Odontogenic effects of two calcium silicate-based biomaterials in human dental pulp cells

Material and methods. Human dental pulp cells isolated from dental pulps were cultured in standard culture conditions in Dulbecco’s Modified Eagle’s Medium (DMEM) and then the effects of Micro-Mega mineral trioxide aggregate (MM-MTA) (Micro-Mega, Besançon, France) and ProRoot MTA (MTA) (Dentsply Sirona, Tulsa, USA) (positive control) were evaluated on HDPCs at 1, 7 and 14 days. Untreated cells were used as a negative control. Odontoblastic differentiation was assessed by alkaline phosphatase (ALP) activity. Runtrelated transcription factor 2 (RUNX2), alkaline phosphatase liver/bone/kidney (ALPL), bone morphogenetic protein 2 (BMP2), dentin sialophosphoprotein (DSPP), and Distal-less homeobox 3 (DLX3), as odontoblastic/ osteoblastic expression markers, were evaluated by semi-quantitative real-time polymerase chain reaction (RT-PCR) analysis. Calcium levels of culture media were also determined.


Introduction
Mineral trioxide aggregate (MTA) was introduced in the early 1990s and has been the material of choice for endodontic therapies due to its high mineralization capacity and relatively few inflammatory reactions in clinical use. 1,2A number of in vivo and in vitro studies concerning cell and tissue interactions with MTA have shown its favorable biological characteristics.−12 Moreover, the placement of MTA on pulp tissue leads to the proliferation, migration and differentiation of odontoblast-like cells, which produce a collagen matrix. 1 Despite many positive characteristics, MTA has a couple of disadvantages, such as its extended setting time, difficulty in handling due to its sandy consistency and high porosity, poor dispersion, and high cost. 13,14fter the development of MTA, many types of endodontic cements were developed, with Micro-Mega MTA (MM-MTA; Micro-Mega, Besançon, France) being one of those materials.It has a similar content as MTA, which is composed of tricalcium silicate, dicalcium silicate, tricalcium aluminate, calcium sulfate dehydrate, bismuth oxide, magnesium oxide, calcium carbonate (CaCO 3 ), and chloride accelerator. 15,16The shorter setting time of MM-MTA is thought to be a result of the addition of CaCO 3 and chloride accelerator to the formula. 16The material has also shown to be safe with respect to acceptable levels of arsenic, lead and metal oxides. 15Additionally, the evaluation of different physical properties of the material that mainly focused on the radiopacity and push-out bond strength has yielded favorable results. 17,18The MM-MTA is pre-dosed and the activated capsule is mechanically mixed, thus ensuring correct proportioning and homogenous mixing.
−21 However, the exact mechanism of dentin regeneration/dentinogenesis after exposure to MTA and MM-MTA has not yet been completely explicated.Thus, the aim of this study was to evaluate and compare the biologic effects of newly developed MM-MTA and MTA on odontogenic differentiation of primary human dental pulp cells (HDPCs).The null hypothesis was that MTA and MM-MTA have similar effects in promoting odontogenic differentiation.

Material and methods
Six human impacted 3 rd molars without any pathology extracted for orthodontic and impacted reasons were collected from 6 donors aged 18−25 years with informed consent, under a protocol approved by the Institutional Review Board and Ethics Committee of Baskent University, Ankara, Turkey (Project No. D-KA13/06).All 3 female and 3 male donors were healthy, without any known disease, were not taking any medications, and were non-smokers and non-alcohol consumers.Immediately after extraction, the extracted molars were cut horizontally at 1 mm below the cementoenamel junction.The carefully extracted pulp tissue was transferred to the laboratory in Dulbecco's Modified Eagle's Medium (DMEM)-F12 (Biochrom AG, Berlin, Germany) containing 1 drop of 1% streptomycin, neomycin and penicillin mixture (Biological Industries, Kibbutz Beit-Haemek, Israel).Physical dissection of the specimens was carried out by a scalpel.Under sterile conditions, the specimens were enzymatically digested with type I collagenase and dispase (Biochrom AG).After centrifugation at 500 g for 9 min, the supernatant was removed and the cells were placed in cell-culture medium (DMEM-F12 containing 10% heat-inactivated fetal bovine serum [FBS]; Biochrom AG), 2% l-glutamine (Biochrom AG), and 1% penicillin-streptomycin mixture (Biological Industries).The cells were cultured at 37°C in a humidified incubator supplemented with 5% CO 2 (Heraeus, Hanau, Germany).The cell culture medium was changed every 2−3 days and cells were subcultured when confluence reached 80%.Fourth to 7 th subculture of cells was used for experimental procedure.Viability of cells was evaluated using the trypan blue exclusion test and up to 95% viable cells containing cultures were included in the experiments.

