- Email: [email protected]
Bioaggregate Inhibits Osteoclast Differentiation, Fusion, and Bone Resorption In Vitro
Basic Research—Biology
Bioaggregate Inhibits Osteoclast Differentiation, Fusion, and Bone Resorption In Vitro Jun Tian, MS,* Wenting Qi, MS,* Yuanhe Zhang, MS,† Michael Glogauer, PhD,‡ Yongqiang Wang, PhD,‡ Zhihui Lai, MS,† and Hongwei Jiang, PhD* Abstract Introduction: Several bioactive bioceramics with osteoconductive effects can inhibit osteoclast formation and bone resorption in vitro, but the exact underlying mechanism has not been fully elucidated. Bioaggregate (BA), a novel calcium silicate nanoparticulate bioceramic, significantly induces bone and periodontal regeneration. In this study, we aimed to explore the effect of BA extracts on osteoclastogenesis and bone resorption. Methods: The RAW264.7 cells were treated with soluble receptor activator of nuclear factor kappa B ligand to osteoclastogenesis. BA extracts were used to investigate the effect of BA on osteoclast differentiation, fusion, and bone resorption. Furthermore, the ions in BA extracts were quantitatively analyzed. Finally, the key molecules in the receptor activator of nuclear factor kappa B ligand–RANK signaling pathway, including receptor activator of nuclear factor kB (RANK), tumor necrosis factor receptor associated factor 6 (TRAF6), nuclear factor-kappa B (NF-kB), and nuclear factor of activated T cells c1 (NFATc1), were explored. Results: BA suppressed osteoclastogenesis and bone resorption in vitro. BA releases Si ions and a small amount of Sr ions and provides alkalinity. Treatment with BA extracts decreased the migration ability and fusion of RAW264.7 cells. We also observed that BA causes a significantly decreased expression of RANK, TRAF6, NF-kB, and NFATc1. Conclusions: Our study provides further insight into the mechanism by which calcium silicate–based bioceramics inhibit osteoclastogenesis and bone resorption and also suggests that BA is a useful material for several clinical situations because it both stimulates osteoblast differentiation and inhibits osteoclast formation. (J Endod 2015;-:1–7)
Key Words Bioaggregate, bone resorption, calcium silicate bioceramic, osteoclastogenesis, osteoclasts, RAW264.7
M
any adult skeletal diseases are caused by excessive osteoclast (OC) activity and bone resorption including periapical periodontitis–, rheumatoid arthritis–, or periodontal disease–associated bone inflammation, osteoporosis, and Paget disease (1, 2). Thus, OC-targeted therapies should be a first-line treatment for excessive bone loss (3). OCs are specialized cells derived from hematopoietic stem cells through the monocyte/macrophage lineage. The formation of mature resorbing OCs comprises multiple steps including the differentiation of OC precursors into mononuclear preosteoclasts (pOCs), the fusion of mononuclear pOCs to form multinucleated OCs, and the activation or maturation of OCs capable of bone resorption (3). The differentiation and activation of OCs are dependent on the receptor activator of nuclear factor kappa B ligand (RANKL), which is produced by osteoblast cells and activated T cells (2). The activation of RANK, a RANKL receptor that is expressed in OC precursors, by RANKL leads, via the initiation of the TRAF6-NF-kB-NFATc1 signaling pathways, to the expression of OC-specific genes during differentiation and the activation of resorption by mature OCs (3–5). Bioaggregate (BA) (Innovative Bioceramix, Vancouver, BC, Canada) is a recently developed bioceramic with a nanocomposition that is mainly composed of calcium silicates, calcium phosphate, amorphous silicon oxide, and tantalum oxide (6). As an innovative bioceramic, BA is a potential alternative to the well-studied mineral trioxide aggregate (MTA, another bioceramic primarily consisting of calcium silicates) that has been used globally in clinical procedures such as root end filling, repair of root perforations, capping, and pulpotomy (7, 8). Compared with MTA, BA is claimed to be aluminum free and contains a significant amount of tantalum oxide as a radiopacifier instead of bismuth oxide, which may lead to tooth discoloration (9). BA and its dissolution products exhibit excellent biocompatibility (7, 10–12) and osteoconductive effects (10). Several silica-based materials, such as bioactive glass 45S5 and MTA, which are biocompatible and bioactive with osteoconductive effects, inhibit OC formation and bone resorption in vitro (8, 13, 14). In addition, BA shows a suppressive effect on OC differentiation and inflammatory bone resorption in vivo (15). However, the mechanisms of the inhibitory effect of BA on osteoclastogenesis (OCG) and bone resorption have not been well explored. The ionic dissolution products from inorganic materials likely play a critical role in the biological behavior of these materials (16). Therefore, in the present study, BA extracts were used to systematically investigate the effect of BA on the differentiation of OC precursors, the fusion of mononuclear pOCs, and bone resorption. Furthermore, the ions in BA extracts were quantitatively analyzed to elucidate the further possible mechanisms of the biological behavior. Finally, the key molecules in the
From the *Guanghua School of Stomatology, Affiliated Stomatological Hospital, Guangdong Province Key Laboratory of Stomatology and †Instrumental Analysis and Research Center, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; and ‡Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Hongwei Jiang, Guanghua School of Stomatology, Affiliated Stomatological Hospital, Sun Yat-sen University, 56 Ling Yuan Xi Road, Guangzhou 510055, PR China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.05.018
JOE — Volume -, Number -, - 2015
Bioaggregate Inhibits Osteoclast Differentiation
1
Basic Research—Biology TABLE 1. Trace Elements Detected within Bioagreggate (BA) mg/kg BA Mean SD
Al
Fe
24.00 2.43 0.66 0.60
K
Mg
Na
Sr
Zn
74.29 574.05 48.46 150.04 15.17 4.45 3.34 2.10 0.71 1.49
SD, standard deviation.
RANKL-RANK signaling pathway, including RANK, TRAF6, NF-kB, and NFATc1, were comprehensively explored.
Materials and Methods Material Preparation and Characterization BA was prepared following the manufacturer’s directions under sterile conditions. Briefly, BA was mixed with sterilized deionized water and packed into sterile plastic molds 8 mm in diameter and 12 mm in height. The specimens were allowed to solidify in a humidified 5% CO2, 95% air atmosphere for 24 hours at 37 C. Then, each specimen was eluted in 40 mL Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 C in a humidified 5% CO2, 95% air atmosphere for 24 hours. The extracts were drawn off and sterilized by passage through a 0.22-mm filter. Various dilutions (final dilutions of 50% and 25%) of the media extracts were then prepared in DMEM. The pH of the extracts was determined using a twin pH meter (Merck, Darmstadt, Germany). The trace elements within the BA powder and the ions released from the BA samples were analyzed using inductively coupled plasma optical emission spectrometry (iCAP 6500 Duo; Thermo Fisher Scientific, Florence, KY) as described previously (17). Supplemental Materials and Methods contains additional information. Cell Culture and In Vitro OCG For OCG, 5 104 RAW264.7 cells (passages 5–15; ATCC, Manassas, VA) were seeded into 6-well culture dishes and cultured with 60 ng/mL soluble recombinant RANKL (sRANKL) (R&D Systems, Minneapolis, MN) for 4 days as described previously (18). At day 2.5 of the OCG induction, cell-cell fusion among the RAW264.7 cells began to occur. Cell Proliferation Assay (CCK-8 Assay) The effect of BA extracts on RAW264.7 cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8) cell proliferation kit (Dojindo, Kumamoto, Japan). Cells were seeded into 96-well plates in sextuplicate in the absence and presence of BA extracts. After 1, 2, 3, and 4 days in culture, 10 mL CCK-8 was added to each well followed by incubation at 37 C for 2.5 hours. Tartrate-resistant Phosphatase Staining At day 4 of in vitro OCG, the cells were stained for tartrate-resistant acid phosphatase (TRAcP) using a commercially available staining kit
(Sigma-Aldrich, St Louis, MO). The TRAcP-stained cells were then counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). TRAcP-positive multinucleated cells containing 3 or more nuclei were categorized as OCs. The number of TRAcP-positive OCs and the number of nuclei within these OCs were counted in 10 random fields of view. The fusion index ([nuclei in the OCs/total nuclei] 100) was calculated (19).
