Effects of Sr2+, BO33−, and SiO32− on Differentiation of Human Dental Pulp Stem Cells into Odontoblast-Like Cells

This study aimed to clarify the effects of strontium (Sr2+), borate (BO33−), and silicate (SiO32−) on cell proliferative capacity, the induction of differentiation into odontoblast-like cells (OLCs), and substrate formation of human dental pulp stem cells (hDPSCs). Sr2+, BO33−, and SiO32− solutions were added to the hDPSC culture medium at three different concentrations, totaling nine experimental groups. The effects of these ions on hDPSC proliferation, calcification, and collagen formation after 14, 21, and 28 days of culture were evaluated using a cell proliferation assay, a quantitative alkaline phosphatase (ALP) activity assay, and Alizarin Red S and Sirius Red staining, respectively. Furthermore, the effects of these ions on hDPSC differentiation into OLCs were assessed via quantitative polymerase chain reaction and immunocytochemistry. Sr2+ and SiO32− increased the expression of odontoblast markers; i.e., nestin, dentin matrix protein-1, dentin sialophosphoprotein, and ALP genes, compared with the control group. BO33− increased the ALP gene expression and activity. The results of this study suggested that Sr2+, BO33−, and SiO32− may induce hDPSC differentiation into OLCs.


Introduction
If reversible pulpitis with deep caries is diagnosed via a thorough examination, vital pulp therapy (VPT) could be an appropriate treatment based on minimal intervention dentistry (MID) [1].The aim of VPT would be achieved by promoting pulp vitality using direct pulp capping, pulp amputation, or indirect pulp capping and protecting the pulp tissue from harmful external stimuli using a long-term secure seal [2,3].The pulp's healing process after applying VPT depends on the odontogenic differentiation ability of human dental pulp stem cells (hDPSCs) in the pulp tissue [4].In other words, pulp irritations caused by bacteria in caries, deep cavity preparation, and pulp capping agents may induce the proliferation of undifferentiated pulp tissue cells and their differentiation into odontoblast-like cells (OLCs) [5].
OLCs form reparative dentin by synthesizing, secreting, and calcifying organic substrates, such as collagenous and noncollagenous proteins (NCPs) [6].Calcium hydroxide preparations and mineral trioxide aggregate (MTA) are commonly used as pulp capping agents [7].Although the chemical stimulation of these pulp capping agents promotes the differentiation of hDPSCs into OLCs and the formation of restorative dentin, their failure to adhere to the dental structure and restorations and low physical strength may increase the risk of dissolution and microleakage of restorations after a long-term prognosis [8].In contrast, it has been reported that adhesive composite resin showed dentin bridge formation when used for direct pulp capping; however, that speed was slower when compared to the cement used for direct pulp capping [9].Conversely, resinous direct pulp capping materials possess advantages such as easy handling for application, control for time setting, and high physical properties.
Currently, composite resins containing surface prereacted glass-ionomer (S-PRG) fillers, showing bioactive effects due to the sustainable release of multi-ions (Na + , Sr 2+ , Al 3+ , F − , SiO 3 2− , and BO 3 3− ), have been used in clinical practice [10].Restorations prepared using composite resins containing S-PRG fillers show lower plaque build-up and bacterial adhesion on the surfaces of restorations compared with those prepared using conventional composite resins [11], and remineralization and antidemineralization are recognized around the restoration prepared using composite resins containing S-PRG fillers [12,13].Furthermore, applying an S-PRG filler-containing pulp capping cement to exposed rat molars has been reported to promote repaired dentin formation [13,14].
Previous studies suggested that Sr 2+ , BO 3 3− , and SiO 3 2− , released from S-PRG fillers, might promote calcification and induce the differentiation of hDPSCs into OLCs [13].Sr 2+ was identified in the dental pulps after applying direct pulp capping to the exposed pulps of the dog teeth using strontium hydroxide [15].Another study reported that the abilities of hDPSCs in cell proliferation, odontogenic differentiation, and mineralization were enhanced by culturing them in a strontium chloride solution.For BO 3  3− and SiO 3 2− , the abilities of hDPSCs in cell proliferation and odontogenic differentiation were enhanced by culturing them in a medium supplemented with sodium pentaborate solution [16] and calcium silicate [17], respectively.
These results indicate the biocompatibility of Sr 2+ , BO 3 3− , and SiO 3 2− with the dental pulp.As mentioned above, the long-term stable biocompatibility of the dental pulp capping agent is necessary for achieving a successful VPT [18].Composite resins containing S-PRG fillers may be utilized as a new direct pulp capping material in the clinic because of their biocompatibility, dentine adhesion, operability, and mechanical strength; however, details of the biological function from various ions produced by S-PRG fillers have not been clarified.We focused on Sr 2+ , BO 3 3− , and SiO 3 2− among various ions slowly released from S-PRG fillers because they indicate dental pulp biocompatibility.No previous studies have compared the effects of these three ions on hDPSCs.Hence, this study aimed to determine how Sr 2+ , BO 3 3− , and SiO 3 2− affect cell proliferative capacity, the induction of differentiation into OLCs, and substrate formation by hDPSCs.The null hypothesis was that Sr 2+ , BO 3 3− , and SiO 3 2− would not affect cell proliferative capacity, the induction of differentiation into OLCs, and substrate formation by hDPSCs.