Material preparation and application
Teflon tubes (10 mm internal diameter, 2 mm height) were sterilized with ethylene oxide gas.White MTA (Pro-Root MTA; Dentsply Sirona, Tulsa, USA, Lot No. 12002493) (positive control) and MM-MTA (Lot No. 7302238) were prepared according to the manufacturer's instructions, put into the tubes and stored in an incubator at 95% ±5% relative humidity at 37ºC for 1 day.
Dental pulp cells and the tubes were incubated in 9 cm 2 Petri dishes.To prevent direct contact of materials with cells, the tubes were placed in a permeable transwell inserts with a pore size of 0.4 µm (Greiner Bio-One GmbH, Frickenhausen, Germany).At the end of the 1, 7 and 14-day time periods, the cells were harvested for gene expression and alkaline phosphates (ALP) activity, and also cell culture media were collected for calcium levels.Untreated cells were used as a negative control.All cell culture experiments were repeated in triplicate.

Determination of alkaline phosphatase activity
Intracellular ALP activities were determined according to the manufacturer's protocol by a commercially available colorimetric kit (Abcam, Cambridge, UK).Briefly, the total cell number was determined by using a Thoma cell counting chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and subsequently 1 × 10 5 cells were used.Cells washed in phosphate buffered saline (PBS) were homogenized in an assay buffer and para-nitrophenyl phosphate (pNPP) solution was added on cells in a 96-well microplate.After 60 min of incubation at room temperature under dark conditions, the stop solution was added.Optical densities were measured at 405 nm in a microplate spectrophotometer (Epoch; BioTek, Swindon, UK).Data analyses were carried out according to the manufacturer's instruction and ALP activity was expressed as U/mL.

Determination of calcium levels
Calcium levels were determined according to the manufacturer's protocol by a commercially available colorimetric kit (Abcam).Then, a chromogenic reagent and an assay buffer were added on 20 µL of culture medium in a 96-well microplate.After incubating for 5−10 min at room temperature under dark conditions, optical densities were measured at 575 nm in a microplate spectrophotometer (Epoch).Optical density of normal medium was used as blank and subtracted from optical density of all samples.Calcium concentrations were calculated according to the manufacturer's instruction and expressed as mg/dL.

Real-time polymerase chain reaction analysis
Total RNA was isolated from MTA and MM-MTA applied cells of 6 patients with TriPure isolation reagent according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany).One microgram of total RNA was reverse transcribed using Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics GmbH).Real-time ready catalog assays, which are short FAM-labeled hydrolysis probes containing locked nucleic acid, were used for real-time polymerase chain reactions (RT-PCR) (Roche Diagnostics GmbH).Expression of runtrelated transcription factor 2 (RUNX2; Assay ID: 113380), alkaline phosphatase liver/bone/kidney (ALPL; Assay ID: 103448), bone morphogenetic protein 2 (BMP2; Assay ID: 104558), dentin sialophosphoprotein (DSPP; Assay ID: 139816), and distal-less homeobox 3 (DLX3; Assay ID: 140745) were determined using semi-quantitative RT-PCR by Light Cycler 480 II system (Roche Diagnostics GmbH) according to the manufacturer's instructions with a preincubation step at 95°C for 10 min, followed by 45 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s.Semiquantitative PCR reactions were run in duplicate, and the relative expression levels of RUNX2, ALPL, BMP2, DSPP and DLX3 transcripts' were calculated by the threshold cycle (Ct) and the 2 -ddCt method. 22Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID: 141139) were used for normalization of the expression data.

Statistical analysis
The Shapiro-Wilk test was used to assess the normality of distributions of the variables, and Levene's test was used to assess the homogeneity of variances among groups.Parametric test assumptions were not available for some variables.Data transformation methods were applied to these variables and then the analysis was performed.Comparisons of group means were performed with the repeated measures analysis of variance (ANOVA).
Data analyses were performed using the Statistical Package for the Social Sciences, v. 17.0 (SPSS Inc., Chicago, USA).A p-value < 0.05 was considered statistically significant.The results of statistical analysis were expressed as number of observations (n), mean ± standard deviation (mean ± SD, median and minimum-maximum values [min-max]).

Alkaline phosphatase activity
Alkaline phosphatase activities levels were increased with MTA and MM-MTA.The most intense ALP activity was exhibited by the MM-MTA group at day 14.However, there was no significant difference in ALP activity among the negative control, MTA-, and MM-MTA-treated groups for all time periods (p > 0.05) (Fig. 1).

Calcium levels
Observation from spectrophotometric analysis at day 7 revealed higher calcium level in HDPCs exposed of MTA and MM-MTA, whereas the calcium levels were not significantly different in negative control, MTA-and MM-MTA-treated groups for all time periods (p > 0.05) (Fig. 2).

