto/osteogenesis signaling pathway in hDPSCs treated with (**B**) Biodentine, (**C**) osteogenic differentiation media (positive control), and (**D**) uninduced cells, cultured in their own growth media (negative control), compared to day 1 of treatment. A cut-off point of two-fold change in expression was utilized. Yellow dots indicate the genes that were upregulated; blue dots indicate the genes that were downregulated; and black dots indicate the unchanged genes. Extracellular Matrix-Related Gene Clusters during Odonto/Osteogenic Differentiation (ECMG)  
To characterize ECMG expression profiles, hDPSCs were compared as three groups; BD-treated, positive control, and negative control for 21 days' culture time.