Transwell Cell Migration Assay Cell migration was evaluated using permeable Transwell supports (Corning Life Sciences, Corning, NY) with 5-mm membrane pores in a 24-well plate as described previously (20). Briefly, 105 cells were seeded into the Transwell inserts. The inserts were placed in 600 mL growth medium containing 60 ng/mL sRANKL and various concentrations of BA extracts as chemoattractants and were incubated for 24 hours at 37 C. The inserts were then stained with DAPI. Resorption Pit Assay RAW264.7 cells (5 103 cells/well) were cultured in dentin discs (Osteosite Dentine Discs; Immunodiagnostic Systems Ltd, Tyne and Wear, UK) in 96-well plates for 4 days with 60 ng/mL sRANKL and extracts containing different concentrations of BA. On day 4, the cells on the discs were removed with 6% sodium hypochlorite. The discs were then stained with 1% (w/v) toluidine blue in 0.5% sodium borate. The areas of the resorption pits were measured using ImageJ software (National Institutes of Health, Bethesda, MD). Western Blot Analysis Western blotting was performed as described previously (18). The nuclear and cytoplasmic proteins were extracted using the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific Pierce, Rockford, IL) according to the manufacturer’s instructions. The following primary antibodies were used: mouse anti-RANK (Abcam, Cambridge, UK), rabbit anti-TRAF6, mouse anti–DC-STAMP (Millipore, Temecula, CA), rabbit anticathepsin K (Abcam), rabbit anti-NF-kB, mouse anti–c-Jun (Millipore), and mouse antiactin (CST, Danvers, MA). Horseradish peroxidase–conjugated goat antirabbit/mouse immunoglobulin G (CST) was used as the secondary antibody. Enzyme-linked Immunosorbent Assay for NFATc1 Activation Detection At the indicated time points, nuclear extracts were obtained using a nuclear extract kit (Nuclear Extract Kit; Active Motif, Carlsbad, CA) following the manufacturer’s instructions. The activation of NFATc1 in 5 mg nuclear extract was evaluated with an enzyme-linked immunosorbent assay using the TransAM NFATc1 transcription factor assay kit (Active Motif).
TABLE 2. Ion (Si, Sr, Ca, and P) Concentrations (mg/mL) and pH of Bioaggregate (BA) Extracts DMEM BA extract BA (50%) BA (25%)
Si
Sr
Ca
P
pH
0.28 0.01 1.26 0.06* 0.70 0.03* 0.52 0.01*
0.010 0.000 0.020 0.001* 0.016 0.001* 0.013 0.000*
67.77 0.69 69.55 1.57 66.96 1.19 66.88 0.51
26.94 0.19 25.54 1.10 26.55 0.86 27.13 0.41
7.71 0.01 8.15 0.01* 8.11 0.01* 8.03 0.02*
DMEM, Dulbecco’s Modified Eagle’s Medium. *P < .01 when compared with data for the same ion or pH in DMEM.
2
Tian et al.
JOE — Volume -, Number -, - 2015
Basic Research—Biology TABLE 3. Other Ion (except Si, Sr, Ca, and P) Concentrations (mg/mL) of Bioaggregate (BA) Extracts DMEM BA extract
Al
Fe
K
Mg
Na
Zn
ND ND
0.06 0.00 0.05 0.01
190.9 0.92 201.5 13.09
16.48 0.01 15.69 0.44
3273 35.36 3223 111.2
0.07 0.00 0.06 0.01
DMEM, Dulbecco’s Modified Eagle’s Medium; ND, not detected.
Statistical Analysis The experiments were performed at least 3 times. The results are expressed as means standard deviation. Statistical analysis was performed using 1-way analysis of variance followed by the Student-Newman-Keul test. A P value <.05 was considered statistically significant.
Results Elements within the BA Powder and Ion Concentrations and pH of BA Extracts The BA powder contained trace amounts of Fe, K, Mg, Na, Sr, and Zn. In addition, the BA was free of metal oxides except for traces of Al although the tested BA was advertised as Al free (Table 1). The concentrations of Si (0.52–1.26 mg/mL) and Sr (0.013–0.020 mg/mL) ions were higher in the BA extracts than in DMEM (0.28 and 0.010 mg/mL, respectively) regardless of dilution. However, Ca, P,
and other contents were nearly identical in the BA extracts and DMEM. The pH values of the BA extracts (8.15) were higher than those of DMEM (7.71) and exhibited a slight decrease in pH with dilution (Tables 2 and 3).
Inhibition of OC Formation by BA Extracts As shown in Figure 1A and B, RAW264.7 cells cultivated in the presence of RANKL only matured into numerous, large, TRAcP+ multinucleated cells. In contrast, BA was observed to dose dependently inhibit OCG as shown by the declining number of OCs. The inhibition of OCG by the BA may be caused by the cytotoxicity of BA or the inhibition of the proliferation of RAW264.7 cells. However, this possibility was excluded by the CCK-8 assay results. BA was not cytotoxic to RAW264.7 cells. The growth rate of the RAW 264.7 cells was not affected by the BA extracts after being cultured for 4 days (Fig. 1C).
Figure 1. BA extracts inhibit RANKL-induced osteoclast formation dose dependently without affecting the proliferation of osteoclast precursors. (A) RAW264.7 macrophages were stimulated for 4 days with 60 ng/mL sRANKL to induce OCG in the presence of various concentrations of BA extracts. The cells were fixed and stained for TRAcP. To visualize nuclei, the cells were stained with DAPI. (B) TRAcP+ multinucleated cells containing 3 and more nuclei were counted as osteoclasts. (C) The cell proliferation ability of RAW264.7 cells exposed to extracts of BA was measured using the CCK-8 assay (**P < .01).
JOE — Volume -, Number -, - 2015
Bioaggregate Inhibits Osteoclast Differentiation
3
Basic Research—Biology A
0
B
25%
**
50
30
Fusion index (%)
No. of Nuclei/FOV (10x)
C
*
40
50%
20 10
(BA)
**
40
**
30 20 10
0
0
0
25%
50% (BA)
0
sRANKL 60 ng/ml
0
25%
50%
BA
DC-STAMP β-actin
DC-STAMP/Actin (Fold Induction)
E
D
25%
50% (BA)
1.5
** 1.0
0.5
0.0
0
25%
50% (BA)
Figure 2. The effect of BA extracts on the migration and fusion of RAW264.7 cells. (A) The migration of RAW264.7 monocytes was evaluated in a Transwell assay. (B) Migrated cells were counted. (C) RAW264.7 cells exposed to various concentrations of the BA extracts were cultured with 60 ng/mL sRANKL for 4 days. The fusion index was calculated as the percentage of nuclei in the TRAcP-positive osteoclasts among the total nuclei. (D) RAW264.7 cells exposed to various concentrations of the BA extracts were cultured with 60 ng/mL sRANKL for 2.5 days. Western blot analysis was performed to determine the protein expression levels of DC-STAMP and actin. (E) The band intensities were normalized to b-actin (*P < .05, **P < .01).