Discussion
Dentinogenesis is the dentin formation process in which odontoblasts are responsible for the organic matrix synthesis and posterior mineral crystal deposition in this matrix.This pattern of formation is similar to that of bone, another mineralized connective tissue. 23For both mineralized tissues, it is of fundamental importance to understand how     In this study, the effect of MTA and MM-MTA on odontogenic differentiation of HDPCs is evaluated by measuring the mRNA levels of various differentiation markers genes and ALP activity.One of these specific markers is DSPP, which is believed to play a regulatory role in the mineralization of reparative dentin; it also serves as a specific marker for the odontoblastic phenotype. 24Bone morphogenetic proteins are a group of cytokines that play an important role in stem cells' activity, adjusting the proliferation and differentiation of cells (e.g., osteoblast), and bone formation. 25The up-regulation of ALPL (also known as tissue-nonspecific) has been associated with the onset of osteogenesis in vitro and its transcription can be up-regulated by BMPs. 26Runt-related transcription factor 2 is a transcription factor that plays an essential role in bone and tooth development.The up-regulation of RUNX2 in early odontoblasts followed by the downregulation in differentiated and functional odontoblasts suggests that it is important for RUNX2 to maintain the developmental stage-specific expression during odontogenic differentiation. 27Homeodomain gene DLX3 has been recently demonstrated as a potent regulator for proliferation and odontoblastic differentiation of HDPCs. 28Alkaline phosphatase activity is frequently used for the evaluation of biologic mineralization. 29This enzyme is crucial for the initiation (but not for the progression/maintenance) of the matrix mineralization process. 30n the present study, we aimed to reflect the clinical conditions of direct pulp capping in which the contact between pulp cells and biomaterials occurred indirectly through blood clots and physiologic fluids, which might not accurately model clinical setting.Seo et al. stated that the use of an interposed membrane only allows for the assessment of the effect of the diffusible components of MTA. 12 The results of the present study showed that ALP activity, calcium levels and mRNA expression levels of the dental pulp cells after treatment with elutes of MTA and MM-MTA were not significantly different compared with those of the untreated cells in the negative control group.This situation may be attributed to the permeable transwell inserts, which could act as a barrier and possibly decrease activation of the dental pulp cells. 31Developing a model that would allow studying the effect of direct interaction between materials and dental pulp cells would be important for future studies.
−21 The results of the present study agree with previous descriptions of these biomaterials in terms of odontogenic activity. 21Our results were also consistent with the study carried out by Chang et al., who reported similar ALP activity and up-regulation of odontoblastic markers such as ALP and DSPP between MTA and MM-MTA at 7 and 14 days. 32In the present study, DSPP gene was significantly down-regulated after day 1 and again significantly up-regulated after day 7 by both biomaterials.This could be attributed to a 14-day culture period, which might be the reason for unexpected differentiation results for both biomaterials. 33Higher gene expression levels might be obtained in a longer period.
Extracellular proteins, particularly family of the transforming growth factor (TGF) proteins, including BMPs, have been explained in stem cell research as potent molecules to induce odontogenic differentiation.Micro-Mega MTA exhibited higher BMP2 expression than MTA at all time periods of the present study.Despite similar calcium levels, the different effects of MM-MTA and MTA   on BMP2 expression may be related to the differences in the speed of ion release in 2 biomaterials during the culture period.A study by Setbon et al., which, which investigated the composition of different tricalcium silicate cements, revealed that MM-MTA possessed a higher concentration of magnesium and sodium than MTA. 34It has been demonstrated that these elements may represent an interest in terms of stimulation of osteodifferentiation. 35 Further pharmacokinetic studies on MM-MTA and MTA are, therefore, required to characterize and compare the ions released from these 2 biomaterials.
Expression patterns of homeobox genes of DLX family have been investigated during early stages of mouse odontogenesis.From that data, DLX genes have been proposed to contribute to odontogenic patterning. 36It is also shown that DLX3 supports the osteogenic differentiation in dental follicle cells via a BMP2 positive feedback loop. 37Moreover, Li et al. stated that DLX3 expression progressively increased during odontoblast differentiation. 28In contrast with those studies, the present study showed no significant up-regulation of DLX3 marker at all time periods of the study.Further studies are required to reveal more details about the relationship between DLX transcription factors and the regulation of the odontogenic differentiation.These studies will also be available to disclose new information about the regulation of the BMP2/DLX3 pathway.

Conclusions
Within the parameters of this study, MM-MTA exhibited higher BMP2 expression than MTA at all time periods of the present study.Overall, MTA and MM-MTA showed similar osteo-/odontogenic-like phenotype differentiation of HDPCs with the mechanisms of up-regulation of the majority of osteo-/dentinogenesis-related genes.In this respect, our results suggest that MM-MTA is a potential alternative to MTA for use as an effective pulp capping material.More clinical studies are needed to assess if the expressions of these markers are clinically relevant.

Fig. 3 .
Fig. 3.The effects of MTA and MM-MTA on BMP2 mRNA levels in HDPCs were determined by semi-quantitative RT-PCR at different time points (day 1, 7 14).Significant differences between test materials are indicated by asterisk and s (* p < 0.05) MTA -mineral trioxide aggregate; MM-MTA -Micro Mega MTA; HDPCs -human dental pulp cells; BMP2 -bone morphogenetic protein 2; RT-PCR -real-time polymerase chain reaction.
are capable of promoting odontogenic differentiation.