# Biocompatibility and Osteo/Odontogenic Potential of Various Bioactive Root-End Filling Materials
## 4 Discussion
Cell viability is reported to be enhanced two- to three-fold with Pure Portland Cement in DPSCs, at days 3 and 7 compared to cells cultured in control media, which confirms the finding of the present study [23]. When MSCs were previously exposed to Well-Root ST, cell viability was found to be similar on days 1 and 7 but significantly increased compared to the control group on day 3, which is in accordance with our findings [10]. Culturing human periodontal ligament stem cells on Well-RootST indicated favorable cell attachment and proliferation potentially due to both chemical composition and the smooth surface of the material [24]. There were no recent reports on stem cell viability when treated with BIOfactor MTA and Medcem MTA. However, it should be noted that osteogenically induced MSCs performed better over the 7-day incubation with a consistent increase in proliferation and were significantly promoted on day 7 in comparison to all tested materials, suggesting cell viability may be more prone to change over a longer course of exposure with the MTA-based materials. Additionally, several factors that may cause disparities between studies need to be considered while interpreting cellular response to MTA-based materials, such as the type of tested cells, cytotoxicity assessment methods, concentration, and exposure type of the tested materials. Despite minor variances between tested materials, the null hypothesis that all tested MTA-based materials would not interfere with the viability of the stem cells was accepted. The early phase of osteoblast differentiation involves an increase in ALP activity which reflects the number of osteogenically committed progenitor cells. A high correlation between ALP induction during in vitro osteogenic differentiation and in vivo bone-forming capacity is demonstrated both in enzyme activity and in mRNA expression [25]. A material with a faster osteogenic induction during early exposure may be more favorable for regenerative pulp therapies as it would immediately stimulate a mineralization barrier. ALP activity results indicated no difference among the tested materials on the immediate exposure measured on day 1. However, Pure Portland Cement exhibited elevated ALP activity on day 3 which continued until the end of cell culture with a significant increase compared to the control group, which may indicate its potential for maintaining osteo/odontogenic activity. The capacity of the tested materials to induce differentiation was further evaluated with the expression of osteogenic and odontogenic gene markers, RUNX2, DMP-1, and DSPP. RUNX2, a major transcription factor in mineralization, is highly expressed through all stages of craniofacial bone development in osteogenic mesenchyme [26]. RUNX2 directs MSCs to differentiate towards osteoblast lineage and supplies immature osteoblasts in forming immature bone, while downregulation is necessary for mature osteoblasts in their terminal differentiation [27]. RUNX2 was significantly upregulated in MSCs cultured in osteogenic media as expected on both day 3 and day 7, which was used as a positive control of the osteogenic differentiation capacity of used cells. Only Medcem MTA indicated significant upregulation of RUNX2 starting on day 3; however, all other tested materials also promoted RUNX2 expression after 7 days in accordance with previous studies [18], indicating their commitment towards osteogenic differentiation. Interestingly, Pure Portland Cement-exposed MSCs demonstrated a nearly six-fold increase in RUNX2 expression, reaching the highest level among all groups. Pure Portland Cement was reported to express higher levels of RUNX2 and elevated ALP activity in DPSCs compared to that of MTA and the control group, which also confirms the findings of the present study to suggest Pure Portland Cement may have an enhanced osteogenic capacity [23].