Interaction of dental pulp stem cells with Biodentine and MTA after exposure to different environments

ABSTRACT Objective: The aim of the present study was to evaluate and compare the cytotoxic effects of Biodentine and MTA on dental pulp stem cells (DPSCs) and to assess cell viability and adherence after material exposure to an acidic environment. Material and Methods: DPSCs were cultured either alone or in contact with either: Biodentine; MTA set for 1 hour; or MTA set for 24 hours. After 4 and 7 days, cell viability was measured using the MTT assay. Biodentine and MTA were also prepared and packed into standardized bovine dentin disks and divided into three groups according to the storage media (n=6/group): freshly mixed materials without storage medium (Group A); materials stored in saline (Group B); materials stored in citric acid buffered at pH 5.4 (Group C). After 24 hours, DPSCs were introduced in the wells and cell adherence, viability, and cellular morphology were observed via confocal microscopy after three days of culture. Cell viability was analyzed using repeated-measures analysis of variance test with Tukey's post hoc tests (α=0.05). Results: Biodentine expressed significantly higher cell viability compared with all other groups after 4 days, with no differences after 7 days. Notably, cell viability was significantly greater in 24-hour set MTA compared with 1-hour set MTA and control groups after 7 days. Material exposure to an acidic environment showed an increase in cell adherence and viability in both groups. Conclusions: Biodentine induced a significantly accelerated cell proliferation compared with MTA. Setting of these materials in the presence of citric acid enhanced DPSC viability and adherence.


INTRODUCTION
Mineral trioxide aggregate (MTA) is a calcium silicate-based material and has attracted considerable attention because of its excellent biocompatibility, sealing ability, and antimicrobial properties 20,29 . Although it was initially introduced as a material for repair of root perforations, it is currently used in vital pulp therapy, as a root-end procedures, and in regenerative endodontic therapy 12,15,28 . Despite its broad spectrum of clinical indications, MTA comes with certain limitations including long setting time, difficult handling, possibility of crown staining, and high cost 4,20 .
Biodentine (Septodont, Saint Maur des Fosses, France), a new calcium silicate-based restorative cement, was recently introduced for endodontic procedures. This bioceramic material is a fast-setting restorative material recommended as a dentin substitute that can be used in similar applications such as MTA 14 . Materials that are intended for endodontic applications should stimulate repair or be biologically neutral in order to promote healing 6 . If, however, endodontic materials are cytotoxic, and cause cell death by apoptosis or necrosis 8 . Although there is evidence about the excellent 2016;24(5):481-6 biocompatibility of MTA 5,25,29 , limited information is available about the possible cytotoxicity of Biodentine 2,13,14,17 .
Clinicians often face the challenge of placing materials in a low pH environment due to the presence of inflammation 18 . Variations in the pH at the time of placement could affect the physical and chemical properties of both MTA and Biodentine 5,19,24,31 . It has been shown that the low pH of the surrounding microenvironment affects the hydration reaction of MTA 16 , and that the more acidic the MTA solution during the setting process is, the more extensive its porosity will be 19 . However, adherence of cells to these materials after the exposure to an acidic environment.
The primary aim of this study was to evaluate the viability of dental pulp stem cells (DPSCs) when in contact with Biodentine in comparison with MTA. The secondary aim was to examine whether the presence of these materials in an acidic environment could have an effect on the DPSCs viability and adherence to these materials.

Cell culture
Human DPSCs were provided by ProCell, Biotechnological Application SA (Athens, Greece). The cells were screened with Flow Cytometry for mesenchymal surface markers. The triple panel of protein CD73, CD90, and CD105, which by consensus is expressed in mesenchymal stem cells, was detected in high levels (>85%). Moreover, the cells were negative for CD45 (hematopoietic cell marker), CD34 (hematopoietic stem cell marker), and CD31 (endothelial cell marker). DPSCs were cultured in basal culture media composed Gibco, Glasgow, UK) supplemented with 10% fetal bovine, 1X L-glutamine (Gibco), penicillin (100 U/ mL; Gibco) and streptomycin (100 mg/mL; Gibco). with 0.05% trypsin (Gibco, Carlsbad, CA, USA) and passed to subsequent culture plates or used in utilized in this study.

