Hydraulic calcium silicate-based root canal sealers mitigate proinflammatory cytokine synthesis and promote osteogenesis in vitro

Background/purpose The mineralized tissue-inductive ability and anti-inflammatory properties of hydraulic calcium silicate-based (HCSB) sealers have not been fully elucidated. This study aimed to evaluate the effects of the HCSB sealers Bio-C sealer (BioC), Well-Root ST (WST), and EndoSequence BC sealer (BC), on osteoblastic differentiation/mineralization and proinflammatory cytokine synthesis by macrophages. Materials and methods Diluted extracts of set sealers or calcium chloride solutions of approximately equivalent Ca2+ concentrations were applied to a mouse osteoblastic cell line (Kusa-A1 cells) and lipopolysaccharide-stimulated mouse macrophage cell line (RAW264.7 cells). Expressions of osteoblastic markers in Kusa-A1 cells and proinflammatory cytokines in RAW264.7 cells were evaluated by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Mineralized nodules were detected by Alizarin red S staining. Cell proliferation was assessed by WST-8 assay and cell attachment on set sealers was examined by scanning electron microscopy. Results The three sealer extracts significantly upregulated osteocalcin and osteopontin mRNA, and promoted significant mineralized nodule formation in Kusa-A1 cells. The three sealer extracts significantly downregulated the mRNA expressions of interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor (TNF)-α and protein levels of IL-6 and TNF-α in RAW264.7 cells. Calcium chloride solutions induced osteoblastic differentiation/mineralization. AH Plus Jet (a control sealer) extract did not. The three HCSB sealers did not interfere with the growth and attachment of Kusa-A1 cells. Conclusion BioC, WST, and BC were biocompatible, upregulated osteoblastic differentiation/mineralization, and downregulated proinflammatory cytokine expression. Ca2+ released from HCSB sealers might be involved, at least in part, in the induction of osteoblastic differentiation/mineralization.


Introduction
The primary objective of endodontic treatment is to eliminate intracanal bacteria mechanically and chemically, which is followed by complete obturation of the root canal space with root canal filling materials. 1,2The essential biological property of root canal filling materials is biocompatibility, and the use of these materials with bioactivity is expected to promote optimal periapical healing. 3,4However, most traditional root canal filling materials were developed with a focus on their physical properties and they rarely possess bioactive properties. 2,5ydraulic calcium silicate-based (HCSB) materials were introduced as endodontic materials with high bioactivity. 6,7he most commonly used HCSB material is mineral trioxide aggregate (MTA), which is formulated from Portland cement combined with bismuth oxide powder for radiopacity. 8,9MTA has shown favorable clinical outcomes for direct pulp capping, root-end filling, and iatrogenic root perforation repair, 8 which may be attributed to its good biocompatibility, 10 mineralized tissue-inductivity 10 and anti-inflammatory properties. 11These biological properties of MTA are related to its continuous release of Ca 2þ . 12CSB materials have been advocated for use as root canal sealers because of their good biocompatibility and bioactivity, and several premixed HCSB sealers formulated with calcium silicates as a major component have been developed.13 EndoSequence BC sealer (BC; Brasseler USA, Savannah, GA, USA) is the most studied premixed HCSB sealer and its composition includes calcium silicates, calcium phosphate monobasic, calcium hydroxide, filler, and thickening agents.BC promotes significant osteoblastic differentiation, 14e16 and exhibits anti-inflammatory effects.17 Bio-C Sealer (BioC; Angelus, Londrina, PR, Brazil), a recently-developed HCSB sealer, contains tricalcium silicate, dicalcium silicate, and tricalcium aluminate as its major ingredients and has mineralization-inducing potential in human periodontal ligament stem cells (hPDLSCs).18 Well-Root ST (WST; Vericom, Gangwon-Do, Korea), another recently-developed HCSB sealer, contains calcium aluminosilicate compound as its major component and is reported to promote alkaline phosphatase activity, osteoblastic marker gene expression, and mineralized nodule formation in hPDLSCs.19 However, the mineralized tissue-inductive ability and anti-inflammatory properties of these recently-developed HCSB sealers have not been fully elucidated.Thus, this study aimed to compare the effects of three HCSB sealers, Bio-C sealer (BioC), Well-Root ST (WST), and EndoSequence BC sealer (BC), as well as AH Plus Jet (AHP; an epoxy resinbased sealer; Dentsply Sirona, York, PA, USA) as a control sealer, on osteoblastic differentiation/mineralization and proinflammatory mediator synthesis in macrophages.

