Effects of CEM cement and emdogain on proliferation and differentiation of human stem cells from the apical papilla: a comparative in vitro study

This study compared the effects of calcium-enriched mixture (CEM) cement, Emdogain (EMD), and their combination (CEM/Emdogain) on the differentiation and proliferation of stem cells from the apical papilla (SCAPs). In this in vitro, experimental study, SCAPs were isolated from two sound immature impacted third molars and cultured. After ensuring their stemness by detecting cell surface markers they were exposed to CEM cement, Emdogain, and CEM cement coated with Emdogain for 24 and 72 h. The control cells did not undergo any intervention. Cell viability [by methyl thiazolyl tetrazolium (MTT) assay], expression of odontogenic differentiation genes [by quantitative reverse-transcription polymerase chain reaction (qRT-PCR)], and alkaline phosphatase (ALP) activity (by ALP staining kit) were evaluated. Data were analyzed by one-way ANOVA, t-test, and Mann–Whitney test (α = 0.05). Cell viability in the CEM cement and CEM/Emdogain groups decreased compared with the control group at 72 h (P < 0.05). Expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) genes, and ALP activity significantly increased in all three experimental groups compared with the control group at both 24 and 72 h. This increase was substantially more significant in CEM/Emdogain group (P > 0.05). The number of mineralized nodules significantly increased in all groups at 72 h, with a higher rate in the CEM/Emdogain group. All biomaterials increased the differentiation of SCAPs, expression of odontogenic differentiation genes, and ALP activity, but CEM/Emdogain was considerably more effective for this purpose.