Effects of BA Extracts on the Migration and Fusion of RAW264.7 Cells The migration of monocytes/preosteoclasts to close proximity before fusion is an important step for cell-cell fusion (21). Our results showed that RAW264.7 cells exposed to 50% BA extracts exhibited decreased migration toward these chemoattractants compared with the control group (Fig. 2A and B). Furthermore, we observed that BA apparently decreased the fusion index in a dose-dependent manner (Fig. 2C). DC-STAMP is required for cellcell fusion in OCs. The suppression of DC-STAMP expression detected by Western blot analysis was consistent with the fusion efficiency (Fig. 2D and E).
Effects of BA Extracts on RANK and TRAF6 Expression As shown in Figure 4A–C and F–H, the expression of RANK and TRAF6 was significantly up-regulated when the cells were exposed to RANKL. Western blotting revealed a decrease in RANK and TRAF6 expression upon 50% BA treatment compared with the control in day 4 OC cultures but not day 1 and day 2.5 cultures. Notably, a gradual decrease in RANK expression was observed on days 2.5 and 4 compared with day 1 in the 50% BA extracts group. In day 4 osteoclast cultures, although a decrease in RANK and TRAF6 expression was observed when the osteoclasts were treated with 25% BA extracts compared with the control, this difference was not significant (P > .05).
Effects of BA Extracts on Bone Resorption Differentiated multinuclear OCs can resorb mineralized bone surfaces, and cathepsin K, the principal protease mediating matrix degradation within the low pH environment of the sealing zone, is essential for this resorptive activity. Our results showed that the BA extracts inhibited mineralized tissue resorption (Fig. 3A and B). In accordance with the resorbed surface area, the expression of cathepsin K was down-regulated by BA in a dose-dependent manner (Fig. 3C and D).
Suppression of Nuclear Translocation of NF-kB by BA Extracts RANKL-RANK signaling enables the translocation of active NF-kB to the nucleus where it stimulates osteoclastogenic genes. Western blotting revealed a decrease in NF-kB content in pOC or OC nuclei in the presence of 50% BA extracts compared with the control, with no difference in the cytoplasmic distribution of NF-kB. Furthermore, BA did not affect the content of nuclear c-Jun, a transcription factor not directly involved in RANK signaling (Fig. 4D and E).
4
Tian et al.
JOE — Volume -, Number -, - 2015
Basic Research—Biology A
50μm
*
**
C
D 1.5
100 sRANKL
80
0
60
Cathepsin K
40
β-actin
20 0
25%
50%
Cathepsin K/Actin (Fold Induction)
Resorpted surface area relative to control
B
*
**
1.0
0.5
0.0
0
25%
50% (BA)
0
25%
50% (BA)
Figure 3. BA extracts suppress the resorption of mineralized tissue and cellular expression of cathepsin K. (A) RAW264.7 cells treated with 60 ng/mL sRANKL and various concentrations of BA extracts were seeded onto dentin slices. On day 4, the resorption pits that had formed on the dentin slices were stained with toluidine blue. (B) The areas of the resorption pits were measured using ImageJ software. (C) Western blot analysis was performed to determine the protein expression levels of cathepsin K and actin. (D) The band intensities were normalized to b-actin. *P < .05, **P < .01 versus the control group (without BA).
Effects of BA Extracts on NFATc1 Expression Under RANKL stimulation, there was an appreciable increase in NFATc1 content in the nuclear extracts of RAW264.7 cells. In day 2.5 and 4 OC cultures, enzyme-linked immunosorbent assay revealed a decrease in NFATc1 expression in the nuclear region upon BA treatment compared with the control in day 2.5 and 4 cultures but not day 1 cultures (Fig. 4I). In addition, a dose-dependent inhibitory effect of the BA extracts on NFATc1 activation was observed (Fig. 4J).
Discussion This study shows that BA directly inhibits both the differentiation of RAW264.7 cells (precursor cells) to TRAcP+ multinucleated OCs and bone resorption in vitro. The formation of OCs is generally divided into 2 key stages: the differentiation of precursors into mononuclear TRAcP+ pOCs and the multinucleation of pOCs into OCs. The migration of pOCs into close proximity of neighboring cells is an important step in OCG before fusion (21), and defective migration alone could contribute to decreased OC formation (20). Using sRANKL as a chemoattractant, we showed that monocytes treated with 50% BA exhibit decreased migration toward these chemoattractants. In addition, a significant decrease in fusion efficiency upon BA treatment compared with the control was observed. DC-STAMP is a key protein responsible for the cell-cell fusion of OCs. DC-STAMP–defective mice exhibit an osteopetrotic phenotype (22, 23). In the present study, we observed that BA suppresses the expression of the pivotal fusion protein DC-STAMP. Therefore, the inhibition of migration and fusion by BA may underlie the negative effect of BA on multinucleated OC formation. BA also impaired cathepsin K expression, thus inhibiting resorptive activity. In addition, the reduction of resorption induced by BA may be because of decreased fusion efficiency. The fusion of pOCs enables multinucleation (24), which enhances the resorption efficiency of OCs both in vivo and in vitro (23, 25). Small decreases JOE — Volume -, Number -, - 2015
in the number of nuclei per cell have a large negative effect on bone resorption (26). However, further studies are required to determine the effects of BA on the resorptive function of mature OCs. The RANKL/RANK signaling pathway is the central regulator of OC differentiation and function. Hung et al (27) reported that a calcium silicate cement inhibited RANKL-induced OCG by perturbing the TRAF6–NF-kB signaling pathways through the release of Si. In the present study, BA suppressed not only TRAF6 expression and the nuclear translocation of NF-kB but also RANK expression in the late stage of OCG. However, the NF-kB signaling pathway is also activated by cytokines, such as interleukin 1, that do not induce OCG. NFATc1, which is the protein most strongly induced by RANKL, is the master transcription factor for OCG (4, 5, 28). We determined that BA inhibited NFATc1 protein expression dose dependently. A number of OC-specific genes, such as cathepsin K (29) and DC-STAMP (30), are the transcriptional targets of NFATc1. The decreased cathepsin K and DC-STAMP expression observed in the present study are consistent with down-regulated NFATc1 expression. Because NFATc1 expression is dependent on the TRAF6–NF-kB axis, we presume that BA hinders RANK–TRAF6–NF-kB–NFATc1 signaling to inhibit OCG and resorptive activity. Ionic dissolution products from inorganic materials are thought to be critical to the biological behavior of these materials (16). The inductively coupled plasma optical emission spectrometry analysis indicated that the BA powder contained only trace amounts of Al without other toxic trace metals, which is consistent with a previous study (17). No toxic elements were detected in the BA extracts under our experimental conditions, which explains the excellent biocompatibility of BA with various types of cells. Compared with DMEM, a significant increase in Si and a moderate increase in Sr were observed within the BA extracts. Biosilica indirectly inhibits OCG by increasing osteoprotegerin expression in osteoblastlike cells (31, 32). In addition, Si was recently shown to directly and significantly inhibit OC formation and bone resorption in vitro (31, 32). Sr also appears to Bioaggregate Inhibits Osteoclast Differentiation
5
Basic Research—Biology
Figure 4. The effect of BA extracts on RANK, TRAF6, NF-kB, and NFATc1 expression in the RANKL-RANK signaling pathway. (A) RAW264.7 cells were cultured with 60 ng/mL sRANKL and 50% BA extracts for 0, 1, 2.5, or 4 days. The cells were lysed for Western blotting with specific antibodies against RANK, TRAF6, and actin. (B and C) The band intensities were normalized to b-actin. (F) RAW264.7 cells exposed to various concentrations of BA extracts were cultured with 60 ng/mL sRANKL for 4 days. Western blot analysis was performed to determine the protein expression levels of RANK, TRAF6, and actin. (G and H) The band intensities were normalized to b-actin. (D) The cells were pretreated with BA extracts for 6 hours before stimulation with 60 ng/mL sRANKL for 25 minutes. Nuclear and cytoplasmic proteins were extracted for Western blotting with specific antibodies against NF-kB (p65), actin, and c-Jun. (E) At 4 days of OCG with treatment with 50% BA extracts, the nuclear and cytoplasmic proteins were extracted for Western blotting with specific antibodies against NF-kB, actin, and c-jun. (I) At 0, 1, 2.5, or 4 days of OCG, nuclear extracts were isolated. Activation of NFATc1 was detected by performing an enzyme-linked immunosorbent assay on the NFATc1 content in the nuclear extracts. (J) The effect of various concentrations of BA extracts on NFATc1 expression in nuclei at day 2.5 of OCG was evaluated by enzyme-linked immunosorbent assay. *P < .05 and **P < .01 versus the group without BA treatment but with sRANKL.
