Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth

Abstract Objective The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA), calcium hydroxide (CH) and BiodentineTM (BD) on stem cells from human exfoliated deciduous teeth (SHED) in vitro. Material and Methods SHED were cultured for 1 – 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL), and tested for viability (MTT assay) and proliferation (SRB assay). Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 μm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1) was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning) and culture medium supplemented with 20% FBS were used as controls. Results MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA) (p<0.05). In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH) (p<0.05). A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Conclusion Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.


Introduction
Vital pulp therapy based on the use of stem cell has promising research and therapeutic applications in dentistry. Stem cells in the dental pulp respond to tooth injuries by migrating, proliferating and differentiating into odontoblasts, which conduct the synthesis and secretion of tertiary dentin 6,9,17,32 . In particular, deciduous teeth contain a population of postnatal "stem cells from human exfoliated deciduous teeth" (SHED), which are capable of extensive proliferation and multipotential differentiation in vitro 8,17,19 . In dental tissues, SHED constitute an autogenous source of cells because of their potential for pulp regeneration in vivo, which could be applied in vital pulp therapy 7,12,25 .
Following the diagnosis of deep carious/traumatic pulp injuries of primary and permanent teeth, vital pulp therapy may be clinically performed by applying a capping material directly onto the pulp tissue to allow pulp/dentine regeneration 2,12 . During conservative pulp treatments such as pulpotomy and direct pulp capping, interactions between pulp cells and the capping material affect stem cell proliferation 6,29 . While the exact nature of interactions between capping materials and the injured pulp tissue (during wound healing and regeneration) remains unclear, in vitro studies in two or three-dimensional culture systems as well as in vivo studies on capping materials have aided in material selection, which is the key to ensure a good treatment outcome 2,6,8,15,26,29 .
An ideal capping material should be highly biocompatible, prevent bacterial microleakage and promote the formation of mineralized tissue 16,18,24 .
Different materials are used in the endodontic treatment of primary and permanent teeth, including calcium hydroxide (CH), mineral trioxide aggregate (MTA), and the recently launched Biodentine TM (BD) 16

Material and methods
Cell culture SHED, isolated and characterized as previously described 19 , were maintained in alpha-MEM medium supplemented with 10% fetal bovine serum (FBS, Certified, Heat-inactivated) and 1% penicillin and streptomycin solution (culture medium components were from Gibco, Invitrogen, Grand Island, NY, USA).
Cells were maintained at 37°C and 5% CO 2 and split at a ratio of 1:3 when they reached 80% confluence.

Results
To estimate the effect of different agents on cell viability, SHED were stimulated with CH (CH group), MTA (MTA group) or BD (BD group) conditioned medium for seven days, and subjected to the MTT assay. The viabilities of SHED treated with either of the three conditioned media were similar after 1, 3 and 5 days of treatment (p>0.05; Figure 1), and lower than that of the positive control after 3, 5 and 7 days (p<0.05). After 7 days of treatment, the viability of cells in the MTA group was similar to that in CH (p>0.05; Figure 1), but higher than that in the negative control and the BD group (p<0.05; Figure 1).
To examine the effect of conditioned media on SHED proliferation, SRB assay was used (Figure 2).  In addition, relatively up-regulated DMP-1 expression when SHED were cultured with MTA was seen on days 7, 14 and 21.

Discussion
In this study, we examined the effect of the capping materials CH, MTA and BD on SHED, by incubating these cells with medium that had been exposed to each of these capping agents ("conditioned medium"). We preferred to use conditioned medium rather than direct incubation with capping agents to simulate a clinical situation in which the dental pulp stem cells residing The bioactivity of proliferation, migration and differentiation of dental pulp stem cells is regulated by networks of signaling molecules, including growth factors and their, receptors and transcriptional control systems 13 . It is likely that differences in SHED responses to the various capping materials are due to differential modulation of growth factor release, signaling pathway activation and transcriptional regulation. While the evaluation of these factors is beyond the scope of this work, these are important aspects for future studies on the response of SHED to capping agents.

Conclusions
The three capping materials -Biodentine TM , MTA and CH -maintained viability and stimulated proliferation, migration and odontogenic-like phenotype differentiation in SHED, which supports the notion that these materials are adequate for pulpotomy treatment of primary teeth, where SHED are autogenous sources for vital pulp therapy.