Strontium ranelate promotes odonto-/osteogenic differentiation/mineralization of dental papillae cells in vitro and mineralized tissue formation of the dental pulp in vivo.

This study examined the effects and mechanisms of strontium ranelate (SrRn)-a drug used to treat osteoporosis-on the proliferation and differentiation/mineralization of cloned dental pulp-like cells (mouse dental papillae cells; MDPs). It also determined whether topical application of SrRn to exposed dental pulp tissue promotes the formation of mineralized tissue in vivo. The MDPs were cultured with or without SrRn, and cell proliferation, odonto-/osteoblastic gene expression, mineralized nodule formation, and Akt phosphorylation were evaluated. The formation of mineralized tissue in SrRn-treated pulp tissue in rat upper first molars was evaluated histologically. The SrRn up-regulated cell proliferation and expression of Alp (alkaline phosphatase), Bsp (bone sialoprotein), Dmp (dentin matrix acidic phosphoprotein)-1, Dspp (dentin sialophosphoprotein), and Oc (osteocalcin) in a dose-dependent manner. Mineralized nodule formation was also enhanced by SrRn. NPS-2143, a calcium-sensing receptor (CaSR) antagonist, and siRNA against the CaSR gene blocked SrRn-induced proliferation, odonto-/osteoblastic gene expression, and mineralized nodule formation. SrRn induced Akt phosphorylation, and this was blocked by NPS-2143. Topical application of SrRn to exposed rat molar pulps induced the formation of osteodentin-like mineralized tissue. Our study revealed for the first time that SrRn promotes proliferation and odonto-/osteogenic differentiation/mineralization of MDPs via PI3K/Akt signaling activated by CaSR in vitro; mineralized tissue forms from the dental pulp in vivo.

hard tissue-inducing mechanisms are similar to those of CH 17 . Currently, CH, MTA, and their derivatives are generally used for vital pulp therapy, but more effective pulp capping agents/materials with active dentin-like tissue-inducing capacity might increase the success rate of vital pulp therapy.
Strontium ranelate (SrRn) contains two strontium atoms coupled by ranelic acid. It is used to treat osteoporosis 18 . Previous clinical trials have shown that SrRn significantly improves bone mass/quality and increases bone strength via changes in the bone matrix properties and bone mineral density in osteoporotic patients 18 . SrRn is promising in the treatment of symptomatic osteoarthritis 19 . SrRn has gained attention in osteoporosis therapy because it offers two mechanisms of action, i.e., induction of osteoblastogenesis and suppression of osteoclastogenesis 20,21 . Furthermore, local application of SrRn with a collagen sponge to artificial bone defects formed in rat calvaria promotes the regeneration of bone tissue 22 . These findings indicate the potent mineralized tissue-forming capacity of SrRn and suggest that SrRn can be used on dentin (indirect pulp capping) and dental pulp tissue (direct pulp capping) as a pulp-capping agent with a potent capacity to form mineralized tissue. Thus, the aims of this study were (1) to examine the effects and mechanisms of SrRn on the proliferation and differentiation/mineralization of cloned dental pulp-like cells (mouse dental papilla cells; MDPs); and (2) to determine whether topical application of SrRn to exposed dental pulp tissue promotes mineralized tissue formation in vivo.