Cell viability
White ProRoot MTA (Dentsply Tulsa Dental Specialties, Memphis, TN, USA) and Biodentine (Septodont, Saint Maur des Fosses, France) were prepared according to the manufacturer's instructions and placed at the bottom of a 48-well plate (n=5/group). Materials were placed at a 2 mm thickness and fully coverage of the bottom of after placement, materials were allowed to set for one hour at 37°C in 5% CO 2 and 100% humidity under sterile conditions and 1x10 4 DPSCs were introduced in each well in direct contact with the materials. To account for the difference in setting time between the testing materials, a group of 24-hour set ProRoot MTA (MTA24h) was also included (n=5). MTA was allowed to set for 24 hours under the same conditions described above before DPSCs were introduced. All groups were incubated at 37°C in 5% CO 2 and 100% humidity for 4 and 7 days. Cells without materials served as control. Cell viability was measured using methylthiazolyldiphenyl-tetrazolium bromide (

S p e c i m e n p r e p a r a t i o n f o r immuno uorescence
36 dentin disks from anterior bovine teeth were horizontally sectioned into 3-mm slices using an Isomet device (Buehler Ltd., Lake Bluff, IL, USA) and the canal space of each dentin slice was enlarged to 2.6 mm in diameter. Biodentine and MTA were prepared according to the manufacturers' instructions under aseptic conditions and packed into the lumen of dentin disks (N=18/group). Specimens from each material group were further divided into three groups according to the storage media and placed in 24-well plates -Group A: freshly mixed materials without storage; Group B: materials with saline as storage medium; Group C: materials with citric acid buffered at pH 5.4 as storage medium (n=6/group). Each specimen was kept in contact with a saline-or citric acid-soaked piece of gauze for 24 hours in room temperature.

I m m u n o f l u o r e s c e n c e a n d c o n f o c a l microscopy
After 24 hours of storage, 2.5x10 4 DPSCs were introduced in the wells in direct contact with the dentin disks. Groups were cultured for 72 hours at 37°C in a 5% CO 2 that adhered to the surface of the samples were followed by 0.1 M PBS washing twice for 10 minutes. The samples were then stained using Phalloidin-Rhodamin solution (Molecular Probes, ThermoFisher manufacturer's protocol to reveal the cytoskeleton 4',6-diamidino-2-phenylindole (DAPI, Vectashield H-1200, Vector Laboratories Inc., CA, USA) was added to stain cell nuclei. Samples were viewed with a microscope with Vectashield's coverslips (VECTOR Laboratories, Peterborough, UK), observed under the confocal microscope Leica TCS SP5 (Leica, Wetzlar, Germany), and processed using Leica software, LAS AF (Leica Microsystems GmbH, Wetzlar, Germany).

Statistical analysis
Data for the cell viability assay were analyzed using one-way repeated analysis of variance (ANOVA) with Tukey's post hoc tests to assess pairwise differences. The level of statistical test was performed to assess equality of group variances prior to performing ANOVA. JMP software (SAS Institute, Cary, NC, USA) and Prism 6 (Graph Pad, La Jolla, CA, USA) were used for data analysis. Mean and standard deviation (mean±SD) were reported for summary statistics. difference between Biodentine and all other groups after 7 days. Intragroup comparisons showed that to 7 days (p=0.014). A more detailed presentation differences is presented in Table 1.