Cell Culture
The hDPSCs used in this study were obtained commercially (Lonza Group, Basel, Switzerland) and cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St Louis, MO, USA) by adding 10% fetal bovine serum (Cytiva, MA, USA), 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B at 37 °C in an atmosphere humidified with 5% CO 2 .Three to five passages were used for all experiments.For every assay, hDPSCs were seeded at 1.0 × 10 4 cells/mL.After 24 h, the medium was replaced to ion-containing medium and changed every 3 days.

Cell Proliferation Assay
Cell proliferation was assessed using alamarBlue® Cell Viability Reagent (Invitrogen, Carlsbad, CA, USA).The hDP-SCs (1 × 10 3 cells/100 µL) were seeded in wells of a 96-well plate and incubated for 3, 7, 10, 14, 21, and 28 days at 37 °C.Then, 10 µL of the alamarBlue® reagent was added in each well and incubated at 37 °C for 3 h (n = 6).Fluorescence radiated from vital cells was measured using a fluorescence plate reader (POWERSCAN MX, DS Pharma Biomedical, Osaka, Japan) at an excitation wavelength of 540 nm and a fluorescence wavelength of 610 nm.Calibration curves were used to convert fluorescence to cell counts.

Alkaline Phosphatase (ALP) Activity Assay
The hDPSCs were incubated for 14, 21, and 28 days and seeded in a 24-well plate at 5 × 10 3 cells/well.The cells were dissolved using 0.05% Triton® X-100 (Wako, Osaka, Japan) and centrifuged for 1 min at 1200 rpm.The lysates were treated with p-nitrophenyl phosphate in a carbonate buffer solution (pH 9.8), and 0.2 mol/L NaOH was used to stop the reaction (n = 6).Absorbance at a wavelength of 405 nm was measured using a microplate reader (Viento®808, DS Pharma Biomedical, Osaka, Japan).The absorbance was defined as ALP activity.

Alizarin Red S Staining
Alizarin Red S staining confirmed calcium deposition.The hDPSCs were seeded in a 24-well plate at 5 × 10 3 cells/well and cultured for 14, 21, and 28 days.After washing the cells with cacodylic acid buffer solution, they were fixed in 100% methanol for 20 min at 4 °C.The fixed cells were stained with Alizarin Red S using a calcification staining kit (AK21, Cosmo Bio, Tokyo, Japan) according to the manufacturer's protocol (n = 6).To quantify the relative amount of calcium, the dye was extracted by incubating deionized water with 10% cetylpyridinium chloride at room temperature for 1 h, and absorbance of the extracted dye in the supernatant was measured at a wavelength of 560 nm.

