# Biocompatibility and Osteo/Odontogenic Potential of Various Bioactive Root-End Filling Materials
## 4 Discussion
On the other hand, RUNX2 is not specific to osteogenic differentiation, as the protein is detectable in the nuclei of pre- and immature odontoblasts during the early stages of tooth development and regulates both tooth and bone-related expressions [27]. The most commonly evaluated phenotypic markers for odontogenic differentiation are DMP-1 and DSPP. DMP-1 acts as an inducer in the odontogenic differentiation of pulp cells whereas DSPP is a non-collagenous protein highly expressed in the dentin matrix and regarded as the terminal differentiation marker of odontoblasts [28]. Overexpression of RUNX2 in odontoblasts inhibits their terminal differentiation and reduces DSPP expression, which indicates RUNX2 is able to induce transdifferentiation of odontoblasts into osteoblasts and needs to be inhibited during odontblast differentiation to promote normal cell maturation and dentinogenesis [29]. Although these interactions overlap and cannot be singled out as a specific marker of osteogenesis or odontogenesis, a predisposition in MSCs towards osteogenic or odontogenic differentiation with the tested materials can be noted. In the present study, RUNX2 and DSPP expression levels were different between tested materials and osteogenic medium. MTA Angelus showed upregulation of DSPP after 7 days, unlike osteogenic medium, whereas RUNX2 was highly expressed in both groups, which confirms the findings of a similar study with DPSCs [30]. Among the tested biomaterials, Medcem MTA demonstrated significant upregulation of DMP-1 on both time points while BIOfactor MTA also indicated a significant increase on day 7. Regarding DSPP expression, MTA Angelus responded with a significant increase early in cell culture whereas Medcem MTA demonstrated a dramatic increase on day 7. In a previous report when MSCs were seeded on MTA-coated cell plates to assess odontogenic differentiation, ALP activity was decreased but relative DSPP gene expression was upregulated [31]. The low ALP activity may be associated with decreased cell viability, possibly due to the direct exposure of cells to MTA; nevertheless, the cells were able to demonstrate odontogenic differentiation capabilities. In the present study, the cells treated with Well-Root ST and Pure Portland Cement responded with a lesser increase in DSPP expression. Tested MTA materials resulted in upregulation of odontogenic gene markers DMP-1 and DSPP whereas Pure Portland Cement and the osteogenic differentiation control group indicated higher ALP levels in addition to upregulation on RUNX2 expression. Although it is not conclusive, this may suggest a predisposition of MTA materials to direct cells towards odontogenic differentiation while the osteogenic medium may promote cells towards osteogenic lineage. Minor differences in gene expression and cell viability between different formulations of tested MTA materials provide little evidence that one material is superior than the other. Therefore, the second null hypothesis that all tested bioactive materials would support osteo/odontogenic differentiation of stem cells similarly was partially accepted. As with all in vitro studies, the major limitation lies in the lack of mimicking host reactions to environmental stimuli compared to animal and human studies. Although the biological properties and potential of bioactive cements, like the ones tested in the present study, have been investigated in numerous studies, there are new materials introduced to the market in accordance with high demand. Updated biological profiling of these materials is necessary before their vast clinical use. Hence, it is crucial to test biocompatibility and bioactivity before moving to in vivo investigations and clinical trials to elucidate underlying mechanisms and cell behaviors. The use of standardized procedures such as cytotoxicity assessment according to ISO-10993-5 guidelines and relative gene expression profiles calculated with \(2^{-\Delta\Delta\text{CT}}\) ensure reporting reliable results.