directly reduce OC differentiation, activity, and bone resorption by inhibiting RANKL-induced nuclear translocation of NF-kB (33). Furthermore, alkalinity has been reported to suppress OC formation
6
Tian et al.
(34). The alkalinity provided by BA may be associated with the hydration reactions of calcium silicates, which generate calcium hydroxide (35). Thus, the potential of BA to antagonize OCG may be
JOE — Volume -, Number -, - 2015
Basic Research—Biology dependent on the joint actions of Si and Sr release (especially Si) and alkalinity. Further studies are required to determine which one of these factors plays the predominant role. In summary, our results showed that BA inhibits OC differentiation, fusion, and bone resorption in vitro. Moreover, we determined that the inhibitory effects of BA may account for the inhibited migration and fusion of RAW264.7 cells, the blockade of RANK–TRAF6–NF-kB–NFATc1 signaling pathways, and the release of Si and small amounts of Sr ions as well as the alkalinity provided by BA.
Acknowledgments Jun Tian and Wenting Qi contributed equally to this study. Supported by the Natural Science Foundation of Guangdong Province (no. S2013010015833 and no. 2014A030313166). The authors deny any conflicts of interest related to this study.
Supplementary Material Supplementary material associated with this article can be found in the online version at www.jendodon.com (http://dx.doi. org/10.1016/j.joen.2015.03.022).
References 1. Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000;289: 1508–14. 2. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–42. 3. Edwards JR, Mundy GR. Advances in osteoclast biology: old findings and new insights from mouse models. Nat Rev Rheumatol 2011;7:235–43. 4. Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007;40:251–64. 5. Kobayashi Y, Udagawa N, Takahashi N. Action of RANKL and OPG for osteoclastogenesis. Crit Rev Eukaryot Gene Expr 2009;19:61–72. 6. Park JW, Hong SH, Kim JH, et al. X-Ray diffraction analysis of white ProRoot MTA and Diadent BioAggregate. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:155–8. 7. Yan P, Yuan Z, Jiang H, et al. Effect of bioaggregate on differentiation of human periodontal ligament fibroblasts. Int Endod J 2010;43:1116–21. 8. Hashiguchi D, Fukushima H, Yasuda H, et al. Mineral trioxide aggregate inhibits osteoclastic bone resorption. J Dent Res 2011;90:912–7. 9. Keskin C, Demiryurek EO, Ozyurek T. Color stabilities of calcium silicate-based materials in contact with different irrigation solutions. J Endod 2015;41:409–11. 10. Yuan Z, Peng B, Jiang H, et al. Effect of bioaggregate on mineral-associated gene expression in osteoblast cells. J Endod 2010;36:1145–8. 11. Jung JY, Woo SM, Lee BN, et al. Effect of Biodentine and Bioaggregate on odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp cells. Int Endod J 2015;48:177–84. 12. Chang SW, Lee SY, Kum KY, et al. Effects of ProRoot MTA, Bioaggregate, and Micromega MTA on odontoblastic differentiation in human dental pulp cells. J Endod 2014;40:113–8. 13. Hashiguchi D, Fukushima H, Nakamura M, et al. Mineral trioxide aggregate solution inhibits osteoclast differentiation through the maintenance of osteoprotegerin expression in osteoblasts. J Biomed Mater Res A 2011;96:358–64.
JOE — Volume -, Number -, - 2015
14. Mladenovic Z, Johansson A, Willman B, et al. Soluble silica inhibits osteoclast formation and bone resorption in vitro. Acta Biomater 2014;10:406–18. 15. Zhang J, Zhu L, Peng B. Effect of BioAggregate on osteoclast differentiation and inflammatory bone resorption in vivo. Int Endod J 2014 Oct 31. doi: 10.1111/iej. 12405. [Epub ahead of print]. 16. Hoppe A, Guldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011;32:2757–74. 17. Kum KY, Kim EC, Yoo YJ, et al. Trace metal contents of three tricalcium silicate materials: MTA Angelus, Micro Mega MTA and Bioaggregate. Int Endod J 2014;47: 704–10. 18. Hassanpour S, Jiang H, Wang Y, et al. The actin binding protein adseverin regulates osteoclastogenesis. PLoS One 2014;9:e109078. 19. Jiang H, Wang Y, Viniegra A, et al. Adseverin plays a role in osteoclast differentiation and periodontal disease-mediated bone loss. FASEB J 2015;29: 2281–91. 20. Leung R, Wang Y, Cuddy K, et al. Filamin A regulates monocyte migration through Rho small GTPases during osteoclastogenesis. J Bone Miner Res 2010; 25:1077–91. 21. Chen EH, Grote E, Mohler W, et al. Cell-cell fusion. FEBS Lett 2007;581: 2181–93. 22. Kukita T, Wada N, Kukita A, et al. RANKL-induced DC-STAMP is essential for osteoclastogenesis. J Exp Med 2004;200:941–6. 23. Yagi M, Miyamoto T, Sawatani Y, et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med 2005;202:345–51. 24. Helming L, Gordon S. Molecular mediators of macrophage fusion. Trends Cell Biol 2009;19:514–22. 25. Miyamoto T. Regulators of osteoclast differentiation and cell-cell fusion. Keio J Med 2011;60:101–5. 26. Ishii M, Saeki Y. Osteoclast cell fusion: mechanisms and molecules. Mod Rheumatol 2008;18:220–7. 27. Hung CJ, Kao CT, Chen YJ, et al. Antiosteoclastogenic activity of silicate-based materials antagonizing receptor activator for nuclear factor kappaB ligandinduced osteoclast differentiation of murine marcophages. J Endod 2013;39: 1557–61. 28. Takayanagi H, Kim S, Koga T, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 2002;3:889–901. 29. Matsumoto M, Kogawa M, Wada S, et al. Essential role of p38 mitogen-activated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1. J Biol Chem 2004;279:45969–79. 30. Kim K, Lee SH, Ha Kim J, et al. NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP). Mol Endocrinol 2008;22:176–85. 31. Wiens M, Wang X, Schroder HC, et al. The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells. Biomaterials 2010;31:7716–25. 32. Schroder HC, Wang XH, Wiens M, et al. Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation. J Cell Biochem 2012;113:3197–206. 33. Caudrillier A, Hurtel-Lemaire AS, Wattel A, et al. Strontium ranelate decreases receptor activator of nuclear factor-KappaB ligand-induced osteoclastic differentiation in vitro: involvement of the calcium-sensing receptor. Mol Pharmacol 2010;78: 569–76. 34. Zhai Z, Qu X, Li H, et al. The effect of metallic magnesium degradation products on osteoclast-induced osteolysis and attenuation of NF-kappaB and NFATc1 signaling. Biomaterials 2014;35:6299–310. 35. Candeiro GT, Correia FC, Duarte MA, et al. Evaluation of radiopacity, pH, release of calcium ions, and flow of a bioceramic root canal sealer. J Endod 2012;38: 842–5.