Sirius Red Staining
Sirius Red staining was performed to substantiate collagen formation.After culturing hDPSCs for 14, 21, and 28 days, the cultured cells were fixed in 4% paraformaldehyde and stained with Sirius Red using a staining kit (Picrosirius Red Stain Kit, Polysciences, Warrington, PA, USA) according to the manufacturer's protocol (n = 6).To quantify the relative mass of collagen formed, the dye was extracted by incubating in 0.1 mol/L NaOH solution for 1 h, and the absorbance of the extracted dye in the supernatant was measured at a wavelength of 560 nm.

Statistical Analysis
According to the dispersibility of data, the Kruskal-Wallis test with Scheffe's post hoc test or the one-way analysis of variance with Tukey's post hoc test was used for data analysis.

Cell Proliferation
The cell proliferation assay findings are shown in Fig. 1.
After incubating for 3 days, the number of cells on SrH was significantly higher than that in SrL, SrM, BL, Si group, and control (p < 0.036), and the number of cells on SrM were significantly lower than that on SiL, SiM, BM, and BH (p < 0.040).The number of cells on SiH significantly increased compared to the number on SiL after 10 days of incubation (p = 0.046).The number of cells on SrL significantly increased, after 21 days of incubation, compared to that on SiM (p = 0.018).The experimental groups did not significantly differ after 7, 10, 14, and 28 days of incubation (p > 0.05).The proliferation of hDPSCs gradually increased during the first 14 days of incubation; however, their increase slowed down after 21 days.The peak number of cells on SiH was identified after 10 days, and that on SiL and BH was identified after 14 days of incubation.

ALP Activity
The results of the ALP activity test are shown in Fig. 2. All experimental groups showed that ALP activity was significantly higher after 21 days of incubation than after 14 days of incubation (p < 0.001).For the SrL, SrM, BL, BM, SiL, and control, the ALP activity significantly decreased after 28 days of incubation compared to after 21 days (p < 0.020).No significant differences were observed among the experimental groups after 14 days of incubation (p > 0.05).After 21 days of incubation, ALP activity on SrL significantly increased compared to other experimental groups (p < 0.020).After 28 days of incubation, ALP activity was significantly higher in BL than that in other experimental groups, except for BM and BH (p < 0.020).

Alizarin Red S staining
The results of staining with Alizarin Red S and representative photographs after staining are shown in Figs. 3 and 4, respectively.The absorbance of Alizarin Red S-stained cells increased over time in almost all experimental groups.The experimental groups did not show any significant differences throughout the incubation period (p > 0.05).SrL, BL, BM, BH, SiL, SiH, and control showed that the absorbance of cells stained with Alizarin Red S was significantly higher after 28 days than after 14 days of incubation (p < 0.047).There were no significant differences in the absorbance of cells stained with Alizarin Red S among all experimental groups between 21 and 28 days of incubation (p > 0.05).The degree of staining with Alizarin Red S in all experimental groups was almost identical.Calcification deposits were not clearly observed.