Preparation of sealer extracts
BioC, WST, BC, and AHP (Table 1) were mixed following the manufacturer's instructions, used to fill polypropylene discs (3 mm height Â 7.5 mm diameter), and incubated at 37 C with 100% humidity for 2 days to achieve the set conditions.AHP, an epoxy resin-based sealer, was used as a control sealer against the three HCSB sealers in this study because AHP contains no typical bioactive components such as calcium silicates and calcium aluminosilicate.The set sealers were immersed in 3 mL of distilled water (DW) under shaking at room temperature for 24 h, and the sealer extracts were filtered (0.45-mm pore size, Sartorius, Go ¨ttingen, Germany) and mixed with a-MEM or D-MEM at a 1:4 ratio.Calcium chloride (CaCl 2 ) was dissolved in DW at concentrations of 0, 5, 10, and 20 mM, and then mixed with a-MEM at a 1:4 ratio.DW was used as a negative control.

Mineralized nodule formation
Kusa-A1 cells (2 Â 10 4 cells/well) were seeded in 48-well plates and cultured in a-MEM containing 10% FBS for 24 h.Then, the medium was replaced with a mineralization medium containing b-glycerophosphate (5 mM; Merck, Rahway, NJ, USA), L-ascorbic acid (0.2 mM; FUJIFILM Wako Pure Chemical), and dexamethasone (1 nM; FUJIFILM Wako Pure Chemical), with or without sealer extracts or CaCl 2 .Mineralized nodules were stained with Alizarin red S (FUJIFILM Wako Pure Chemical) on days 3 and 6.For quantification, images were taken and the number of pixels in a region of interest was quantified using ImageJ software (ver.1.53; National Institutes of Health, Bethesda, MD, USA).

Reverse transcription-quantitative polymerase chain reaction
Kusa-A1 cells (5 Â 10 4 cells/well) were seeded in 24-well plates and cultured without sealer extracts for 24 h followed by the application of sealer extracts or CaCl 2 for 24 h.RAW264.7 cells (1 Â 10 5 cells/well) were seeded in 12-well plates and cultured in D-MEM containing 10% FBS for 24 h followed by serum starvation for 24 h.Then, RAW264.7 cells were stimulated with lipopolysaccharide (LPS, 100 ng/mL, Escherichia coli O111: B4, Merck) with or without sealer extracts for 3 h.Total RNA was extracted with a QuickGene-RNA extraction kit (FUJIFILM Wako Pure Chemical) and converted into cDNA with a PrimeScript cDNA synthesis kit (Takara Bio, Kusatsu, Japan).Quantitative polymerase chain reaction (qPCR) was performed with GoTaq qPCR MasterMix (Promega, Madison, WI, USA) and specific primers (Table 2) using the CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA).Relative normalized expression of the target genes was calculated following the DDCt method against the housekeeping gene, b-actin.

Scanning electron microscopy
Mixed sealers used to fill in polypropylene discs (2 mm height Â 9 mm diameter) were allowed to set for 2 days followed by rinsing with DW for 24 h, sterilization with 70% ethanol for 15 min, and washing with phosphate-buffered saline for 5 min.Then, samples were placed in a 24-well plate  with 2 mL a-MEM for 24 h, and Kusa-A1 cells (2.5 Â 10 4 cells/ well) were seeded on the samples and cultured for 72 h.Following fixation in 2.5% glutaraldehyde (FUJIFILM Wako Pure Chemical), the samples were dehydrated, dried in a critical-point dryer (HCP 2, Hitachi, Tokyo, Japan), sputtercoated with platinum using an ion-coater (E102, Hitachi), and scanned with an electron microscope (JSM-7900, JEOL, Japan) at an accelerating voltage of 15 kV.