Introduction
Conventional root canal therapy is performed in case of loss of pulp vitality. However, outstanding success may be achieved only by tissue engineering to replace the lost tissue (dental pulp, dentin, or cementum) in case of the absence of adequate remaining tooth structure, which is referred to as regenerative endodontics. Endodontic regenerative procedures benefit from the differentiation potential of dental pulp stem cells (DPSCs) or the progenitor cells of the mature pulp (Tziafas and Kodonas 2010). Regenerative endodontic procedures are the treatment of choice for immature permanent teeth with necrotic pulp and open apex, allowing the completion of root development and increasing the chance of normal physiological responses .
A certain number of mesenchymal stem cells are isolated and collected from the apical tissue for regenerative treatment of mature and immature teeth (Chrepa et al. 2015). These cells are derived from the apical papilla (Chrepa et al. 2017) but are also produced in bone, periodontal ligament, and granuloma. The stem cells of the apical papilla (SCAPs) are a population of dental mesenchymal stem cells with high proliferation potential, self-renewal capacity, and low immunogenicity (Sonoyama et al. 2006). The SCAPs are more suitable for tissue regeneration than DPSCs because the latter group has a higher proliferation rate and mineralization capacity (Zhai et al. 2019). It has been reported that SCAPs isolated from immature impacted third molars have a proliferation rate three times higher than that of DPSCs . Also, SCAPs can express dentin sialophosphoprotein (DSPP), which indicates their applicability for regenerative procedures Sonoyama et al. 2006). The SCAPs can differentiate into different cell types including osteoblasts, odontoblasts, neurons, adipocytes, chondroblasts, and hepatocytes. (Dong et al. 2013;Sonoyama et al. 2008). Scaffolds provide an optimal network for the correct positioning of cells and regulation of their proliferation, metabolism, and differentiation in endodontic regeneration (Nakashima and Reddi 2003).
In endodontic regenerative procedures, biomaterials are directly exposed to a blood clot containing stem cells such as SCAPs. Bioceramic materials, especially calcium silicate, are the material of choice for regenerative treatments due to optimal properties such as favorable biocompatibility, and antimicrobial activity (Parirokh and Torabinejad 2010). Stem cells such as SCAPs have complex interactions with bioactive materials and dentin (Smith et al. 2016).
Endodontic regenerative procedures are a successful alternative to old techniques for root development and apex closure (especially in cases of dental pulp injury due to traumatic dental injury) by differentiation of stem cells, pulp regeneration, and continuation of mineralization (Lee et al. 2015). In contrast to the apexification procedure (artificial induction of apical barrier formation), which requires several sessions of calcium hydroxide injection into the root canal, regenerative treatments are short and enable physiological growth and development of the root structure and increase the root length and thickness (Shah et al. 2008). Apexification treatments do not increase the root dentin thickness; thus, the root walls remain thin and fragile, and the tooth remains susceptible to fracture. Also, it has been reported that long-term use of calcium hydroxide weakens the dentin structure (Andreasen et al. 2002). Despite the higher success rate of mineral trioxide aggregate (MTA) in singlesession apexification treatment, the roots remain thin and fragile due to incomplete root development (Parirokh and Torabinejad 2010). Thus, novel revascularization techniques are preferred for immature necrotic teeth (Dong et al. 2013).
Calcium-enriched mixture (CEM) cement was first introduced in 2006, as an endodontic filling material and contains different calcium compounds (Asgary et al. 2006). CEM cement can result in the formation of greater hydroxyapatite crystals in phosphate-buffered saline (PBS), which simulates the interstitial tissue fluid (Lee et al. 2015). Also, CEM cement has optimal physical, chemical, and clinical properties such as shorter setting time, causing less discoloration, better handling properties, and lower film thickness than MTA (Karkehabadi et al. 2021). CEM cement includes several calcium compounds such as calcium oxide, calcium phosphate, calcium carbonate, calcium silicate, calcium sulfate, calcium hydroxide, and calcium chloride. Its clinical application is similar to that of MTA, and it sets in aqueous environments. It has easy handling and provides an optimal seal (Balto 2004). Since CEM cement can produce hydroxyapatite using intrinsic ions, it is used in contact with the necrotic pulp in endodontic regenerative procedures (Bonson et al. 2004).
Emdogain is an enamel matrix derivative, which is produced from the developing porcine enamel matrix. Amelogenin is the main component of Emdogain. It also contains a low concentration of metalloproteinases and growth factors. It reportedly affects the migration, attachment, and proliferation potential of periodontal ligament cells (Amin et al. 2013). Amelogenin particles also participate in the process of maturation and growth of DPSCs (Ye et al. 2006). Emdogain was recently suggested as a pulp-capping agent which enhances the formation of reparative dentin and odontoblastic differentiation. Since it has a gel consistency, it does not rapidly diffuse in the pulp tissue (Utneja et al. 2015).
The benefits of CEM cement have been demonstrated in previous studies, including the antibacterial effect (Asgary et al. 2007), non-significant cytotoxic effects of CEM cement on human stem cells from the apical papilla (Saberi et al. 2016), and promoting an alkaline pH, as well as releasing calcium and phosphate (Ghazvini et al. 2009). Considering the significance of endodontic regenerative procedures and the gap of information regarding the efficacy of CEM cement coated with Emdogain for regenerative procedures, this study aimed to assess the effects of CEM cement, Emdogain, and their combination (CEM/ Emdogain) on the differentiation and proliferation of SCAPs, However, this has not yet been considered.

Materials and methods
This in vitro experimental study was conducted on SCAPs to assess their viability percentage, expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) genes, and alkaline phosphatase (ALP) activity after 24 and 72 h of exposure to CEM cement, Emdogain, and CEM cement coated with Emdogain.

Isolation of SCAPs
SCAPs were isolated from two sound immature impacted third molars of two patients presenting to the School of Dentistry of Hamadan University of Medical Sciences. The teeth were scheduled for extraction for orthodontic reasons. The patients signed informed consent forms and consented to the use of their extracted teeth for research purposes. Considering the in vitro study design and the sample size of previous studies (Bakopoulou et al. 2011;Pereira et al. 2012), three repetitions were considered for each of the experimental and control groups at each time point. The study was also approved by the Ethics Committee of Hamadan University of Medical Sciences (IR.UMSHA.REC.1398.796).