Sirius Red Staining
The results of staining with Sirius Red and representative photographs after staining are shown in Figs. 5 and 6, respectively.In all groups, the absorbance of cells stained with Sirius Red increased over time.After 14 days of incubation, the absorbance of cells stained with Sirius Red on BM and BH was significantly higher than that of cells stained with Sirius Red on control (p < 0.005).After 21 days of incubation, the absorbance of cells stained with Sirius Red on SrH was higher than that of cells stained with Sirius Red on SiL and SiM (p < 0.029).The absorbance of cells stained with Sirius Red showed significant increases between 14 and 21 days of incubation on SrM, SrH, SiH, and BH (p < 0.034) and between 21 and 28 days of incubation on BH and SiH (p < 0.001).All experimental groups showed significant differences in the absorbance of cells stained with Sirius Red between 14 and 28 days of incubation (p < 0.005).The degree of staining with Sirius Red in all experimental groups increased over time. of incubation, were significantly higher than in the control (p < 0.039).After 28 days of incubation, the nestin expression ratio in the SrH showed significant increases when compared to the SiL and control (p < 0.038).Throughout the incubation period, the SrH showed the highest nestin expression ratio, and the value was four times higher than that of the control.The SrH also showed a significantly higher nestin expression ratio compared with the BL after 14 days, the B and Si groups after 21 days, and the SiL after 28 days of incubation (p < 0.004).Almost all experimental groups, including the control, showed peak expression ratios of nestin after 21 days of incubation.The nestin expression ratios in the SrH, BH, and SiM tended to increase over time, whereas those in the SrM and SiL tended to decrease over time.DMP-1 expression ratios are shown in Fig. 8.After 14 days of incubation, the DMP-1 expression ratio was significantly higher in the SiL than in the BL and BH (p < 0.038).After 21 days of incubation, DMP-1 expression ratios were significantly higher in the Sr, SiM, and SiH than in the control (p < 0.049).Furthermore, the SrM showed the highest DMP-1 expression ratios with significant differences in the SiL and B group (p < 0.002).Significant differences were also noted between SiH and B group (p < 0.026).After 28 days of incubation, the DMP-1 expression ratio was significantly higher in the SrL than in the SiH and control (p < 0.001) and in the BL than in the control (p = 0.049).The peak expression in almost all experimental groups was observed after 21 days of incubation; however, that in the SiL and control was observed after 14 days of incubation.Throughout the incubation period, the DMP-1 expression in the B group and control tended to be lower than that in the Sr and Si groups.
DSPP expression ratios are shown in Fig. 9.After 14 days of incubation, the DSPP expression ratio in the SiL and SiH showed significant increases compared to that in the control (p < 0.049), and that in the SiL significantly increased compared to that in the BH (p = 0.021).After 21 days of incubation, the DSPP expression ratio was significantly higher in the SrL, SrH, SiM, and SiH than in the control (p < 0.042) and in the SrL than in the B group (p < 0.044).After 28 days of incubation, no significant differences were observed between all experimental groups (p > 0.05).The DSPP expression ratio in the Si and SrM decreased over time, whereas that in the BM increased over time, and the peak DSPP expression ratio in the SrL, SrH, BH, and control was observed after 21 days of incubation.Throughout the incubation period, the DSPP expression was lower in the B group and control than in the Sr and Si groups, similar to the DMP-1 expression.ALP expression ratios are shown in Fig. 10.The ALP expression ratio was significantly higher in the SiL than in the BM and control after 14 days of incubation (p < 0.005).The ALP expression ratio was significantly higher in the BL and SrL than in the SiL and control after 21 days of incubation (p < 0.035).After 28 days of incubation, the ALP expression ratio was significantly higher in the SrH than in the BM, SiM, and SiH, and in the BH than in the SiH (p = 0.038).The peak ALP expression ratio in the Si and Sr groups was observed after 14 days of incubation, whereas that in the BL and BM was observed after 21 days of incubation.The ALP expression ratio in the SiM, SiH, SrL, and SrM decreased over time.
All experimental groups showed increasing expression ratios of genes associated with differentiation into OLCs compared with the control.The kinds and concentrations of ions added into the culture medium influenced the amount and time of gene expression.

Immunocytochemistry
Table 4 shows the experimental groups that expressed positive reactions for DMP-1 and DSPP after immunocytochemical staining; representative images are shown in Fig. 11.The control showed no obvious expression for DMP-1 and DSPP throughout the incubation period.A positive reaction for DMP-1 was observed in the SrL and SrH after 14 days, the SrM and SrH after 21 days, and the BL, BM, Sr, and Si groups after 28 days of incubation.A positive reaction for DSPP was observed in the SiH, BM, and Sr group after 14 days; the BL, BH, and Sr and Si groups after 21 days; and the BL, BH, and Sr and Si groups after 28 days of incubation.DMP-1 tended to be expressed slowly at the cell nucleus and around it in some groups.On the other hand, the positive reaction for DSPP was observed in the entire cells in most cells.In the Sr group, the positive reaction for DSPP was detected around the cell nucleus after 14 days of incubation and throughout the cell after 21 days of incubation.The positive reaction for DMP-1 and DSPP in SiL after 28 days of incubation was observed in cells focally.Cells in the Sr group reacted positively to immunocytochemical staining after 14 days whereas those in the Si group reacted positively to immunocytochemical staining after 21 days of incubation.In the experimental group, only SrH showed a positive reaction for DMP-1 and DSPP in all incubation periods.BH showed a positive reaction only for DSPP after 21 days of incubation.

