Combined effects of bone morphogenetic protein-7 and mineral trioxide aggregate on the proliferation, migration, and differentiation of human dental pulp stem cells

Abstract Bioactive molecules present the potential to be used along with biomaterials in vital pulp therapy and regenerative endodontic treatment. Objective The aim of this study was to assess the effects of the combined use of bone morphogenetic protein-7 (BMP-7) and mineral trioxide aggregate (MTA) on the proliferation, migration, and differentiation of human dental pulp stem cells (DPSCs). Methodology For the proliferation analysis, DPSCs were incubated with a growth medium and treated with MTA and/or BMP-7 at different concentrations. For the following analyses, DPSCs were incubated with a differentiation medium and treated with MTA and/or BMP-7. Moreover, there were groups in which DPSCs were incubated with the growth medium (control), the differentiation medium, or DMEM/F12 containing fetal bovine serum, and not treated with MTA or BMP-7. Cell proliferation was analyzed using the WST-1 assay. The odontogenic/osteogenic differentiation was evaluated by immunocytochemistry, alkaline phosphatase (ALP) activity assay, alizarin red staining, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was evaluated using a wound-healing assay. Data were analyzed using analysis of variance and Tukey test (p=0.05). Results The use of BMP-7 with MTA presented no significant effect on cell proliferation in comparison with the treatment with MTA alone (p>0.05), but showed higher ALP activity, increased mineralization, and higher expression of DMP1 and DSPP when compared with other groups (p<0.05). Nestin expression was higher in the control group than in groups treated with MTA and/or BMP-7 (p<0.05). The cell migration rate increased after treatment with MTA when compared with other groups in all periods of time (p<0.05). At 72 hours, the wound area was smaller in groups treated with MTA and/or BMP-7 than in the control group (p<0.05). Conclusion The use of BMP-7 with MTA increased odontogenic/osteogenic differentiation without adversely affecting proliferation and migration of DPSCs. The use of BMP-7 with MTA may improve treatment outcomes by increasing repair and regeneration capacity of DPSCs.


Introduction
The aim of vital pulp therapy (VPT), which includes indirect pulp capping, direct pulp capping, partial pulpotomy, and complete pulpotomy, is to preserve the vitality and health of the pulp tissue that may be affected by trauma, caries, or dental procedures. 1 The success of VPT depends on the properties of the materials used in close proximity to the pulp. 2 In recent years, the use of biomaterials in VPT has gained importance due to their high biocompatibility, bioactivity, and good sealing ability. 3 Mineral trioxide aggregate (MTA), a calcium silicatebased biomaterial, is preferred by dentists to perform VPT due to its good sealing ability, biocompatibility, and bioactivity. 4 MTA is associated with good clinical outcomes. 3 However, the success of MTA varies depending on the pulp diagnosis and type of VPT performed. [5][6][7] According to a previous systematic review, the success rate of direct pulp capping with MTA ranged from 61% to 100%, 5 while the success rate of pulpotomy with MTA ranged from 83% to 100%. [8][9][10] Despite the promising results, there is an ongoing search for materials and applications that achieve the best biological effects to improve the clinical outcomes of VPT.
Bioactive molecules present the potential to be used along with biomaterials in both VPT and regenerative endodontic treatment (RET). 11 RET is generally performed by inducing apical bleeding to form a blood clot in the root canal and placing a biomaterial on the clot. 12 These procedures release bioactive molecules from the dentin extracellular matrix that regulate intercellular signal transduction and play an important role in the repair and regeneration processes of the dentin-pulp complex. 13 Bone morphogenetic protein-7 (BMP-7), also called osteogenic protein-1 (OP-1), is one of these bioactive molecules and can induce dental pulp stem cells (DPSCs) to undergo odontogenic differentiation. 14 MTA can also induce odontogenic differentiation of DPSCs. 15 This effect of MTA is not only related to its alkaline pH and the release of calcium ions, but also to its ability to induce the release of these bioactive molecules from dentin. 16 The use of BMP-7 with MTA could improve the biological response and increase the clinical success of VPT and RET by enhancing the repair and regeneration processes. Therefore, the aim of this study was to assess the effects of the combined use of BMP-7 and MTA on the proliferation, migration, and differentiation of DPSCs. Group MTA+BMP-7 (100 ng/mL) Group BMP-7 (25 ng/mL) Group BMP-7 (50 ng/mL) Group BMP-7 (100 ng/mL) The prepared MTA samples were placed in transwells with 8 µm pore polycarbonate membrane inserts (Corning, Sigma-Aldrich, USA), which were immediately placed in culture plates containing the growth medium and DPSCs. The growth medium and recombinant human BMP-7 were refreshed every two days. After the incubation periods, each insert was removed, and the medium was withdrawn from each well and transferred to 96-well plates. An amount of 10 μL of WST-1 (Roche Applied Science, Penzberg, Germany) was added to each well and incubated for four hours at 37°C with 5% CO 2 . The optical density