Ca 2D concentration measurement
Ca 2þ concentration in the sealer extracts was measured by inductively coupled plasma atomic emission spectrometry (IC-7000 ver.2; Shimadzu, Kyoto, Japan).

Statistical analysis
GraphPad Prism (ver.9;GraphPad, La Jolla, CA, USA) was used for statistical analyses.Following the verification of data normality and variance homogeneity using the Shapiro-Wilk test and Levene's test, respectively, data were analyzed using one-way analysis of variance followed by the Tukey-Kramer test.A P-value less than 0.05 was considered statistically significant.

Effects of sealer extracts on osteogenic differentiation and mineralization in Kusa-A1 cells
We first evaluated the mRNA expression of osteopontin (Opn) and osteocalcin (Oc), which are typical osteoblastic markers, 23,24 especially Oc is reported to be involved in mineralization. 24mRNA expression of Opn was significantly upregulated by BioC and BC in Kusa-A1 cells compared with the negative control (DW) and AHP (P < 0.001, Fig. 1a), and the mRNA expression of Oc was significantly upregulated by WST in Kusa-A1 cells compared with the negative control (P < 0.01, Fig. 1a).AHP failed to promote the mRNA expressions of Opn and Oc (Fig. 1a).Clear mineralized nodules were detected in Kusa-A1 cells approximately 6 days after culture in mineralization medium (Fig. 1b).The application of BioC, WST, and BC promoted significant Alizarin red S staining at 3 and 6 days compared with the negative control (DW) and AHP (P < 0.001, Fig. 1b).

Effects of sealer extracts on Kusa-A1 cell proliferation
The proliferation of Kusa-A1 cells was increased significantly by BioC and BC at 48 and 72 h (P < 0.05 and 0.001, respectively; Fig. 2a) and by WST at 72 h (P < 0.01, Fig. 2) compared with the negative control (DW) and AHP.BioC and BC promoted significant cell proliferation compared with WST at 72 h (P < 0.01, Fig. 2a).

Attachment of Kusa-A1 cells to set sealers
Kusa-A1 cells cultured on set BioC, WST, and BC were wellspread and attached to the sealers by numerous filopodia or pseudopodia.However, well-attached cells were rarely observed on AHP at 72 h of cell culture (Fig. 2b).

Effects of Ca 2D on osteogenic differentiation/ mineralization and proliferation of Kusa-A1 cells
The mean amount of Ca 2þ released from BioC, WST, and BC was 917, 293, and 990 mg/L, respectively (Table 3).The mRNA expression of Opn in Kusa-A1 cells was increased significantly by 10 and 20 mM CaCl 2 compared with the negative control (DW) (P < 0.05, Fig. 3a).The mRNA expression of Oc was not affected by CaCl 2 (Fig. 3a).Mineralized nodule formation was enhanced in Kusa-A1 cells by CaCl 2 at 3 days (Fig. 3b), and the intensity of Alizarin red S staining related to CaCl 2 application was significant at 3 and 6 days compared with the negative control (P < 0.001, Fig. 3b).
Ten and 20 mM CaCl 2 significantly upregulated the proliferation of Kusa-A1 cells compared with the negative control at 72 h (P < 0.05 and 0.01, respectively; Fig. 3c).