Proliferation Assays
A previous study demonstrated that hDPSCs could differentiate into five different cell types, neurogenic, osteogenic/ odontogenic, adipogenic, myogenic, and chondrogenic, under stimulation with appropriate induction media [19].Another study clarified that OLCs, one of the final differentiated forms, showed a longer doubling time compared to hDPSCs, which are capable of self-proliferation and multilineage differentiation as a stem cell function [20].Therefore, conditions for inducing differentiation may not always correspond to those for inducing cell proliferation.In the present study, hDPSC generally reached confluence during incubation between 14 and 21 days, and then their proliferation ability tended to decline.In general, cell proliferation slows down as the cell population becomes confluent.Several studies have shown that the nodules formed via the aggregation of multilayered hDPSCs are a necessary condition for differentiation into OLCs and the generation of a mineralized matrix [21][22][23][24][25].In particular, cell-to-cell interactions could be important for differentiating hDPSCs into OLCs.The interaction between cell proliferation and differentiation has not been elucidated; however, cell-to-cell contact likely inhibits cell proliferation but promotes differentiation.Hence, all sequence assays in this study were performed after 14 days of incubation when cells might reach confluence.

Collagen and Calcification Formation
Dentin collagen represents the most abundant protein in the dentin matrix and is fundamental in biomineralization [26].Therefore, assessments of collagen formation and calcification are important for elucidating the functional and morphological differentiation of hDPSCs into OLCs.Results of the Sirius Red staining test showed that the collagen formation ability was significantly higher in the SrM, SrH, BM, and BH than in other experimental groups.Hence, it is speculated that Sr 2+ and BO 3 3− may promote hDPSCs' differentiation into OLCs.Conversely, the amount of collagen formation increased over time in all groups, including the control.hDPSCs may possess collagen formation abilities because they are adherent cells that require an extracellular matrix (ECM) rich in collagen to adhere to the surrounding substrate.The Alizarin Red S staining test showed that the calcification level in all experimental groups cannot be determined qualitatively; however, quantitative data of absorbance were increased  throughout the experimental period; this suggested that almost all experimental groups tended to calcify.We suppose that the process of calcification occurs after differentiation into OLCs.Therefore, Alizarin Red S staining did not show clear deposition.It is clinically important to investigate the different effects of ions on calcification and to assess the functionality of differentiated OLCs.However, as the observation of organization is limited by 28 days cell culture, this should be confirmed by more cell culture period.We will investigate the effect of each ion on the differentiation of hDPSCs into OLCs in the next research project.
During the calcification process, amorphous calcium phosphate (ACP) is deposited on collagen fibers, followed by its conversion to octacalcium phosphate, and, finally, hydroxyapatite formation [27].In vivo experiments have shown that osteoblasts delivered the amorphous progenitor cell phase to collagen fibers at the early stages of bone growth [28].This report posed the question of how ACP penetrates collagen and emerges as an important factor affecting the calcification speed.Another in vitro study showed that the smaller the size of ACP aggregates, the faster their penetration into collagen [29].It is speculated that the penetration rate of Sr 2+ into collagen may be slow due to its large molecular weight compared to Ca 2+ .

