## 4 Discussion
For collagen synthesis and formation, COL10A was significantly downregulated, except in the BD-treated group, as the downregulation was insignificant (fold change less than two), compared to the other treated groups. The type X collagen gene (COL10A1) influences deposition of other matrix molecules to this region, thereby providing a proper environment for hematopoiesis, mineralization and modeling, which are essential for endochondral ossification [45]. Col10A (Col X) plays a critical role through this process in addition to its structural support of the matrix, as 45% of the total collagen producedrepresents Col 10A. Moreover, Col10A initiates the biomineralization through the increase in the Ca2+ influx into the matrix [35]. Hence, it is very important during tissue engineering, as it is essential for both MSC-mediated cartilage and bone formation [46]. Additionally, the BD-treated group has shown an increase in the expression levels of Col14 and Col1. These two collagen types have key roles with respect to the architecture of the extracellular matrix, and bone quality. Moreover, these genes have a direct relationship with other molecules, such as IFG molecules, which are important to start the induction of new bone formation [47; 48]. On the other hand, the BD-treated group has shown downregulation of the expression of COL1A1, COL1A2, COL3A1 and COL5A1. This could be explained by the fact that Collagen 1 and 3 are considered early markers for odontogenic and osteogenic differentiation in which these genes are important for the phenotypical appearance of odontoblasts and osteoblasts, and play key roles as regulatory molecules for the differentiation and proliferation of human osteoblastic cells [49], as the organic phase of dental tissues is composed of about 30% collagen, types I, III, and V [50]. Thus, their upregulated expression levels can be detected at early stages of odontogenesis: days 4 to 7 [49]. Regarding the expression of collagen genes, our results could be explained by previously published studies, where it was shown that calcium silicate biomaterials have a negative effect on the expression of collagen type I, as these biomaterials can downregulate its expression and stimulate the degradation of collagen fibers in the matrix [51; 52]. Furthermore, BD has downregulated the expression of MMP2 and stimulated the expression of MMP9, which have key roles in different cellular mechanisms, such as proliferation, migration, differentiation and the healing process of pulp tissue [23]. These results are similar to the previously published data [40; 53]. For other extracellular matrix proteins, BD-treated cells showed upregulation of the major ECM proteins, such as MMP9, MMP10, ITGA2, ITGA3, ITGAM, FLT, and PHEX. These upregulated expression patterns were observed to be similar to other groups: positive and negative control groups. However, the untreated group showed an elevated expression level of the ECMG proteins. This could be explained as previously described [45], that this upregulation could be considered an artifact that appears to be greater or similar to treated groups: either BD or the positive control group [45]. Additionally, based on previously published results, the undifferentiated MSCs highly express these ECM genes; upon the osteogenic differentiation, a downregulation in the expression of the derived genes was detected. This could be a result of either the fact that the viability of cells was decreased at late phases of osteogenic induction, or that the long-term culture of un-induced cells may provoke cells to start their own differentiation. [45; 46; 47]. For genes that are involved in the Ca2+ signaling pathway, the BD-treated group has a stimulatory effect on the expression levels of these growth factors: IGF1 and 2, VEGF, TGF\(\beta\), BMP2, and BMP4. Since this pathway has a very important role in the odontoblast and osteoblast differentiation processes, the high level of cytoplasmic Ca2+ promotes the upregulation of the previously mentioned growth factors.