Materials and Methods
Cell culture and chemicals. The MDPs were derived from the incisor apical buds of ICR mice and were immortalized by transfection of human papillomavirus type 16 E6 gene missing the PDZ domain binding motif 23,24 . They were cultured in alpha-modified minimum essential medium (α-MEM, Wako Pure Chemical Industries, Osaka, Japan) with 10% fetal bovine serum (FBS, HyClone/GE Healthcare, UT, USA) and an antibiotic and anti-fungal solution (penicillin-streptomycin-amphotericin B suspension; Wako Pure Chemical Industries) at 37 °C/5% CO 2 /100% humidity. SrRn (LKT Laboratories, St Paul, MN, USA) and calcium chloride (CaCl 2 ; Wako Pure Chemical Industries) were dissolved in α-MEM directly and sterilized with a syringe filter (Millex-HA filter, Merck Millipore, Darmstadt, Germany). NPS-2143 hydrochloride (dissolved in dimethylsulfoxide, 1.0 µM; Cayman Chemicals, Ann Arbor, MI, USA) and LY294002 (dissolved in dimethylsulfoxide, 1.0 µM; Cayman Chemicals) were used as selective and potent calcium-sensing receptor (CaSR) antagonists 25 along with a potent pan-PI3K/Akt inhibitor 26  Odonto-/osteoblastic gene expression. MDPs (5 × 10 4 cells/well) were seeded in 12-well plates. After 24 h of culture, the media was changed to include the test agents, and cells were cultured for 72 h. The total RNA was extracted by QuickGene RNA cultured cell kit S (Wako Pure Chemical Industries). The cDNA was converted from extracted RNA using Primescript (Takara, Shiga, Japan). The qPCR assays were performed with specific primers (Table 1) and GoTaq qPCR Master Mix (Promega, Madison, WI, USA) using a CFX96 Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA). Gyceraldehyde-3-phosphate dehydrogenase (Gapdh) was the internal control.
Mineralized nodule formation. MDPs (1 × 10 4 cells/well) were seeded in 48-well plates and cultured in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS. After 24 h of culturing, the media was changed to an osteogenic medium containing L-ascorbic acid (0.2 mM; Wako Pure Chemical Industries) and β-glycerophosphate (5.0 mM; Sigma Aldrich, St. Louis, MO, USA) with or without test agents. Mineralized nodules were stained with alizarin red S (Wako Pure Chemical Industries) after 7 d of culture. The density of the mineralized nodules was measured by ImageJ software v2 (https://imagej.net/ImageJ2).
Western blotting. MDPs (2 × 10 5 cells/well) were seeded in 24-well plates. Test agents were added to the media after 24 h of culture. Cells were lysed with a RIPA buffer (20 mM Tris/HCl pH 7.4, 150 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1% NP-40, 5% glycerol) containing cOmplete Ultra (Merck Millipore) and PhosSTOP EASY (Merck Millipore). Cell lysates were applied to SDS-PAGE (10%), and the gel was transferred to a PVDF membrane (Immobilon-P, Merck Millipore) using a semi-dry transfer system (0.15 mA, 1 h; WSE-4040, ATTO, Tokyo, Japan). Transferred membranes were incubated with anti-Akt (1:1000; polyclonal, GTX121936, GeneTex, Los Angeles, CA, USA) and anti-p-Akt (1:500; polyclonal, GTX121936, GeneTex) antibodies overnight at 4 °C followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1000; Jackson Immuno Research Labs, In vivo study. All animal experiments were approved by the Animal Care and Use Committee of TMDU, all surgical methods were performed in accordance with relevant ethical guidelines and regulations (#A2017-155A). Wistar rats (n = 12, male, 5-wk-old; Clea Japan, Tokyo, Japan) were given ad libitum access to food and water prior to the experiment. The rats were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). The cavity preparation and pulp exposure were performed in the upper first molars of both sides with #1/2 round bars using a dental handpiece motor under a stereoscopic microscope (Dental Microscope Z;  Mani, Tochigi, Japan). Bleeding in the cavities following the pulp exposure was removed with sterile cotton pellets. The SrRn (mixed with sterile water at 2 mg/µl), mineral trioxide aggregate (ProRoot MTA, Dentsply Sirona, Ballaigues, Switzerland; mixed according to the manufacturer's instructions), or CaCl 2 (mixed with sterile water at 2 mg/µl) was dressed over the exposed pulp (n = 4 in each group). No application of SrRn, MTA, and CaCl 2 was used as a control. The samples applied to each cavity were randomly chosen from SrRn, MTA, CaCl 2 , and no application; there was no rat in which the same samples were applied contra-laterally. The cavities were sealed with glass ionomer cement (Ionosit-Baseliner, DMG, Hamburg, Germany). The rats were sacrificed by CO 2 euthanasia after 3 weeks. The upper jaws were dissected from the maxilla and fixed with 4% paraformaldehyde/ PBS for 24 hours at 4 °C. Samples were then demineralized using 17% EDTA (Dojindo Molecular Technologies) for 3 weeks. After demineralization, the glass ionomer cement was removed, and the samples were embedded in paraffin. Hematoxylin and eosin staining was performed on 5 µm-thick sections, and the stained sections were observed under a light microscope (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany).