Cell culture
The teeth were rinsed with sterile phosphate-buffered saline (PBS; Gibco BRL, Grand Island, NY, USA) immediately after extraction and stored in sterile PBS. Stem cells were isolated from the radicular pulp tissue by enzymatic digestion using 2 mg/mL of type I collagenase (Worthlington Biomedical, Lakewood, NJ, USA) and transferred to Dulbecco's modified Eagle's medium (Gibco, GrandIsland, NY, USA). The cells were re-cultured in the culture medium supplemented with 15% fresh bovine serum(Gibco, Invitrogen, NY, USA), and then in alpha-minimum essential medium supplemented with 10% fetal bovine serum in sterile cell culture flasks (SPL Life Science, Gyeonggi-do, South Korea), (incubated at 37 °C, 5% CO 2 and 85% moisture). The culture medium was refreshed every 2-3 days, and the cells were passaged after 1 week. Four passages were done to reach the adequate confluence of stem cells, and 4-μm insert plates (SPL Life Science, Gyeonggi-do, South Korea) were used for the treatment of the cells with the respective biomaterials.

Characterization of SCAPs
After reaching 80% confluence, the culture medium was removed from the flask, and the cells were rinsed with PBS twice. The cells were detached by using trypsin/EDTA, and the culture medium was added to the flask. The culture medium and the cells were then transferred into a 15-mL Falcon tube and centrifuged at 1200×g for 6 min. The cell sediment was rinsed with PBS twice, and the cells were evaluated for the presence of specific stem cell surface markers (CD105 and CD90), and hematopoietic cell surface markers (CD45 and CD34) by flow cytometry. The cells exhibited mesenchymal cell surface markers and were negative for hematopoietic cell surface markers (Ishizaki et al. 2003). The FACScalibur cytometer (Becton Dickinson) and CellQuest software were used to assess data. Of all, 97.9% of the cells expressed the CD-105 marker and 95.8% of them expressed the CD-90 marker. Also, 99.8% of the cells did not express the CD-34 or the CD-45 markers.

Study groups
The cells were evaluated in four groups: no intervention (control), treatment with Emdogain, treatment with CEM cement, and treatment with the combination of CEM cement and Emdogain. Three repetitions were considered for each test in each group (Pereira et al. 2012).
CEM cement (BioniqueDent, Tehran, Iran) was prepared in sterile conditions according to the manufacturer's instructions. The mixture was applied in a paraffin wax mold, condensed, and allowed 10 min to set at 37 °C in 96% humidity. Specimens with 10 mm diameter and 1 mm thickness were fabricated as such.

Assessment of cell viability
The methyl thiazolyl tetrazolium (MTT) assay was used as a quantitative analysis to assess cell viability and proliferation. For this purpose, the cells were cultured in a 96-well plate. The cells in the first row were treated with 100 µg/mL Emdogain. The cells in the second row were treated with CEM cement, and the cells in the third row were treated with a mixture of CEM cement and Emdogain. The control cells did not receive any treatment. At 24 and 72 h, 10 λ of the MTT solution was added to all wells, and the plate was incubated at 37 °C. After 2 h, the contents of the wells were removed, and 100 λ of dimethyl sulfoxide was added to the cell sediments. After 20 min, the percentage of viable cells was calculated by reading the optical density of the suspension by an ELISA Reader (Bio-Rad, Hercules, CA) at 570 nm wavelength.

Assessment of odontogenic-osteogenic differentiation
For this purpose, the cells were cultured in a 24-well plate and after exposure to CEM cement, Emdogain, and a combination of the two, osteogenic odontogenic medium i.e., regular medium containing 10 mM betaglycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 50 mg/mL ascorbic acid was added to the wells. The culture medium was refreshed every 72 h. After 21 days, the cells were fixed with 4% paraformaldehyde, rinsed with PBS, and incubated with Alizarin Red stain at room temperature for 15 min. The differentiated cells stained red after this period due to the presence of calcium deposits (Qu et al. 2014).

Assessment of the expression of odontogenic differentiation genes
After treating the cells with Emdogain, CEM cement, and a combination of both, RNA was extracted using Trizol. The extracted total RNA was quantified by NanoDrop at 260 nm and 280 nm wavelengths. Next, the cDNA was synthesized by Superscript II firststrand cDNA synthesis kit (Invitrogen, CA, USA) according to the manufacturer's instructions. The expression of BSP, DMP1, and DSPP genes in the presence of the beta-actin gene as the housekeeping gene was quantified, and the results were normalized. The cDNA samples along with specific primers underwent real-time polymerase chain reaction (PCR).