ALP Activity
ALP activity increases when cells differentiate into calcification cells [30].The ALP on the cell surface is important in cell differentiation and calcification as it regulates the phosphate concentration around the cells [31].In this study, ALP activities in the Sr and B groups were significantly higher than those in other groups.These ALP activities increased in the first 21 days of incubation and decreased thereafter.A previous study evaluating hDPSC differentiation reported that ALP activity increased as the pulp cells differentiated [32].The study in which confluent hDPSCs were induced for differentiation showed that the activity of hDPSCs decreased after 28 days of culture [33].These findings are consistent with those of the present study and suggest that the progression of hDPSC differentiation into OLCs may correspond to ALP activity changes.In the present study, the ALP activity of the control and experimental groups increased over time.A previous study also showed that extending the culture period and increasing the cell density increased the ALP activity in undifferentiated hDPSCs [34].Therefore, culturing in vitro may cause hDPSCs to differentiate into calcification-forming cells on the prescribed pathway.
High ALP activity is also a common feature of stem cells [35].Various isotypes of ALP exist, and ALP expressed on stem cells possesses a similar molecular-biological target to that expressed on osteocytes [36].Hence, whether the change in hDPSC behavior causes a shift to a differentiation phase or is a feature of stem cells based on ALP activity alone cannot be determined.Multiple perspectives are necessary to clarify the differentiation of hDPSCs into OLCs.Therefore, we investigated how the genes that participate in the differentiation of hDPSCs into OLCs are expressed using qPCR to capture hDPSC changes in more detail.

qPCR
ALP is an important marker at the early stages of odontoblast differentiation [37].This study found that the ALP gene emerged after 14 or 21 days of incubation in most groups and then tended to decline rapidly.Previous studies that attempted to induce the differentiation of hDPSCs reported ALP gene expression increased at the early stages of differentiation, followed by decreased expression at the start of the calcification process [32,[38][39][40], consistent with the results of the present study.Hence, it is speculated that ALP gene expression may peak at the early stages of differentiation into OLCs and that a subsequent decline in gene expression may be caused by accelerated differentiation.
Nestin, a component of the intermediate filament protein forming the cytoskeleton, increasingly emerges in neural stem cells [41].It is also a well-known marker gene for odontoblasts.Previous studies have shown that the upregulation stage of nestin expression in dental pulp cells was limited during the tooth's growth, and nestin disappeared when the tooth's growth was completed.Furthermore, nestin has been reported to re-emerge during the formation of repair dentin and disappeared upon the completion of the repair dentin formation [42].These findings indicate that nestin is a gene associated with odontoblast differentiation and dentin-forming capacity, regardless of the prevailing normal or pathological state.Therefore, hDPSCs differentiating into OLCs appear to express nestin continuously, until the actual function is initiated.In this study, significantly higher nestin expression was observed in the Sr and Si groups, suggesting that the addition of Sr 2+ and SiO 3 2− to the medium might induce the differentiation of hDPSCs into OLCs.
In the present study, the Sr and Si groups showed increased DMP-1 and DSPP expression, indicating that Sr and Si ions may be pertinent to the differentiation of hDPSCs to OLCs and substrate formation.Odontoblasts form dentin by secreting collagenous and NCPs involved in regulating crystal nucleation and mineral-phase growth [43].DMP-1 and DSPP in the NCPs are important markers because, during tooth development and dentin formation, they are highly expressed in the odontoblasts [44].Recent studies revealed that DMP-1 and DSPP are factors contributing to substrate calcification and the induction of hDPSC differentiation [45].DMP-1 is an acid phosphoprotein and is highly expressed in cells with calcifying functions, such as osteoblasts, odontoblasts, cementoblasts, ameloblasts, osteocytes, and chondrocytes [46].The expression of DMP-1 precedes that of DSPP in hDPSCs [47].Ling et al. reported that DMP-1 directly or indirectly regulates DSPP expression [48].DMP-1 is important in odontoblast differentiation and ECM calcification [46,49].
DSPP expression is linked to cell polarization, ECM deposition, and the initiation of ECM calcification [50].Heterogeneous mutations in the human DSPP gene cause dentinogenesis imperfecta (DGI) types II and III and dentin dysplasia type II [51][52][53][54].Furthermore, DSPP-null mice exhibit tooth defects similar to DGI type II in humans, such as enlarged pulp cavities, increased predentin width, hypocalcification, and pulp exposure [55].These reports indicate that DSPP is important for differentiation into OLC and dentin maturation.