Statistical Analysis. All in vitro experiments were carried out in triplicates. The data were submitted to one-way ANOVA followed by Tukey's test. The level of significance was established at * P < 0.05 or ** p < 0.001 using Prism software v7 (GraphPad, San Diego, CA, USA).

CaSR is involved in the up-regulation of cell proliferation and differentiation/mineralization of MDPs induced by SrRn.
Next, we investigated the possibility that CaSR acts as a targets of SrRn in MDPs, because Sr 2+ is known to activate CaSR 27,28 , which is involved in the control of many important cellular functions such as proliferation and differentiation 29 . The promoted cell proliferation and expression of Alp, Bsp, Dmp-1, Dspp, and Oc induced by SrRn on MDPs were disrupted by NPS-2143-a selective and potent CaSR antagonist ( Fig. 2A,B). The CaSR siRNA down-regulated the mRNA expression of CaSR in MDPs and also suppressed the expression of Alp, Bsp, Dmp-1, Dspp, and Oc induced by SrRn in MDPs (Fig. 2C). Mineralized nodule formation promoted by SrRn in MDPs was blocked by NPS-2143 (Fig. 2D).
SrRn promoted mineralization of the exposed rat molar pulp in vivo. Finally, the impact of topical application of SrRn on exposed dental pulp tissue including the formation of mineralized tissue was examined in vivo. The application of SrRn to the exposed pulp tissue of rat upper first molars induced mineralized tissue formation in the pulp tissue of all specimens (Fig. 5A,B). The newly formed mineralized tissue exhibited an atubular, osteodentin-like structure with sparse or no cellular inclusions in the inner (pulp side) portion. This new tissue completely separated the pulp tissue from the pulp chamber exposed to the oral cavity. The pulp tissue appeared mostly normal with a few inflammatory cells. MTA also induced mineralized tissue formation in the exposed pulp tissue in all specimens; the mineralized tissue had irregularly aligned tubules and fewer cellular inclusions compared to the SrRn-applied specimens. The underlying pulp tissue appeared essentially normal (Fig. 5C,D). In contrast, application of CaCl 2 failed to induce continuous mineralized tissue formation; some islets of mineralized tissues were formed in the dental pulp tissue. Abscess formation was detected in the exposed area of the pulp tissue (Fig. 5E,F). Some of the CaCl 2 -applied samples showed deposition of mineralized tissues on the surface of the root canal wall dentin. Controls without treatment (Fig. 5G,H) failed to form mineralized tissue in the exposed pulp tissue. Four specimens were examined, and the results were reproducible.

Discussion
In this study, SrRn up-regulated the proliferation of MDPs (Fig. 1A), which possess dental pulp cell properties 23,24 . SrRn also increased the production of human dental pulp cells (see Supplemental Fig. 2). This is consistent with prior findings showing that SrRn up-regulates the proliferation of MC3T3-E1 cells (a mouse calvaria derived osteoblastic cell line 28,31 ) and a primary osteoblast cell line 32 . However, the opposite results have also been reported for MC3T3-E1 cells 33 . The reason(s) for such discrepancy remains unclear, but there is a large variation among the sub-clones of MC3T3-E1 34 , and differences in the properties of different MC3T3-E1 sub-clones may result in different results during cell proliferation. The SrRn also promoted the odonto-/osteoblastic gene expression in a dose-dependent manner (Fig. 1B) and induced mineralization in MDPs (Fig. 1C). Osteoblastic differentiation by SrRn is reported in primary murine bone cells 21 , murine marrow stromal cells 35 , and MC3T3-E1 cells 36 . SrRn also induces commitment of osteoblastic differentiation from mesenchymal stem cells 37 .
