## 5 Discussion
In this case, the genes of interest are those implicated in osteogenic differentiation, from the first stages, such as APL or Col I [29], to those expressed in the late phase of this process and considered markers of osteoblastic cell differentiation [30], for example OCN. Among these genes, the RUNX2 transcription factor is known to play a key role in osteoblastic differentiation [31] because it can directly stimulate transcription of OCN, Col I, OPN and collagenase III genes binding to the sequence PuCCPuCA. Beyond this, the molecular mechanism of RUNX2 action is still unknown. Although RUNX2 is expressed exclusively in mineralized tissues and their precursors, in many cases there may be no correlation between their expression and the expression of osteoblast-related genes. It has been demonstrated that inconsistencies between Runx2 mRNA or protein levels and its transcriptional activity suggests that posttranslational modification and/or protein-protein interactions may regulate this factor [32]. The results of the qRT-PCR experiments show that the RUNX2 gene is not upregulated in cells seeded on SCP-c materials or the control. The addition of the OM does not increase RUNX2 expression, but OPN or IBSP expression increases in samples incubated with the materials, indicating the participation of RUNX2 in guiding the expression of these markers. RUNX2 mRNA variations are not correlated with OPN or IBSP levels, probably because the RUNX2 protein is active after post-transcriptional or post-translational modifications [33; 34]. ALP activity increased in cells incubated with SCP-c materials 28 days after the seeding with GM and OM, which is concordant with the maximum release of silicon to the medium previously stated by our group [14]. It has been widely proved that silicon plays an important role promoting cell proliferation [11], ALP expression and mineralization [35]. On the other hand, ALP activity can be influenced by calcium levels, which in small amounts cause the increase of ALP activity and cell differentiation. Moreover, osteogenic supplements, like ascorbic acid and \(\beta\)-glycerophosphate, up-regulate ALP gene expression [36]. Osteogenic supplements in OM, low concentrations of Ca and increasing levels of Si from 21 days to 28 days may create a synergistic effect on the stimulation of ALP activity. The formation and maturation of the extracellular matrix (ECM) are also studied as markers of osteoblastic differentiation. Col I and SPARC are both markers of early osteoblastic differentiation [37]. Their mRNA expression seemed to be influenced by the Si-Ca-P monophasic ceramic in this study from first week to the last week by adding OM at 28 days. _ah_MSCs express high levels of Col I in their in vivo natural environment [38]. qRT-PCR results for Col I were confirmed by Col I immunofluorescence images. In fact, Col I seemed to be synthesized since the first stages of ECM deposition in all the samples considered, and it increased after adding OM at 28 days. During the fourth week, SCP-c also was able to upregulate the markers of a more mature extracellular matrix, such as OPN and IBSP1, whose role is to influence and regulate mineralization processes. The qRT-PCR results showed that cells seeded under the influence of SCP-c reached the maximum expression of these mRNA markers during the fourth week with the best performance in presence of the OM. These data are in accordance with the results stated by [PERSON] et al. [39] in the genetic expression profiles obtained for _ah_MSCs during osteoblastic differentiation studies. OPN qRT-PCR results are confirmed by the results of the immunofluorescence study. During the last week of culture, OPN proteins are highly expressed in the samples incubated with the material, demonstrating that the ECM is maturing, as expected. HA and \(\beta\)-TCP are highly biocompatible, but differ in their biological response at the implantation site.