This study assessed the effects of Biodentine, Emdogain and Biodentine coated with Emdogain (Biodentine/Emdogain) on proliferation and differentiation of SCAP. Assessment of cell viability by the MTT assay, which is highly reliable for this purpose [25], revealed that after the control group, Emdogain had the highest biocompatibility followed by Emdogain/Biodentine. The Emdogain group had no significant difference with the control group in this regard. However, the Emdogain/Biodentine and Biodentine groups had significantly lower biocompatibility than the control group. Also, Biodentine had significantly lower biocompatibility than Emdogain. Thus, it appears that Biodentine is cytotoxic early after use; however, this effect seems to be alleviated by addition of Emdogain due to its proliferative effect. The biocompatibility of Emdogain at 24 hours was lower than that at 72 hours, which indicates that Emdogain has some cytotoxic effects at first, which are neutralized after 72 hours due to chemical stabilization of the cement.
Emdogain has properties similar to that of extracellular matrix, and regulates the proliferation, migration and differentiation of osteoblasts [26]. Wang et al. [27] demonstrated that Emdogain enhanced the mineralization of pulp cells [27]. The effects of Emdogain in combination with Biodentine are probably due to the possible molecular mechanisms and release of growth factors [28]. Karkehabadi et al. [29] indicated that addition of Emdogain to different biomaterials did not affect the cell viability after 24 and 48 hours; however, it significantly enhanced the cell viability at 7 days. Their results were different from the present findings since a reduction in cell viability occurred at 72 hours in the Emdogain/Biodentine group in the present study. It appears that cell proliferation neutralized the cytotoxic effects at 7 days. Difference between the two studies may be due to the fact that Karkehabadi et al. [29] evaluated DPSCs; while, SCAP were assessed in the present study. The present results regarding no cytotoxicity of biomaterials at 24 hours were in accordance with the findings of Saberi et al, [30] who found no significant difference in cytotoxicity of different biomaterials and the control group at 24, 48 and 168 hours. However, in the present study, Biodentine and Biodentine/Emdogain showed cytotoxicity at 72 hours, which was different from their results at 168 hours, and may be due to the fact that cell proliferation and release of calcium ions are considerably lower at 72 hours compared with 168 hours. The present results were also different from the findings of Mohamed and Fayyad [31] who reported a reduction in cell viability at 24 hours, which was compensated by cell proliferation on the next day. This difference may be due to the use of different cell types (DPSCs).
The rate of release of calcium ions at different time points may explain the variations in cell viability in presence of different cements. Calcium silicate-based cements (such as Biodentine) continuously release calcium ions [32]. Calcium silicate hydrate is then formed, and calcium carbonate phosphate deposits. Release of calcium ions can induce inflammatory toxic reactions [33]; however, it is also critical for the viability of mesenchymal stem cells [34]. Calcium ions play a fundamental role in signaling pathways and regulation of cellular activities such as cell migration [35].
ALP activity was also evaluated in this study since ALP is the primary marker of osteogenic differentiation [36]. The present results indicated a significant increase in ALP activity in all experimental groups at 24 and 72 hours. The maximum ALP activity was noted in the Emdogain/Biodentine group followed by Emdogain, Biodentine and finally the control group. These findings highlight the role of Emdogain and Biodentine (alone or in combination) in enhancement of pulp and dentin regeneration. Similar to the present study, Li et al. [37] indicated a significant increase in ALP activity of DPSCs after 7 days of incubation with Emdogain compared with the control group, and Miller et al. [38] reported higher ALP activity of SCAP induced by some bioceramic materials. Moreover, Wu et al. [36] reported significant enhancement of ALP activity for 3 hours caused by Emdogain. Min et al. [39] demonstrated that Emdogain + MTA significantly increased the ALP activity, which was in line with the present findings regarding the use of Biodentine/Emdogain.
Osteogenic-odontogenic differentiation of SCAP was also evaluated in this study by assessment of the expression of DSPP, BSP, and DMP genes, which play a fundamental role in odontoblastic differentiation and dentin mineralization [40]. The results indicated a significant increase in BSP expression at both 24 and 72 hours in all experimental groups, compared with the control group. This up-regulation was significantly greater in the Biodentine/Emdogain group followed by the Emdogain group. Min et al. [39] reported up-regulation of BSP gene in the MTA and MTA/Emdogain groups, which reached its maximum level after 3 days. Wang et al. [27] reported the up-regulation of odontoblast-like and osteoblast-like cell markers by Emdogain. Also, Jue et al. [41] demonstrated over-expression of BSP by human mesenchymal stem cells exposed to Emdogain. The abovementioned findings all support the present results.
The current results also revealed up-regulation of DMP1 at both time points in all experimental groups compared with the control group. The expression of DMP1 was maximum in the Emdogain/Biodentine group followed by the Emdogain group. DMP1 has a regulatory role in the mineralization process of reparative dentin, and is an odontoblastic marker [42]. The present results regarding the over-expression of DMP1 was in agreement with the findings of Asgary et al, [43] although they evaluated DPSCs.
Expression of DSPP indicates the presence of mature osteoblasts, and is correlated with dentinogenesis [44, 45]. Up-regulation of DSPP was also noted in the experimental groups, compared with the control group at both time points, which was maximum in the Biodentine/Emdogain group followed by the Emdogain group. This finding was in accordance with the results of Miller et al, [38] who reported the over-expression of DSPP by SCAP in presence of Biodentine and EndoSequence, Hajizadeh et al, [46] who reported the over-expression of DSPP by SCAP after 3 weeks of using different biomaterials, and Saberi et al, [47] who showed the up-regulation of osteogenic markers by SCAP due to exposure to Biodentine and MTA.
The mechanism of action of Emdogain in odontoblastic-osteoblastic differentiation has not been well elucidated. It may directly stimulate the odontoblasts or pulp cells to produce collagen matrix [48]. Alternatively, presence of transforming growth factor B1 or amelogenin peptides in Emdogain may induce cell signaling and matrix formation, and lead to subsequent mineralization [49].
Alizarin Red staining of odontoblast-like cells was also performed in this study. This test reveals calcium deposits in the extracellular matrix [50]. The results indicated staining of all experimental groups.
Overall, Biodentine has some toxic effects on the stem cells, which subsides over time. In the long-term, use of Emdogain can help alleviate this effect by induction of cell proliferation. On the other hand, combined use of Emdogain and Biodentine can have a synergistic effect on expression of odontoblastic markers and formation of calcified nodules, which is probably due to the release of calcium ions from Biodentine and growth factors from Emdogain.
This study evaluated cell viability, gene expression, and mineralization after 24 and 72 hours of exposure of SCAP to biomaterials. Future studies are required to assess the effects of biomaterials over longer periods of time. Also, the possible synergistic effects of other biomaterials should be investigated in future studies on different types of stem cells.