One of the targets of Sr 2+ is CaSR-a Class C G-protein coupled receptor 29 expressed on various cells including osteoblasts and their precursors 38 . Osteoblasts from the conditional knockouts of CaSR exhibit delayed differentiation, reduced mineralization capacity, and altered expression of regulators of mineralization 39 . This suggests that CaSR signaling in osteoblasts is essential for their differentiation and mineralization. The enhanced cell proliferation, odonto-/osteogenic differentiation and mineralization induced by SrRn in MDPs were blocked by NPS-2143 (a potent CaSR antagonist) and CaSR siRNA ( Fig. 2A-D). The NPS-2143 also inhibited the SrRn-induced proliferation of human dental pulp cells (see Supplemental Fig. 2). Furthermore, SrRn promoted CaSR expression in MDPs (see Supplemental Fig. 3). These results indicate that SrRn may promote the proliferation and differentiation/mineralization of MDPs via CaSR signaling.
Involvement of the PI3K/Akt pathway is also reported in SrRn-induced osteogenic differentiation of human osteoblasts 30 . The PI3K/Akt signaling stimulates canonical Wnt signaling 37 , and this may be involved in promoted proliferation, differentiation, and mineralization of dental pulp cells due to SrRn. We previously reported that Wnt signaling upregulates odontoblast marker expression in MDPs 45 , and Wnt signaling activated by SrRn via CaSR/PI3K/Akt signaling may induce odonto-/osteoblastic gene expressions of MDPs. The PI3K/Akt signaling activates the production of BMP2 46 , which may also be involved in the differentiation and mineralization of MDPs. Ca 2+ is an agonist of the CaSR with a higher affinity than Sr 2+41 ; CaCl 2 (2 mM) promotes the proliferation of MC3T3-E1 cells 28 . However, CaCl 2 (1 mM) did not enhance the proliferation and differentiation/mineralization of MDPs in our experiment (see Supplemental Fig. 1) suggesting that 1 mM of Ca 2+ concentration may be too low to induce proliferation and differentiation/mineralization of MDPs.
In vivo application of SrRn to rat upper first molars induced tissue mineralization along the exposed pulp tissue (Fig. 5A,B). Mineralized tissues formed in CH-treated pulp tissue have been reported to be irregular with tubular openings or canalicular lumina containing vessels and cells 11 . The mineralized tissue induced by SrRn in this study had an atubular, osteodentin-like structure with sparse or no cellular inclusions in its mineralized structure indicating that it might have sufficient capacity to protect the underlying pulp tissue from the oral environment. The MTA induced dentin-like mineralized tissue (Fig. 5C,D) as previously reported 14,15 , and it offers slow-release of Ca 2+47 . In contrast, CaCl 2 failed to induce homogeneous mineralized tissue did form islets of mineralized bodies in the exposed pulp tissue (Fig. 5E,F). Ca 2+ is reported to have mineralized tissue formation capacity 28,47 , which agrees with these findings. However, CaCl 2 did not induce a continuous mineralized barrier to separate the intact pulp tissue from the oral cavity suggesting that an application method resulting in continuous and slow release of Ca 2+ might be essential to the formation of a mineralized dentin-like barrier.
Systemic side effects should always be considered in developing new treatment agents. Lithium chloride (LiCl 2 )-a GSK3b inhibitor and potent activator of Wnt canonical signaling-has recently been reported to induce mineralized tissue formation following direct application to the rat pulp tissue 48 ; however, a high intake of LiCl 2 can cause a risk of confusion and speech impairment including a risk of death at 20 mg/L of LiCl 2 49 .
The systemic side effects of SrRn are also of note and include cerebrovascular disease, peripheral vascular disease, and prior myocardial infarction. These were reported in patients given a 2,000 mg daily systemic dose treatment of SrRn for a year 50 . However, it is reasonable to suppose that topical application during vital pulp therapy may pose a much lower risk of side effects compared to systemic application 32,37,51,52 ; in vivo studies using rat models reported that systemic use of SrRn (625 g/kg) is well tolerated and safe with no adverse side effects 53,54 . However, it is necessary to evaluate the systemic side effects induced by the local application of SrRn before its clinical application.
In conclusion, we revealed for the first time that SrRn increases the proliferation, odonto-/osteoblastic gene expression, and mineralized nodule formation of dental pulp-like cells. This may be partially mediated by CaSR/ PI3K/Akt signaling (Fig. 6). Our in vivo study revealed that topical application of SrRn induced a continuous barrier of osteodentin-like mineralized tissue on the rat exposed pulp tissue highlighting the potential utility of SrRn as a new pulp-capping material.