Bioaggregate Inhibits Osteoclast Differentiation
7
Bioaggregate Inhibits Osteoclast Differentiation, Fusion, and Bone Resorption In Vitro Jun Tian, MS,* Wenting Qi, MS,* Yuanhe Zhang, MS,† Michael Glogauer, PhD,‡ Yongqiang Wang, PhD,‡ Zhihui Lai, MS,† and Hongwei Jiang, PhD* Abstract Introduction: Several bioactive bioceramics with osteoconductive effects can inhibit osteoclast formation and bone resorption in vitro, but the exact underlying mechanism has not been fully elucidated. Bioaggregate (BA), a novel calcium silicate nanoparticulate bioceramic, significantly induces bone and periodontal regeneration. In this study, we aimed to explore the effect of BA extracts on osteoclastogenesis and bone resorption. Methods: The RAW264.7 cells were treated with soluble receptor activator of nuclear factor kappa B ligand to osteoclastogenesis. BA extracts were used to investigate the effect of BA on osteoclast differentiation, fusion, and bone resorption. Furthermore, the ions in BA extracts were quantitatively analyzed. Finally, the key molecules in the receptor activator of nuclear factor kappa B ligand–RANK signaling pathway, including receptor activator of nuclear factor kB (RANK), tumor necrosis factor receptor associated factor 6 (TRAF6), nuclear factor-kappa B (NF-kB), and nuclear factor of activated T cells c1 (NFATc1), were explored. Results: BA suppressed osteoclastogenesis and bone resorption in vitro. BA releases Si ions and a small amount of Sr ions and provides alkalinity. Treatment with BA extracts decreased the migration ability and fusion of RAW264.7 cells. We also observed that BA causes a significantly decreased expression of RANK, TRAF6, NF-kB, and NFATc1. Conclusions: Our study provides further insight into the mechanism by which calcium silicate–based bioceramics inhibit osteoclastogenesis and bone resorption and also suggests that BA is a useful material for several clinical situations because it both stimulates osteoblast differentiation and inhibits osteoclast formation. (J Endod 2015;-:1–7)
Key Words Bioaggregate, bone resorption, calcium silicate bioceramic, osteoclastogenesis, osteoclasts, RAW264.7
M
any adult skeletal diseases are caused by excessive osteoclast (OC) activity and bone resorption including periapical periodontitis–, rheumatoid arthritis–, or periodontal disease–associated bone inflammation, osteoporosis, and Paget disease (1, 2). Thus, OC-targeted therapies should be a first-line treatment for excessive bone loss (3). OCs are specialized cells derived from hematopoietic stem cells through the monocyte/macrophage lineage. The formation of mature resorbing OCs comprises multiple steps including the differentiation of OC precursors into mononuclear preosteoclasts (pOCs), the fusion of mononuclear pOCs to form multinucleated OCs, and the activation or maturation of OCs capable of bone resorption (3). The differentiation and activation of OCs are dependent on the receptor activator of nuclear factor kappa B ligand (RANKL), which is produced by osteoblast cells and activated T cells (2). The activation of RANK, a RANKL receptor that is expressed in OC precursors, by RANKL leads, via the initiation of the TRAF6-NF-kB-NFATc1 signaling pathways, to the expression of OC-specific genes during differentiation and the activation of resorption by mature OCs (3–5). Bioaggregate (BA) (Innovative Bioceramix, Vancouver, BC, Canada) is a recently developed bioceramic with a nanocomposition that is mainly composed of calcium silicates, calcium phosphate, amorphous silicon oxide, and tantalum oxide (6). As an innovative bioceramic, BA is a potential alternative to the well-studied mineral trioxide aggregate (MTA, another bioceramic primarily consisting of calcium silicates) that has been used globally in clinical procedures such as root end filling, repair of root perforations, capping, and pulpotomy (7, 8). Compared with MTA, BA is claimed to be aluminum free and contains a significant amount of tantalum oxide as a radiopacifier instead of bismuth oxide, which may lead to tooth discoloration (9). BA and its dissolution products exhibit excellent biocompatibility (7, 10–12) and osteoconductive effects (10). Several silica-based materials, such as bioactive glass 45S5 and MTA, which are biocompatible and bioactive with osteoconductive effects, inhibit OC formation and bone resorption in vitro (8, 13, 14). In addition, BA shows a suppressive effect on OC differentiation and inflammatory bone resorption in vivo (15). However, the mechanisms of the inhibitory effect of BA on osteoclastogenesis (OCG) and bone resorption have not been well explored. The ionic dissolution products from inorganic materials likely play a critical role in the biological behavior of these materials (16). Therefore, in the present study, BA extracts were used to systematically investigate the effect of BA on the differentiation of OC precursors, the fusion of mononuclear pOCs, and bone resorption. Furthermore, the ions in BA extracts were quantitatively analyzed to elucidate the further possible mechanisms of the biological behavior. Finally, the key molecules in the
From the *Guanghua School of Stomatology, Affiliated Stomatological Hospital, Guangdong Province Key Laboratory of Stomatology and †Instrumental Analysis and Research Center, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; and ‡Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Hongwei Jiang, Guanghua School of Stomatology, Affiliated Stomatological Hospital, Sun Yat-sen University, 56 Ling Yuan Xi Road, Guangzhou 510055, PR China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.05.018
JOE — Volume -, Number -, - 2015
Bioaggregate Inhibits Osteoclast Differentiation
1
Basic Research—Biology TABLE 1. Trace Elements Detected within Bioagreggate (BA) mg/kg BA Mean SD
Al
Fe
24.00 2.43 0.66 0.60
K
Mg
Na
Sr
Zn
74.29 574.05 48.46 150.04 15.17 4.45 3.34 2.10 0.71 1.49
SD, standard deviation.
RANKL-RANK signaling pathway, including RANK, TRAF6, NF-kB, and NFATc1, were comprehensively explored.