RT-qPCR
The total RNA was isolated from cells using the

R : G C C T G T T C C T C T G A G C T A A C T T ) , D S P P ( F : G G G A A T A T T G A G G G C T G G A A , R : T C A T T G T G A C C T G C A T C G C C ) , n e s t i n ( F : T C A A G A T G T C C C T C A G C C T G G A , R :
A A G C T G A G G G A A G T C T T G G A G C ) , a n d G A P D H ( F : C AT C A C C AT C T T C C A G G A G , R : AGGCTGTTGTCATACTTCTC

Statistical analysis
Analyses were performed using a software program (SPSS 22 for Windows, SPSS Inc., Chicago, USA).
Data were presented as mean ± standard deviation.
Two-way analysis of variance (ANOVA) and Tukey test were used to compare the cell proliferation assay data.
Data of the remaining assays were analyzed using one-way ANOVA and Tukey test. The p-value<0.05 was considered statistically significant. Figure Figure 4C shows ALP activity assay results.

ALP activity
Group MTA+BMP-7 presented the highest ALP activity (p<0.05). There was no significant difference regarding ALP activity between groups MTA and BMP-7 (p>0.05), but they showed higher activity than group DM (p<0.05). Groups GM and DMEM/F12 showed the lowest ALP activity, but there was no difference between them (p<0.05).

RT-qPCR
The RT-qPCR analysis showed that the expression of DMP1 and DSPP was higher in differentiated groups when compared with group GM (p<0.05) (Figures 4D and E).  The selection of markers is essential for the assessment of the odontogenic/osteogenic differentiation, and a combination of markers, such as dentin sialoprotein (DSP), dentin phosphoprotein (DPP), DMP1, and nestin, has been recommended for this assessment. 21 Therefore, DSPP, DMP1, and nestin were selected as odontogenic/osteogenic differentiation markers in this study. DMP1 is an acidic phosphoprotein mainly present in dentin, bone, and cementum. 21 DSPP, a phosphorylated non-collagenous protein, which is cleaved into DSP and DPP, is highly expressed in odontoblasts. 21 Nestin is an intermediate filament predominantly expressed in the developing nervous system. 22 It has been reported that DPSCs that highly express nestin present a significant ability to show neurogenic differentiation. 23 On the other hand, according to previous studies, nestin presents a potential role in odontoblast differentiation, as functional odontoblasts also express nestin. 22,24 In this study, although the nestin expression was present, it was lower in groups cultured in the differentiation medium. DMP1 and DSPP expression, in turn, were higher in these groups, showing the On the other hand, the findings of this study regarding the wound healing assay showed that the incubation of cells with BMP-7 did not significantly affect cell migration when compared to the control group at 24 and 48 hours, similar to a previous study. 29 Moreover, the use of BMP-7 with MTA resulted in slower cell migration in comparison with the use of MTA alone.
This finding suggests that BMP-7 may greatly promote Therefore, MTA discs were placed in culture inserts to prevent direct physical interaction between MTA and cells, similar to previous studies. 34,35 The insert system used in this study presented large pores that allowed soluble compounds from MTA to reach DPSCs over time.
The proliferation analysis was performed using WST-1, which is a tetrazolium salt that produces a highly water-soluble formazan by mitochondrial dehydrogenase enzymes. 36 The WST-1 assay is a widely used colorimetric test to assess cellular viability and proliferation. 36 The BMP-7 dose was selected according to the proliferation assay results. In differentiation assays, lower doses of BMP-7 may be insufficient to produce the aimed effects, while higher doses may trigger competing pathways that cause opposing effects on the downstream signaling cascades. 14 As the BMP-7 dose did not significantly affect cell proliferation, 50 ng/mL was chosen as an optimum dose in this study.
Regardless the BMP-7 dose, there was a decrease in cell proliferation at 72 hours of incubation with MTA, similar to previous studies. 37,38 The decrease in cell proliferation may be the release of calcium hydroxide due to the hydration reaction of MTA. 33 Although the release of calcium hydroxide causes an initial chemical irritation, it initiates the hard tissue production process with stem cell recruitment and differentiation. 33 According to a previous study, a single application of recombinant BMP-7 can be insufficient to induce dentin formation. 39 Proteins, including BMP-7, usually present low stability and degrade rapidly. 40