Discussion
The current study revealed that all the tested HCSB materials but not AHP promoted osteoblastic proliferation and differentiation/mineralization in Kusa-A1 cells.The three HCSB materials released relatively high amounts of Ca 2þ while AHP rarely released it (Table 3), indicating that Ca 2þ is responsible for these properties.This was supported by the finding that 10 and 20 mM CaCl 2 promoted the proliferation and differentiation/mineralization of Kusa-A1 cells (Fig. 3).The Ca 2þ concentrations of 4-times diluted 10 and 20 mM CaCl 2 were almost equivalent to 100 and 200 mg/L, respectively, which are approximate to those in the 4-times diluted extracts of BioC, WST, or BC (229, 73, and 248 mg/L, respectively).Ca 2þ at 2e4 mM (80e160 mg/L) is suitable for the proliferation and survival of osteoblasts, and 6e8 mM (240e320 mg/L) Ca 2þ favors osteoblast differentiation and matrix mineralization. 25Because a-MEM contains 72 mg/L Ca 2þ , final Ca 2þ concentrations in the culture medium containing 4-times diluted BioC, WST, or BC extracts approximated 301, 145, and 320 mg/L, respectively.Therefore, promotion of the proliferation and differentiation/mineralization of Kusa-A1 cells induced by the HCSB sealer extracts may be associated with Ca 2þ release from the HCSB sealers, although factors other than Ca 2þ might also be involved.BioC and BC upregulated the mRNA expression of Opn (Fig. 1a), a typical osteoblastic marker, 23 which was also induced by CaCl 2 (10 and 20 mM, Fig. 3a), suggesting that Ca 2þ released from BioC and BC are involved in the upregulation of Opn mRNA expression.In contrast, the mRNA expression of Oc, an osteoblastic marker closely related to mineralization, 24 was promoted by WST (Fig. 1a) but not by CaCl 2 (Fig. 3a).Thus, the upregulation of Oc mRNA may involve factor(s) other than Ca 2þ released from WST.The three HCSB sealers may release different concentrations of aluminum and/or silicon, which were reported to be inhibitory 26 and stimulatory, 27 respectively, to osteoblastic differentiation.Further analysis is required to determine whether and how aluminum and silicon are involved in the mineralized tissueinductive ability of these materials.This study demonstrated that the marked downregulation of proinflammatory cytokine synthesis was induced by the application of all three HCSB sealer extracts to LPS-stimulated RAW264.7 cells (Fig. 4).IL-1a, IL-1b, and TNF-a are essential factors for osteoclastogenesis and pathogenesis in periapical lesions. 28TNF-a regulates receptor activator NF-kB ligand expression, which is an essential signaling factor that induces osteoclast formation 29 via IL-1 that directly stimulates the differentiation of osteoclast precursors. 30IL-6 has pathological effects on chronic inflammation. 31Therefore, the downregulation of these proinflammatory mediators might contribute to the healing of periapical lesions accompanied by bone regeneration.The three HCSB sealers released high concentrations of Ca 2þ (Table 3), which may contribute to their antiinflammatory effects.On the contrary, AHP, which rarely released Ca 2þ (Table 3), failed to induce the antiinflammatory effects in LPS-stimulated RAW264.7 cells (Fig. 4).Ca 2þ released from MTA was reported to induce Ca 2þ influx via the calcium-sensing receptor (CaSR), and activate calcineurin/nuclear factor of activated T-cells (NFAT) signaling in RAW267.4cells. 11CaSR/NFAT signaling then induces early growth response 2, which inhibits the mRNA expressions of IL-1a and IL-6, and promotes the expression of IL-10, a typical anti-inflammatory cytokine, 32 and suppressor of cytokine signal 3, a major regulator of inflammation. 33In addition, Ca 2þ released from MTA stimulates CaSR/NFAT signaling, which further stimulates the osteogenic differentiation of human dental pulp cells. 34It is tempting to speculate that the activation of CaSR/NFAT signaling is also involved in the osteoblastic differentiation/ mineralization of Kusa-A1 cells induced by the three HCSB sealers.