Materials and Methods Material Preparation and Characterization BA was prepared following the manufacturer’s directions under sterile conditions. Briefly, BA was mixed with sterilized deionized water and packed into sterile plastic molds 8 mm in diameter and 12 mm in height. The specimens were allowed to solidify in a humidified 5% CO2, 95% air atmosphere for 24 hours at 37 C. Then, each specimen was eluted in 40 mL Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 C in a humidified 5% CO2, 95% air atmosphere for 24 hours. The extracts were drawn off and sterilized by passage through a 0.22-mm filter. Various dilutions (final dilutions of 50% and 25%) of the media extracts were then prepared in DMEM. The pH of the extracts was determined using a twin pH meter (Merck, Darmstadt, Germany). The trace elements within the BA powder and the ions released from the BA samples were analyzed using inductively coupled plasma optical emission spectrometry (iCAP 6500 Duo; Thermo Fisher Scientific, Florence, KY) as described previously (17). Supplemental Materials and Methods contains additional information. Cell Culture and In Vitro OCG For OCG, 5 104 RAW264.7 cells (passages 5–15; ATCC, Manassas, VA) were seeded into 6-well culture dishes and cultured with 60 ng/mL soluble recombinant RANKL (sRANKL) (R&D Systems, Minneapolis, MN) for 4 days as described previously (18). At day 2.5 of the OCG induction, cell-cell fusion among the RAW264.7 cells began to occur. Cell Proliferation Assay (CCK-8 Assay) The effect of BA extracts on RAW264.7 cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8) cell proliferation kit (Dojindo, Kumamoto, Japan). Cells were seeded into 96-well plates in sextuplicate in the absence and presence of BA extracts. After 1, 2, 3, and 4 days in culture, 10 mL CCK-8 was added to each well followed by incubation at 37 C for 2.5 hours. Tartrate-resistant Phosphatase Staining At day 4 of in vitro OCG, the cells were stained for tartrate-resistant acid phosphatase (TRAcP) using a commercially available staining kit
(Sigma-Aldrich, St Louis, MO). The TRAcP-stained cells were then counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). TRAcP-positive multinucleated cells containing 3 or more nuclei were categorized as OCs. The number of TRAcP-positive OCs and the number of nuclei within these OCs were counted in 10 random fields of view. The fusion index ([nuclei in the OCs/total nuclei] 100) was calculated (19).
Transwell Cell Migration Assay Cell migration was evaluated using permeable Transwell supports (Corning Life Sciences, Corning, NY) with 5-mm membrane pores in a 24-well plate as described previously (20). Briefly, 105 cells were seeded into the Transwell inserts. The inserts were placed in 600 mL growth medium containing 60 ng/mL sRANKL and various concentrations of BA extracts as chemoattractants and were incubated for 24 hours at 37 C. The inserts were then stained with DAPI. Resorption Pit Assay RAW264.7 cells (5 103 cells/well) were cultured in dentin discs (Osteosite Dentine Discs; Immunodiagnostic Systems Ltd, Tyne and Wear, UK) in 96-well plates for 4 days with 60 ng/mL sRANKL and extracts containing different concentrations of BA. On day 4, the cells on the discs were removed with 6% sodium hypochlorite. The discs were then stained with 1% (w/v) toluidine blue in 0.5% sodium borate. The areas of the resorption pits were measured using ImageJ software (National Institutes of Health, Bethesda, MD). Western Blot Analysis Western blotting was performed as described previously (18). The nuclear and cytoplasmic proteins were extracted using the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific Pierce, Rockford, IL) according to the manufacturer’s instructions. The following primary antibodies were used: mouse anti-RANK (Abcam, Cambridge, UK), rabbit anti-TRAF6, mouse anti–DC-STAMP (Millipore, Temecula, CA), rabbit anticathepsin K (Abcam), rabbit anti-NF-kB, mouse anti–c-Jun (Millipore), and mouse antiactin (CST, Danvers, MA). Horseradish peroxidase–conjugated goat antirabbit/mouse immunoglobulin G (CST) was used as the secondary antibody. Enzyme-linked Immunosorbent Assay for NFATc1 Activation Detection At the indicated time points, nuclear extracts were obtained using a nuclear extract kit (Nuclear Extract Kit; Active Motif, Carlsbad, CA) following the manufacturer’s instructions. The activation of NFATc1 in 5 mg nuclear extract was evaluated with an enzyme-linked immunosorbent assay using the TransAM NFATc1 transcription factor assay kit (Active Motif).
TABLE 2. Ion (Si, Sr, Ca, and P) Concentrations (mg/mL) and pH of Bioaggregate (BA) Extracts DMEM BA extract BA (50%) BA (25%)
Si
Sr
Ca
P
pH
0.28 0.01 1.26 0.06* 0.70 0.03* 0.52 0.01*
0.010 0.000 0.020 0.001* 0.016 0.001* 0.013 0.000*
67.77 0.69 69.55 1.57 66.96 1.19 66.88 0.51
26.94 0.19 25.54 1.10 26.55 0.86 27.13 0.41
7.71 0.01 8.15 0.01* 8.11 0.01* 8.03 0.02*
DMEM, Dulbecco’s Modified Eagle’s Medium. *P < .01 when compared with data for the same ion or pH in DMEM.
2
Tian et al.
JOE — Volume -, Number -, - 2015
Basic Research—Biology TABLE 3. Other Ion (except Si, Sr, Ca, and P) Concentrations (mg/mL) of Bioaggregate (BA) Extracts DMEM BA extract
Al
Fe
K
Mg
Na
Zn
ND ND
0.06 0.00 0.05 0.01
190.9 0.92 201.5 13.09
16.48 0.01 15.69 0.44
3273 35.36 3223 111.2
0.07 0.00 0.06 0.01
DMEM, Dulbecco’s Modified Eagle’s Medium; ND, not detected.
Statistical Analysis The experiments were performed at least 3 times. The results are expressed as means standard deviation. Statistical analysis was performed using 1-way analysis of variance followed by the Student-Newman-Keul test. A P value <.05 was considered statistically significant.
Results Elements within the BA Powder and Ion Concentrations and pH of BA Extracts The BA powder contained trace amounts of Fe, K, Mg, Na, Sr, and Zn. In addition, the BA was free of metal oxides except for traces of Al although the tested BA was advertised as Al free (Table 1). The concentrations of Si (0.52–1.26 mg/mL) and Sr (0.013–0.020 mg/mL) ions were higher in the BA extracts than in DMEM (0.28 and 0.010 mg/mL, respectively) regardless of dilution. However, Ca, P,
and other contents were nearly identical in the BA extracts and DMEM. The pH values of the BA extracts (8.15) were higher than those of DMEM (7.71) and exhibited a slight decrease in pH with dilution (Tables 2 and 3).
Inhibition of OC Formation by BA Extracts As shown in Figure 1A and B, RAW264.7 cells cultivated in the presence of RANKL only matured into numerous, large, TRAcP+ multinucleated cells. In contrast, BA was observed to dose dependently inhibit OCG as shown by the declining number of OCs. The inhibition of OCG by the BA may be caused by the cytotoxicity of BA or the inhibition of the proliferation of RAW264.7 cells. However, this possibility was excluded by the CCK-8 assay results. BA was not cytotoxic to RAW264.7 cells. The growth rate of the RAW 264.7 cells was not affected by the BA extracts after being cultured for 4 days (Fig. 1C).