Assessment of ALP activity
The ALP activity was quantified by using the ALP staining kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. The cells in all groups were rinsed with PBS twice and lysed overnight using 0.2% TritonX-100 (Jiancheng, Nanjing, China) at 37 °C. Next, the working solution was added, and the optical density of each sample was read at 520 nm wavelength using an automatic microplate reader (BioTek, Winooski, VT, USA).

Statistical analysis
Data were analyzed by Statistical Package of the Social Sciences (SPSS Version 25; IBM Corp, Armonk, NY). One-way ANOVA was used to compare the four groups, while pairwise comparisons were performed by t-test (for normally distributed data) or the Mann-Whitney test (for non-normally distributed data). P ≤ 0.05 was considered significant.

Cell viability
As shown in Fig. 1, treatment with CEM cement, Emdogain, and CEM/Emdogain had no cytotoxic effect on SCAP at 24 h, and the cells in the three experimental groups had no significant difference with the control group regarding the viability percentage. The difference among the three experimental groups was not significant regarding the cell viability at 24 h (P > 0.05).
At 72 h, Emdogain had no significant difference with the control group regarding cell viability (P > 0.05). However, treatment with CEM cement and CEM/Emdogain decreased the cell viability compared with the control group.according to the One-way ANOVA analysis, the difference in cell viability was significant among the groups at 72 h. Pairwise comparisons by Tukey's test revealed no significant difference between CEM cement and CEM/Emdogain groups regarding the cell viability at 72 h (P > 0.05). However, the Emdogain group showed significantly higher cell viability than CEM and CEM/Emdogain at this time point.
Expression of osteogenic/odontogenic genes: BSP: As shown in Fig. 2, One-way ANOVA revealed that treatment of SCAPs with CEM cement, Emdogain, and CEM/Emdogain significantly increased the expression of the BSP gene at 24 and 72 h, compared with the control group. This increase was greater in the CEM/Emdogain group than in the other two experimental groups at both time points. The expression of BSP gene was not significantly different in the Emdogain and CEM cement groups (P > 0.05).

DMP1:
As shown in Fig. 3, One-way ANOVA revealed that the expression of DMP1 in the three experimental groups was significantly higher than that in the control group (P < 0.05). At 24 h, no significant difference was noted among the experimental groups. However, at 72 h, the CEM/Emdogain group showed significantly higher expression of DMP1 than the CEM cement and Emdogain groups. The difference between the Emdogain and CEM cement groups was not significant in this regard (P > 0.05).

DSPP:
As shown in Fig. 4, One-way ANOVA revealed that the expression of DSPP in the three experimental groups was significantly higher than that in the control group at both 24 and 72 h (P < 0.05). At 24 and 72 h, the difference between the CEM cement Fig. 2 Expression of BSP gene in the three groups after 24 and 72 h of exposure. ***P < 0.001 compared with the control group. # P < 0.001 compared with the CEM cement and Emdogain groups. **P < 0.01 compared with the control group Fig. 3 Expression of DMP1 gene in the three groups after 24 and 72 h of exposure. ***P < 0.001 compared with the control group. # P < 0.001 compared with the CEM cement and Emdogain groups and Emdogain groups was not significant (P > 0.05) but CEM/Emdogain significantly increased the expression of DSPP gene compared with the other two experimental groups (P < 0.05).

ALP activity
As shown in Fig. 5, One-way ANOVA revealed that ALP activity significantly increased in the three experimental groups compared with the control group at both 24 and 72 h (P < 0.05). The difference Fig. 4 Expression of DMP1 gene in the three groups after 24 and 72 h of exposure. ***P < 0.001 compared with the control group. # P < 0.001 compared with the CEM cement and Emdogain groups

Fig. 5 ALP activity in the three groups after 24
and 72 h of exposure. ***P < 0.001 compared with the control group. # P < 0.001 compared with the CEM cement and Emdogain groups in this respect was not significant between the CEM cement and Emdogain groups at 24 or 72 h. However, the ALP activity was greater in the CEM/Emdogain group compared with the other two experimental groups.

Odontogenic-osteogenic differentiation
In Alizarin Red staining, the orange-red color indicates the initiation of mineralization as shown in Fig. 6. At 72 h, the frequency of mineral nodules increased in all groups. Greater mineralization was noted in the CEM/Emdogain group compared with other groups at both time points.