Good biocompatibility is an essential property for endodontic sealers; however, a direct comparison of biocompatibility between BioC, WST, and BC has not been reported.This study demonstrated that the three sealers did not inhibit cell growth and that Kusa-A1 cells attached well to set BioC, WST, and BC by numerous filopodia/ pseudopodia (Fig. 2b).35e38,40,41 BioC has lower toxicity than AHP in hPDLSCs 18 and exhibits similar toxicity to that of BC in a lethality assay involving brine shrimp, Artemia salina. 35urthermore, subcutaneously implanted BioC induces lower infiltration of inflammatory cells and IL-6 synthesis compared to AHP. 36 WST has been reported to be more biocompatible and less cytotoxic than AHP in human dental pulp stem cells, 37 human tooth germ stem cells, 37 hPDLSCs, 37,38 and MC3T3-E1, a typical osteoblastic cell line. 39,40BC also shows good biological properties related to hPDLSCs 16,41 and a murine osteoblast precursor cell line, IDG-SW3 compared with AHP. 15 Here, we showed that BioC, WST, and BC were equally biocompatible and less toxic compared with AHP.
In conclusion, the three HCSB sealers, BioC, WST, and BC exhibited high biocompatibility with Kusa-A1 cells and promoted their osteoblastic differentiation/mineralization probably by the release of Ca 2þ .Moreover, BioC, WST, and BC reduced the synthesis of proinflammatory cytokines IL-1a, IL-1b, IL-6, and TNF-a from LPS-stimulated RAW264.7 cells.These properties are considered beneficial for the healing of periapical lesions accompanied by new alveolar bone formation and apical closure with newlyformed mineralized tissue(s).Our data provide basic evidence supporting the notion that these HCSB sealers possess suitable biological properties for use as root canal filling materials.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1
Fig. 1 Effect of BioC, WST, BC, and AHP on osteogenic gene expression (a) and mineralized nodule formation (b) in Kusa-A1 cells.(a) mRNA expression of osteopontin (Opn) was significantly upregulated by BioC and BC, and mRNA expression of osteocalcin (Oc) was significantly upregulated by WST in Kusa-A1 cells.AHP had no effect on the mRNA expressions of Opn and Oc.Data are shown as the mean and SD (n Z 3).(b) Mineralized nodule formation of Kusa-A1 cells was significantly increased by BioC, WST, and BC.AHP had no effect on mineralized nodule formation.Data are shown as a ratio relative to the negative control (distilled water) at day 3 (mean and SD, n Z 5).AHP: AH Plus Jet, BC: EndoSequence BC sealer, BioC: Bio-C sealer, CT: negative control, WST: Well-Root ST. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2 (
Fig. 2 (a) Proliferation of Kusa-A1 cells was significantly increased by BioC and BC at 48 and 72 h.WST significantly upregulated the proliferation of Kusa-A1 cells at 72 h.AHP had no effect on the proliferation of Kusa-A1 cells.Data are shown as the mean and SD (n Z 4).(b) Well-attached Kusa-A1 cells were observed on set BioC, WST, and BC, but attached cells were rarely observed on AHP.Representative images from three separate experiments are shown.AHP: AH Plus Jet, BC: EndoSequence BC sealer, BioC: Bio-C sealer, CT: negative control (distilled water), WST: Well-Root ST. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3
Fig. 3 Effects of Ca 2þ on osteogenic gene expression (a), mineralized nodule formation (b), and proliferation (c) of Kusa-A1 cells.(a) mRNA expression of Opn was significantly upregulated by 10 and 20 mM CaCl 2 .Data are shown as the mean and SD (n Z 3).(b) Mineralized nodule formation of Kusa-A1 cells was significantly increased by 5, 10, and 20 mM CaCl 2 .Data are shown as a ratio relative to the negative control (distilled water) at day 3 (mean and SD, n Z 4).(c) Proliferation of Kusa-A1 cells was significantly increased by 10 and 20 mM CaCl 2 at 72 h.Data are shown as the mean and SD (n Z 4).CT: negative control.*P < 0.05, **P < 0.01, ***P < 0.001.

Table 1
Compositions of sealers.

Table 3
Ca 2þ concentration in the sealer extracts.
Data are shown as the mean AE SD (n Z 3).AHP: AH Plus Jet, BC: EndoSequence BC sealer, BioC: Bio-C sealer, WST: Well-Root ST.