Figure 1. BA extracts inhibit RANKL-induced osteoclast formation dose dependently without affecting the proliferation of osteoclast precursors. (A) RAW264.7 macrophages were stimulated for 4 days with 60 ng/mL sRANKL to induce OCG in the presence of various concentrations of BA extracts. The cells were fixed and stained for TRAcP. To visualize nuclei, the cells were stained with DAPI. (B) TRAcP+ multinucleated cells containing 3 and more nuclei were counted as osteoclasts. (C) The cell proliferation ability of RAW264.7 cells exposed to extracts of BA was measured using the CCK-8 assay (**P < .01).
JOE — Volume -, Number -, - 2015
Bioaggregate Inhibits Osteoclast Differentiation
3
Basic Research—Biology A
0
B
25%
**
50
30
Fusion index (%)
No. of Nuclei/FOV (10x)
C
*
40
50%
20 10
(BA)
**
40
**
30 20 10
0
0
0
25%
50% (BA)
0
sRANKL 60 ng/ml
0
25%
50%
BA
DC-STAMP β-actin
DC-STAMP/Actin (Fold Induction)
E
D
25%
50% (BA)
1.5
** 1.0
0.5
0.0
0
25%
50% (BA)
Figure 2. The effect of BA extracts on the migration and fusion of RAW264.7 cells. (A) The migration of RAW264.7 monocytes was evaluated in a Transwell assay. (B) Migrated cells were counted. (C) RAW264.7 cells exposed to various concentrations of the BA extracts were cultured with 60 ng/mL sRANKL for 4 days. The fusion index was calculated as the percentage of nuclei in the TRAcP-positive osteoclasts among the total nuclei. (D) RAW264.7 cells exposed to various concentrations of the BA extracts were cultured with 60 ng/mL sRANKL for 2.5 days. Western blot analysis was performed to determine the protein expression levels of DC-STAMP and actin. (E) The band intensities were normalized to b-actin (*P < .05, **P < .01).
Effects of BA Extracts on the Migration and Fusion of RAW264.7 Cells The migration of monocytes/preosteoclasts to close proximity before fusion is an important step for cell-cell fusion (21). Our results showed that RAW264.7 cells exposed to 50% BA extracts exhibited decreased migration toward these chemoattractants compared with the control group (Fig. 2A and B). Furthermore, we observed that BA apparently decreased the fusion index in a dose-dependent manner (Fig. 2C). DC-STAMP is required for cellcell fusion in OCs. The suppression of DC-STAMP expression detected by Western blot analysis was consistent with the fusion efficiency (Fig. 2D and E).
Effects of BA Extracts on RANK and TRAF6 Expression As shown in Figure 4A–C and F–H, the expression of RANK and TRAF6 was significantly up-regulated when the cells were exposed to RANKL. Western blotting revealed a decrease in RANK and TRAF6 expression upon 50% BA treatment compared with the control in day 4 OC cultures but not day 1 and day 2.5 cultures. Notably, a gradual decrease in RANK expression was observed on days 2.5 and 4 compared with day 1 in the 50% BA extracts group. In day 4 osteoclast cultures, although a decrease in RANK and TRAF6 expression was observed when the osteoclasts were treated with 25% BA extracts compared with the control, this difference was not significant (P > .05).
Effects of BA Extracts on Bone Resorption Differentiated multinuclear OCs can resorb mineralized bone surfaces, and cathepsin K, the principal protease mediating matrix degradation within the low pH environment of the sealing zone, is essential for this resorptive activity. Our results showed that the BA extracts inhibited mineralized tissue resorption (Fig. 3A and B). In accordance with the resorbed surface area, the expression of cathepsin K was down-regulated by BA in a dose-dependent manner (Fig. 3C and D).
Suppression of Nuclear Translocation of NF-kB by BA Extracts RANKL-RANK signaling enables the translocation of active NF-kB to the nucleus where it stimulates osteoclastogenic genes. Western blotting revealed a decrease in NF-kB content in pOC or OC nuclei in the presence of 50% BA extracts compared with the control, with no difference in the cytoplasmic distribution of NF-kB. Furthermore, BA did not affect the content of nuclear c-Jun, a transcription factor not directly involved in RANK signaling (Fig. 4D and E).
4
Tian et al.
JOE — Volume -, Number -, - 2015
Basic Research—Biology A
50μm
*
**
C
D 1.5
100 sRANKL
80
0
60
Cathepsin K
40
β-actin
20 0
25%
50%
Cathepsin K/Actin (Fold Induction)
Resorpted surface area relative to control
B
*
**
1.0
0.5
0.0
0
25%
50% (BA)
0
25%
50% (BA)
Figure 3. BA extracts suppress the resorption of mineralized tissue and cellular expression of cathepsin K. (A) RAW264.7 cells treated with 60 ng/mL sRANKL and various concentrations of BA extracts were seeded onto dentin slices. On day 4, the resorption pits that had formed on the dentin slices were stained with toluidine blue. (B) The areas of the resorption pits were measured using ImageJ software. (C) Western blot analysis was performed to determine the protein expression levels of cathepsin K and actin. (D) The band intensities were normalized to b-actin. *P < .05, **P < .01 versus the control group (without BA).
Effects of BA Extracts on NFATc1 Expression Under RANKL stimulation, there was an appreciable increase in NFATc1 content in the nuclear extracts of RAW264.7 cells. In day 2.5 and 4 OC cultures, enzyme-linked immunosorbent assay revealed a decrease in NFATc1 expression in the nuclear region upon BA treatment compared with the control in day 2.5 and 4 cultures but not day 1 cultures (Fig. 4I). In addition, a dose-dependent inhibitory effect of the BA extracts on NFATc1 activation was observed (Fig. 4J).