Immunocytochemistry
The immunochemical staining for cells showed that DMP-1 tended to be expressed slowly at the cell nucleus and around it in some groups, whereas DSPP tended to be expressed uniformly in most cells.These tendencies observed in the present study were similar to those reported by Baldion et al., who observed the differentiation of hDPSCs into OLCs [20].Hao et al. reported that DMP-1 expression was restricted to the odontoblasts actively secreting substrate and reduced to basal levels in mature odontoblasts [56].When cells differentiated from hDPSCs are considered a community, only a few of them express DMP-1; however, the signal may cause the surrounding cells to express DSPP.The characteristic localization of DMP-1 expression observed in the present study has not been reported; however, it could be related to cell polarization, which is important for the morphological assessment of odontoblast differentiation [57].The protein expression results in the present study were similar to those in previous reports [48,56] and may reflect the differentiation and developmental stages of OLCs induced by the respective ions used in the present study.

Summary
A diagram of the differential gene and protein expression during the maturation process of hDPSCs to OLCs is shown in Fig. 12.In the present study, the ALP activity did not coincide with gene expression and increased during gene expression.This may cause delayed protein synthesis for gene expression.Generally, there is a time lag between gene expression and protein synthesis due to posttranslational modifications [58].Kang et al. reported that ALP gene expression in osteoblasts was remarkably elevated without a concurrent increase in DMP-1 and DSPP expression [59].
DMP-1, DSPP, and ALP gene expression changes in the present study distinguished the differentiation of hDPSCs into osteoblasts.Then, the Sr and Si groups showed a significant upregulation of the ALP gene and odontoblast marker expression compared to the control, which suggested the differentiation of hDPSCs into OLCs.The B group showed a relatively low expression of odontoblast markers and increased ALP gene expression and activity, indicating that BO 3 3− may be involved in inducing calcification and maintaining the differentiation and self-renewal of hDPSCs.
The concentration differences in the same ion caused changes in gene expression levels and timing.However, the types of genes induced by each ion were generally consistent.ALP, nestin, DMP-1, and DSPP expressions were enhanced in the Sr and Si groups, and those of nestin and ALP were enhanced in the B group.Therefore, when provided in nontoxic concentrations, these ions may induce the differentiation of hDPSCs into OLCs.They may also alter the speed of dentin formation by odontoblasts or OLCs.There is considerable heterogeneity in the genotype and phenotype of hDPSC subpopulations in vivo [60].Thus, a single cell population may be formed by a mixture of high-proliferative and low-proliferative cells, pluripotent cells, and unipotent cells, which may imply therapeutic potential.
Elucidating the ionic action for cell differentiation will contribute to the further development of bioactive materials in the future.Other pulp capping materials with ionic effects on pulp cells include MTA cement, calcium hydroxide preparations, and resin materials such as The-raCal [61,62].The common property of these materials is the calcium ions that exhibit bioactive effects.However, no calcium ion is released from the S-PRG filler.The clinical application of the S-PRG filler to the resinous direct pulp capping material would be expected to provide high cavity sealing, excellent physical properties, and easy handling.Additionally, our basic data showing the differentiation-inducing effects of Sr 2+ , SiO 3 2− , and BO 3 3− released from S-PRG fillers offer new prospects for their application as a pulp capping agent.Therefore, the current study's findings could be highly useful for developing bioactive materials for dental pulp in the future.However, synergistic effect of the ions should be proved.The previous data showed that experimental selfadhesive resin including S-PRG filler of 9.1 wt% released Sr 2+ of 1.32 ppm, BO 3 3− of 48.76 ppm, and SiO 3 2− of 31.09ppm [63].We would like to make a complex solution of Sr 2+ , BO 3 3− , and SiO 3 2− and investigate the synergistic effects of these ions in the future.