Results
DPSCs adhered to the surface of dentin disks ( Figure 2

DISCUSSION
Materials introduced in procedures, such as vital pulp therapies, regenerative endodontic therapies, or perforation repairs, should primarily possess biocompatibility. MTA is commonly used in such procedures, since it is considered highly biocompatible 25 . Biodentine, a new calcium silicatebased material, has demonstrated biocompatibility when tested on various cell lines with better handling properties and a shorter setting time when compared with MTA 2,13,14,17 . Nevertheless, limited evidence is available regarding Biodentine interactions with dental pulp stem cells 21,33 . This study aimed to investigate the biocompatibility of Biodentine in comparison with MTA on DPSCs in a time course of 4 and 7 days as well as the cell adherence to these materials after exposure to an acidic environment.
Among various advantageous properties of Biodentine is the faster setting time compared with MTA 22 . To compensate for the differences in setting time between the two materials and have comparable results in cell viability, setting-time points of 1 hour and 24 hours were applied for MTA. Results from the cytotoxicity assays after 4 days contact with Biodentine as compared with the other groups. Our data are in consensus with Widbiller, et al. 32 (2016) who showed that DPSC viability days. Nevertheless, other studies have compared the cell viability of human pulp fibroblasts, human gingival fibroblasts, or osteoblast-like cells exposed to Biodentine or MTA and observed differences 2,13,14 . Interestingly, one-hour set MTA after 4 days and MTA set for 24 hours demonstrated agree with results from a previous study, which showed that apical papilla stem cells expressed 24-hour set MTA as compared with 1-hour set MTA 26 . One possible explanation is that initial release of calcium-ions as well as the presence of leachable and toxic components from fresh MTA may affect the behavior of the cells. It is reported that freshly mixed calcium-silicate based cements may form continuously calcium-silicate hydrates and precipitate calcium-phosphate and calcium carbonate 7 . Nonetheless, MTA and Biodentine had no differences in cell viability after 7 days.
Cell adherence and viability, when in contact with Biodentine compared with MTA, were further microscopy. It has been shown that contact of dental materials with dentin may alter their properties 9 . Thus, the interaction between cement and surrounding dentin was taken into consideration in this study utilizing dentin disks from anterior bovine teeth, since they could be considered an appropriate substitute for human teeth 3 . Confocal micrographs showed that DPSCs attached on the materials stored in citric acid were more sprindleshaped compared with the materials stored in saline. This fact is indicative of a good cell substrate interaction signifying that both calcium-silicate based materials provide a significantly better substrate for cell adhesion when they set in the presence of citric acid 6,23 . A possible explanation is that the acidic conditions of the citric acid induced the release of Ca-ions, and subsequently the relative concentration of Si increased 30 . Furthermore, the acid-etching effect leads to microstructural changes that could affect the adhesion and proliferation of cells on calcium silicate-based materials 1,10,19,27 . The results of this study agree, despite the differences in methodology, with the results of Kang, et al. 11 (2013), who reported that MTA mixed with citric acid showed favorable biocompatibility. Importantly, our study shows that Biodentine promoted greater cell adherence and viability compared with MTA. Nevertheless, the mechanisms that are responsible for these effects are not completely understood and further research is required to elucidate them. These data may be applied in future studies to modify the surface of the materials to promote better adhesion and sprouting of cells.

CONCLUSION
cell proliferation compared with MTA and control groups. Furthermore, 24-hour set MTA allowed for greater cell viability compared with 1-hour set MTA after 7 days. Both 1-hour and 24-h set MTA were initially more cytotoxic compared with Biodentine, 7-day time point. Exposure of MTA and Biodentine to an acidic environment showed an increase in the number of DPSCs adhered to their surface.

INTRODUCTION
Microbiological sampling and examination of teeth with failed root canal treatments have shown of gram-positive organisms. Enterococcus faecalis (E. faecalis) is considered a predominant organism that is frequently isolated from persistently infected root canal 18 . In vivo model, oral bacteria can penetrate up to 200 mm into dentinal tubules, which may make the bacteria resistant to antimicrobial agents 3 inaccessible areas of the root canal anatomy is the main cause of persistent periapical infections. Because of anatomical complexities that cannot