Discussion
There has been a recent rise in interest in the use of emdogain for capping dental pulp. Due to the gellike nature of emdogain, it does not readily penetrate into the pulp tissue. According to research, emdogain accelerates the formation of reparative dentin during pulp capping procedures and enhances odontoblastic differentiation (Ishizaki et al. 2003;Nakashima and Akamine 2005). The study of Karkehabadi et al. (2019) demonstrated that the addition of emdogain to MTA and CEM cement enhanced the viability of human dental pulp stem cells. Since SCAPs play a significant role in the success of endodontic regenerative treatments, the effects of CEM cement, Emdogain, and CEM/Emdogain on the proliferation and differentiation of SCAPs at different time points were evaluated in this study. To the best of our knowledge, this has not been examined previously. Cell viability was evaluated by the MTT assay, which is a standard reliable technique for this purpose and has high accuracy (Chang et al. 2014). The current results showed that none of the products had any toxic effect on the cells at 24 h, and there was no significant difference in this respect among the three experimental groups. At 72 h, Emdogain had no toxic effect on the cells, similar to the control group, but treatment with CEM/ Emdogain decreased the cell viability compared with the control group. Also, Emdogain had significantly lower cytotoxicity than the other two experimental groups at 72 h. This finding indicates that CEM cement, as a silicate cement, has cytotoxic effects early after setting. It appears that this effect is alleviated by covering the CEM cement with Emdogain due to the proliferative effects of Emdogain. The interaction effect of Emdogain in combination with CEM cement depends on molecular mechanisms and their effects on release of growth factors (Paduano  Karkehabadi et al. (2019) reported that the addition of Emdogain had no significant effect on cell viability in presence of other biomaterials at 24 and 48 h, but the addition of Emdogain to CEM cement significantly increased the cell viability at 7 days, which was in contrast to the present findings because both CEM cement and CEM/Emdogain caused a reduction in cell viability at 72 h, with no significant difference with each other. It appears that in the present study, cell proliferation after 7 days neutralized the cytotoxic effects of CEM cement alone and in combination with Emdogain. The Difference between the present results and those of Karkehabadi et al. (2019) may also be due to the use of different cell types (SCAPs in the present study and DPSCs in their study). Saberi et al. (2016) evaluated the cytotoxicity of different biomaterials for SCAPs and reported no significant difference in cytotoxicity of biomaterials and the control group at 24, 48, and 168 h. The present results at 24 h were in agreement with their findings; however, CEM cement and CEM cement plus Emdogain decreased the cell viability at 72 h. Saberi et al. (2016) reported that the cytotoxicity of CEM cement at 168 h was lower than that of other biomaterials. Mohamed and Fayyad (2017) evaluated the effects of nano-hydroxyapatite, MTA, and CEM cement on proliferation of DPSCs, and reported that all biomaterials primarily decreased the viability and number of cells at 1 day. Their results were in agreement with the present results although they used DPSCs. An increase in cell proliferation was then noted in all groups in their study (Mohamed and Fayyad 2017). Variations in cell viability in presence of different types of cement at different time points can be attributed to the release rate of calcium ions. Calcium silicate cements continuously release calcium (Hwang et al. 2008), and CEM cement is no exception to this rule. Also, calcium silicate hydrate continuously forms and calcium carbonate phosphate continuously deposits in the use of calcium silicatebased cements. Release of calcium leads to toxic inflammatory reactions (Carnio et al. 2002). On the other hand, release of calcium is important for the viability of mesenchymal stem cells (Hammarström et al. 1997). Calcium ions are capable of signaling and play fundamental roles in the regulation of cellular activities such that the migration of stem cells is influenced by the calcium ions (Castellanos et al. 2006). The cell viability in presence of CEM cement depends on the calcium release rate and binding of calcium to phosphorus for the formation of hydroxyapatite crystals (Saberi et al. 2016). This cement can change the enzymatic activity of the cells and their permeability. This process eventually increases the reparative procedures (Utneja et al. 2015).
