## 4 Discussion  
Biocompatibility is one of the most important characteristics for dental products [27; 28]. In vitro biocompatibility tests are regulated by ISO 7405 and ISO 10993-5 standards [29]. Although many studies have evaluated the biocompatibility of dental materials [19; 30; 31], the use of different in vitro models (different cell cultures, medium or exposure time) precludes the meaningful comparison of the results reported in those studies [29]. The osteosarcoma-derived MG-63 cells have been used occasionally for evaluating the biocompatibility of calcium silicate cements, due to their similar physiologic and adhesive properties as human osteoblasts [32],as well as their reliability in biocompatibility testing [19; 30; 31; 33]. All the cements tested in the present study demonstrated high biocompatibility with MG-63 cells. The osteogenic response of MG-63 cells was evaluated by ALP activity, an important marker of bone matrix mineralization. The two bioceramic materials RRMPU and FSP, both composed mainly of calcium silicates, zirconium oxide, tantalum oxide and calcium phosphate monobasic, exhibited higher ALP activity compared to MTA, suggesting a better capability of these two materials to induce mineralization. Indeed, MTA is composed by similar elements (calcium, silicate, phosphate, carbon and oxygen), but shows a different overall composition (tricalcium silicate, dicalcium silicate, bismuth oxide, and small proportions of tricalcium aluminate and calcium sulfate). The capability of mineralization potential of bioceramic materials was confirmed by the observation of mineralized nodules in cell cultures and by Alizarin Red S staining. The FSP has been claimed to have optimal mineralizing properties [34]. The previously reported result is in agreement with findings from the present study. The osteogenic differentiation of mesenchymal stem cells is regulated by osteocytes and osteoblasts in a simplified bone niche. To monitor the potential effects of the biomaterials on  
Figure 4: Evaluation of gene expression by RT-qPCR. The osteogenic differentiation process was monitored by RT-qPCR: gene expression of alkaline phosphatase (_ALPL_), bone sialoprotein (_IBSP_), osteoclastin (_BGLAP_), interleukin 1 alpha (_IL-1a_) and interleukin 6 (_IL-6_) was investigated. _ALPL_ expression was significantly downregulated in all the conditions tested (top, left). _IBSP_ expression was increased in culture with RRMPU, but the differences were not statistically significant (top, middle). _BGLAP_ expression was downregulated in all the conditions tested, without any statistically significant difference (top, right). Upregulation of _IL-1A_ (bottom, left) and _IL-6_ was detected in all the conditions tested (bottom, right). osteogenic differentiation, expressions of _ALPL_, _IBSP_ and _BGLAP_ and _ALPL_ genes were used as markers of different phases of osteogenesis. After an initial peak, _ALPL_ levels started to decline as other genes (e.g., BGLAP) were upregulated [35]. In the present study, the expression of these markers was determined after 15 days of culture in an osteogenic differentiation medium. _ALPL_ expression was downregulated in all the conditions tested. The results are in line with the notion that the early osteogenesis phase declines after 15 days of culture. Accordingly, even if the ALP enzyme is still active, gene expression is diminished. As expected, MG-63 cells cultured in DM expressed high levels of _IBSP_, as this gene is involved in the middle phase of osteogenesis. However, high levels of _BGLAP_ expression were not detected. The results suggest that the MG-63 cells in DM had not reached the last phase of osteogenesis after culturing for 15 days. The absence of statistically significant differences in _ALPL_, _IBSP_ and _BGLAP_ expressions between FSP and the other bioceramic materials suggests that the effect of FSP on osteogenic induction is comparable to the other bioceramic materials tested.