Conclusion
In the in vitro cell culture model, hDPSCs stimulated by Sr 2+ , BO 3 3− , or SiO 3 2− exhibited OLC characteristics in morphological, functional, and genetic evaluations, which implied that these ions possess the action of inducing the differentiation of hDPSCs into OLCs.
Nestin F:CAG GGG CAG ACA TCA TTG GT 77 R:CAC TCC CCC ATT CAC ATG CT DMP-1 F:CAA GAC AGA GAG CTA TGA ACA CGA TAT 115 R:TGC AAC CTT CCA ACT CCA ATG DSPP F:GGG CAA AGG CAA TGT CAA GA 160 R:TCC TTG CAT GGA CTT ATC ATCAA ALP F:CCG TCT GTG ACC CAT CTC ATG 110 R:AGG GCA GCC TCT GTC ATC TC GAPDH F:GAC AGT CAG CCG CAT CTT CT 104 R:GCG CCC AAT ACG ACC AAA TC Immunocytochemistry After 14, 21, and 28 days of culture, hDPSCs were fixed for 10 min in 4% paraformaldehyde, washed twice in phosphate-buffered saline (PBS) for 10 min, and permeabilized for 10 min in 0.1% Triton®X-100.After washing the cells in PBS twice for 5 min, the cells were treated with 3% bovine serum albumin for 30 min to block nonspecific binding to antibodies.Anti-human DSPP mouse monoclonal antibody (LFMb-21, Santa Cruz Biotechnology, Dallas, TE, USA) (1:50) and anti-human DMP-1 mouse monoclonal antibody (LMFb-31, Santa Cruz Biotechnology) (1:100) were used as primary antibodies.The cells were treated with each antibody at 4 °C overnight and then washed twice with PBS at room temperature.The cells were incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG (Thermo Fisher Scientific) (1:1000) for 1 h at room temperature in the dark.After washing the cells with PBS twice, the nuclei were stained with DAPI-containing encapsulant (Thermo Fisher Scientific) (n = 2).A confocal laser microscope (LSM710, Carl Zeiss Jena GmbH, Jena, Germany) was used to observe immunofluorescence staining for DSPP and DMP-1.To assess the existence of protein expression, image analysis of the immunofluorescence staining was performed using ImageJ (https:// imagej.nih.gov/ ij/) or Zeiss microscopy ZEN (Carl Zeiss).

Fig. 1
Fig. 1 Results of the cell proliferation assay.The same lowercase letters indicate no significant difference (p > 0.05) between the experimental groups during each incubation period.The same uppercase

qPCRFig. 2
Fig. 2 Results of the ALP activity test.The same lowercase letters indicate no significant difference (p > 0.05) between the experimental groups during each incubation period.The same uppercase let-

Fig. 3 Fig. 4
Fig. 3 Results of Alizarin Red S staining.The same lowercase letters indicate no significant difference (p > 0.05) between the experimental groups during each incubation period.The same uppercase let-

Fig. 5 Fig. 6 Fig. 7 Fig. 8
Fig. 5 Results of Sirius Red staining.The same lowercase letters indicate no significant difference (p > 0.05) between the experimental groups during each incubation period.The same uppercase let-

Fig. 9
Fig. 9 Results of DSPP expression ratios The same lowercase letters indicate no significant difference (p > 0.05) between the experimental groups during each incubation period.The same uppercase letters

Fig. 10
Fig. 10 Results of ALP expression ratios The same lowercase letters indicate no significant difference (p > 0.05) between the experimental groups during each incubation period.The same uppercase letters

Fig. 11
Fig. 11 Representative images of immunocytochemical staining.The positive reaction for DMP-1 tended to be partially found in cells, whereas that for DSPP tended to be entirely shown in cells.In the Sr group, a positive reaction for DSPP was detected around the nucleus of a cell after 14 days of incubation but was detected in the entire

Table 1
Formulation table and pH of the standard ionic solution(50 mM)

Table 2
Concentration of each ion added

Table 3
Nucleotide sequence and amplicon size for each gene