DMP1 and DSPP belong to the family of integrinbinding ligand glycoproteins. They are extracellular matrix proteins that are expressed in the process of odontoblastic differentiation (Feng et al. 1998). These glycoproteins are specific mineralization markers in osteoblasts and odontoblast-like cells. The extracellular matrix compounds in the dental pulp include collagens and non-collagenous proteins. In the present study, DSPP, BSP, and DMP1 proteins were used as markers of odontoblastic-like differentiation. The expression of these genes is correlated with the dentinogenesis process and occurs following the formation of a pre-dentin collagen matrix (Feng et al. 1998). BSP is a main non-collagenous protein specifically expressed in remineralized tissues such as bone, mineralized cartilage, dentin, and cementum (Mizuno et al. 2000). BSP is primarily produced by osteoblasts and is considered as an osteoblastic phenotypic marker (Mizuno et al. 2000) although it has been found in dentin as well (About et al. 2000). In the present study, treatment of SCAPs with the biomaterials in all three experimental groups significantly increased the expression of BSP gene at both 24 and 72 h compared with the control group, and this increase was greater in the CEM/Emdogain group. Min et al. (2009) reported an increase in the expression of BSP and DSPP, and ALP activity by MTA and MTA/Emdogain, which reached the maximum level at 3 days. Their results were in agreement with the present findings regarding the up-regulation of these genes irrespective of the type of biomaterial used. Similarly, Hwang et al. (2008) reported the expression of BSP by odontoblast-like cells of reparative dentin. Wang et al. (2014) indicated that Emdogain enhanced the mineralization of human DPSCs. They also demonstrated the up-regulation of odontoblast and osteoblast-like cell markers following treatment with Emdogain, which was in line with the present results. Accordingly, the mineralization induced by this biomaterial may have osteogenic properties (Min et al. 2009). Similarly, Jue et al. (2010) reported over-expression of BSP in human mesenchymal stem cells due to the effect of Emdogain. DMP1 regulates the process of reparative dentin mineralization and is a specific marker of odontoblasts (Papagerakis et al. 2002). Furthermore, Karkehabadi et al. (2022) reported a significant enhancement of BSP expression after both 24 h and 72 h due to the effect of biodentine and Emdogain. It is present in the extracellular dentin and bone matrix as well (Bozic et al. 2012). In the present study, the expression of DMP1 in the three experimental groups was significantly higher than in the control group. The three experimental groups had no significant difference in this regard at 24 h. However, at 72 h, Emdogain/CEM cement group showed significantly higher expression of DMP1 than the other two experimental groups. Asgary et al. (2014) showed that both MTA and CEM cement increased the phenotypic osteo/odontogeniclike differentiation of DPSCs. Their results regarding the effects of biomaterials on the expression of DSPP and DMP1 were in agreement with the present findings although they evaluated DPSCs. Jae-Hwan et al.
(2019) evaluated the combined effect of MTA and propolis on odontoblastic differentiation of DPSCs and signaling pathways. They reported that this mixture caused up-regulation of DSPP and DMP1 and enhanced the formation of mineralized nodules, as well as the odontogenic differentiation and mineralization of DPSCs. Their results were in line with the present findings, irrespective of the type of biomaterials and target cells. Expression of DSPP gene occurs when pre-dentin collagen matrix has formed and its expression indicates the presence of mature osteoblasts, and is related to dentinogenesis (Rathinam et al. 2015). The expression of DSPP was also higher in the experimental groups at both 24 and 72 h in the present study, and combined use of Emdogain and CEM cement caused significantly greater expression of DSPP than other experimental groups. Miller et al. (2018) evaluated the effects of bioceramic materials on the proliferation and differentiation of SCAPs and showed that Biodentine and EndoSequence enhanced the viability and differentiation of SCAPs and caused up-regulation of DSPP odontoblastic marker. Their findings were in accordance with the present results although different types of biomaterials were used. Hajizadeh et al. (2018) assessed the effects of MTA and CEM cement on odontogenic differentiation and mineralization of SCAPs and reported up-regulation of ALP, SP7, DSPP, and OSC genes after 3 weeks, compared with the control group, which was in line with the present results. Saberi et al. (2019) evaluated the effects of CEM cement, Biodentine, MTA, octacalcium phosphate, and Atlantik on proliferation and odontogenic/osteogenic differentiation of SCAP, and production of pro-inflammatory cytokines. They reported that the biomaterials increased cell proliferation, ALP activity, calcified nodule formation, and expression of odontogenic/osteogenic marker genes in comparison with the control group, which supported the present findings.
Osteogenic and odontogenic differentiation of DPSCs, assessed by the expression of related markers, plays a fundamental role in odontoblastic differentiation and dentin mineralization (Siew Ching et al. 2017). Differentiation and mineralization of osteoblasts and odontoblasts occur early in the proliferation phase and biosynthesis of the extracellular matrix before cell differentiation (Stein et al. 1990). Early in this process, the matrix is matured and some proteins related to pulp cell phenotype such as ALP are detectable. ALP is an index for new bone formation, and its high expression rate has been reported during the primary phase of osteogenic differentiation (Cao et al. 2016). Thus, ALP is the primary marker of osteogenic differentiation. ALP activity increased at both time points in all experimental groups. Also, the combined use of Emdogain and CEM cement increased the ALP activity compared with the use of each biomaterial alone. Accordingly, it appears that a combination of CEM cement and Emdogain may enhance pulp and dentin regeneration. Li et al. (2017) reported a significant increase in ALP activity of DPSCs after 7 days of incubation with Emdogain, compared with the control group, which was in accordance with the present results. Wu et al. (2014) reported a significant increase in ALP activity in human gingival mesenchymal stem cells for 3 h following treatment with Emdogain. Also, Min et al. (2009) showed a significant increase in ALP activity of DPSCs treated with MTA plus Emdogain. The abovementioned results all confirmed our findings.
The mechanism of the effect of Emdogain on odontoblastic/osteoblastic differentiation has yet to be clearly understood. Emdogain may directly stimulate the odontoblasts or pulp cells to produce collagen matrix (Ishizaki et al. 2003). The presence of transforming growth factor B1 or amelogenin peptides in Emdogain may also induce cellular signaling, matrix formation, and mineralization (Iwata et al. 2002).
Alizarin Red staining has long been used for the assessment of calcium deposits by the cells in the culture medium (Gronthos et al. 2000). It indicates calcium deposits in the extracellular matrix. The process of differentiation of odontoblast-like cells is similar to that of osteogenic cells in the bone marrow, leading to the formation of calcified nodules (Wang et al. 2011). Thus, the expression of one of these genes may play a role in dentinogenesis (Liu et al. 2004).
In total, it appears that CEM cement has toxic effects on stem cells similar to all silicate cements, and this effect decreases with time. In the long term, Emdogain may decrease this effect by stimulating cell proliferation. On the other hand, the combined use of CEM cement and Emdogain increased the expression of odontoblastic markers and the formation of dentinlike calcified nodules compared with the use of each biomaterial alone. It appears that the release of calcium ions from the CEM cement and growth factors from the Emdogain as well as their cumulative effect on the expression of different markers resulted in significantly superior odontogenic differentiation in the combined use of these biomaterials.
The effects of biomaterials on SCAP viability and differentiation were assessed at 24 and 72 h. Future studies are required to assess these effects over a longer period of time. Also, the effects of these biomaterials should be compared with those of Biodentine and MTA. Assessment of the synergistic effects of other biomaterials on the viability and differentiation of SCAP and the use of other types of stem cells are also recommended.

Conclusion
All the tested biomaterials increased the differentiation of SCAPs, expression of odontogenic differentiation genes, and ALP activity. However, the efficacy of CEM/Emdogain for the latter two was considerably higher than each product alone.
Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by EK, LG, RN and, HK. The first draft of the manuscript was written by LG and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding This study was financially supported by the School of Dentistry, Hamadan University of Medical Sciences, Hamadan, Iran (Grant Number: 9811298971).

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations
Conflict of interest Elham Khoshbin, Leila Ghasemi, Rezvan Najafi and, Hamed Karkehabadi declare that they have no conflict of interest. The authors have NO affiliations with or involvement in any organization or entity with any financial interest (such as Honoria; educational grants;participation in speaker's bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest(such as personal or professional relationships, affiliations, knowledge or beliefs)in the subject matter or materials discussed in this manuscript. The authors have no relevant financial or nonfinancial interests to disclose. The authors have no financial or proprietary interests in any material discussed in this article.
Ethical approval All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Ethics Committee of Hamadan University of Medical Sciences (IR.UMSHA. REC.1398.796).
Informed consent Informed consent was obtained from all individual participants included in the study.