Discussion This study shows that BA directly inhibits both the differentiation of RAW264.7 cells (precursor cells) to TRAcP+ multinucleated OCs and bone resorption in vitro. The formation of OCs is generally divided into 2 key stages: the differentiation of precursors into mononuclear TRAcP+ pOCs and the multinucleation of pOCs into OCs. The migration of pOCs into close proximity of neighboring cells is an important step in OCG before fusion (21), and defective migration alone could contribute to decreased OC formation (20). Using sRANKL as a chemoattractant, we showed that monocytes treated with 50% BA exhibit decreased migration toward these chemoattractants. In addition, a significant decrease in fusion efficiency upon BA treatment compared with the control was observed. DC-STAMP is a key protein responsible for the cell-cell fusion of OCs. DC-STAMP–defective mice exhibit an osteopetrotic phenotype (22, 23). In the present study, we observed that BA suppresses the expression of the pivotal fusion protein DC-STAMP. Therefore, the inhibition of migration and fusion by BA may underlie the negative effect of BA on multinucleated OC formation. BA also impaired cathepsin K expression, thus inhibiting resorptive activity. In addition, the reduction of resorption induced by BA may be because of decreased fusion efficiency. The fusion of pOCs enables multinucleation (24), which enhances the resorption efficiency of OCs both in vivo and in vitro (23, 25). Small decreases JOE — Volume -, Number -, - 2015
in the number of nuclei per cell have a large negative effect on bone resorption (26). However, further studies are required to determine the effects of BA on the resorptive function of mature OCs. The RANKL/RANK signaling pathway is the central regulator of OC differentiation and function. Hung et al (27) reported that a calcium silicate cement inhibited RANKL-induced OCG by perturbing the TRAF6–NF-kB signaling pathways through the release of Si. In the present study, BA suppressed not only TRAF6 expression and the nuclear translocation of NF-kB but also RANK expression in the late stage of OCG. However, the NF-kB signaling pathway is also activated by cytokines, such as interleukin 1, that do not induce OCG. NFATc1, which is the protein most strongly induced by RANKL, is the master transcription factor for OCG (4, 5, 28). We determined that BA inhibited NFATc1 protein expression dose dependently. A number of OC-specific genes, such as cathepsin K (29) and DC-STAMP (30), are the transcriptional targets of NFATc1. The decreased cathepsin K and DC-STAMP expression observed in the present study are consistent with down-regulated NFATc1 expression. Because NFATc1 expression is dependent on the TRAF6–NF-kB axis, we presume that BA hinders RANK–TRAF6–NF-kB–NFATc1 signaling to inhibit OCG and resorptive activity. Ionic dissolution products from inorganic materials are thought to be critical to the biological behavior of these materials (16). The inductively coupled plasma optical emission spectrometry analysis indicated that the BA powder contained only trace amounts of Al without other toxic trace metals, which is consistent with a previous study (17). No toxic elements were detected in the BA extracts under our experimental conditions, which explains the excellent biocompatibility of BA with various types of cells. Compared with DMEM, a significant increase in Si and a moderate increase in Sr were observed within the BA extracts. Biosilica indirectly inhibits OCG by increasing osteoprotegerin expression in osteoblastlike cells (31, 32). In addition, Si was recently shown to directly and significantly inhibit OC formation and bone resorption in vitro (31, 32). Sr also appears to Bioaggregate Inhibits Osteoclast Differentiation
5
Basic Research—Biology
Figure 4. The effect of BA extracts on RANK, TRAF6, NF-kB, and NFATc1 expression in the RANKL-RANK signaling pathway. (A) RAW264.7 cells were cultured with 60 ng/mL sRANKL and 50% BA extracts for 0, 1, 2.5, or 4 days. The cells were lysed for Western blotting with specific antibodies against RANK, TRAF6, and actin. (B and C) The band intensities were normalized to b-actin. (F) RAW264.7 cells exposed to various concentrations of BA extracts were cultured with 60 ng/mL sRANKL for 4 days. Western blot analysis was performed to determine the protein expression levels of RANK, TRAF6, and actin. (G and H) The band intensities were normalized to b-actin. (D) The cells were pretreated with BA extracts for 6 hours before stimulation with 60 ng/mL sRANKL for 25 minutes. Nuclear and cytoplasmic proteins were extracted for Western blotting with specific antibodies against NF-kB (p65), actin, and c-Jun. (E) At 4 days of OCG with treatment with 50% BA extracts, the nuclear and cytoplasmic proteins were extracted for Western blotting with specific antibodies against NF-kB, actin, and c-jun. (I) At 0, 1, 2.5, or 4 days of OCG, nuclear extracts were isolated. Activation of NFATc1 was detected by performing an enzyme-linked immunosorbent assay on the NFATc1 content in the nuclear extracts. (J) The effect of various concentrations of BA extracts on NFATc1 expression in nuclei at day 2.5 of OCG was evaluated by enzyme-linked immunosorbent assay. *P < .05 and **P < .01 versus the group without BA treatment but with sRANKL.
directly reduce OC differentiation, activity, and bone resorption by inhibiting RANKL-induced nuclear translocation of NF-kB (33). Furthermore, alkalinity has been reported to suppress OC formation
6
Tian et al.
(34). The alkalinity provided by BA may be associated with the hydration reactions of calcium silicates, which generate calcium hydroxide (35). Thus, the potential of BA to antagonize OCG may be
JOE — Volume -, Number -, - 2015
Basic Research—Biology dependent on the joint actions of Si and Sr release (especially Si) and alkalinity. Further studies are required to determine which one of these factors plays the predominant role. In summary, our results showed that BA inhibits OC differentiation, fusion, and bone resorption in vitro. Moreover, we determined that the inhibitory effects of BA may account for the inhibited migration and fusion of RAW264.7 cells, the blockade of RANK–TRAF6–NF-kB–NFATc1 signaling pathways, and the release of Si and small amounts of Sr ions as well as the alkalinity provided by BA.
Acknowledgments Jun Tian and Wenting Qi contributed equally to this study. Supported by the Natural Science Foundation of Guangdong Province (no. S2013010015833 and no. 2014A030313166). The authors deny any conflicts of interest related to this study.
Supplementary Material Supplementary material associated with this article can be found in the online version at www.jendodon.com (http://dx.doi. org/10.1016/j.joen.2015.03.022).
References 1. Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000;289: 1508–14. 2. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–42. 3. Edwards JR, Mundy GR. Advances in osteoclast biology: old findings and new insights from mouse models. Nat Rev Rheumatol 2011;7:235–43. 4. Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007;40:251–64. 5. Kobayashi Y, Udagawa N, Takahashi N. Action of RANKL and OPG for osteoclastogenesis. Crit Rev Eukaryot Gene Expr 2009;19:61–72. 6. Park JW, Hong SH, Kim JH, et al. X-Ray diffraction analysis of white ProRoot MTA and Diadent BioAggregate. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:155–8. 7. Yan P, Yuan Z, Jiang H, et al. Effect of bioaggregate on differentiation of human periodontal ligament fibroblasts. Int Endod J 2010;43:1116–21. 8. Hashiguchi D, Fukushima H, Yasuda H, et al. Mineral trioxide aggregate inhibits osteoclastic bone resorption. J Dent Res 2011;90:912–7. 9. Keskin C, Demiryurek EO, Ozyurek T. Color stabilities of calcium silicate-based materials in contact with different irrigation solutions. J Endod 2015;41:409–11. 10. Yuan Z, Peng B, Jiang H, et al. Effect of bioaggregate on mineral-associated gene expression in osteoblast cells. J Endod 2010;36:1145–8. 11. Jung JY, Woo SM, Lee BN, et al. Effect of Biodentine and Bioaggregate on odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp cells. Int Endod J 2015;48:177–84. 12. Chang SW, Lee SY, Kum KY, et al. Effects of ProRoot MTA, Bioaggregate, and Micromega MTA on odontoblastic differentiation in human dental pulp cells. J Endod 2014;40:113–8. 13. Hashiguchi D, Fukushima H, Nakamura M, et al. Mineral trioxide aggregate solution inhibits osteoclast differentiation through the maintenance of osteoprotegerin expression in osteoblasts. J Biomed Mater Res A 2011;96:358–64.
JOE — Volume -, Number -, - 2015
14. Mladenovic Z, Johansson A, Willman B, et al. Soluble silica inhibits osteoclast formation and bone resorption in vitro. Acta Biomater 2014;10:406–18. 15. Zhang J, Zhu L, Peng B. Effect of BioAggregate on osteoclast differentiation and inflammatory bone resorption in vivo. Int Endod J 2014 Oct 31. doi: 10.1111/iej. 12405. [Epub ahead of print]. 16. Hoppe A, Guldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011;32:2757–74. 17. Kum KY, Kim EC, Yoo YJ, et al. Trace metal contents of three tricalcium silicate materials: MTA Angelus, Micro Mega MTA and Bioaggregate. Int Endod J 2014;47: 704–10. 18. Hassanpour S, Jiang H, Wang Y, et al. The actin binding protein adseverin regulates osteoclastogenesis. PLoS One 2014;9:e109078. 19. Jiang H, Wang Y, Viniegra A, et al. Adseverin plays a role in osteoclast differentiation and periodontal disease-mediated bone loss. FASEB J 2015;29: 2281–91. 20. Leung R, Wang Y, Cuddy K, et al. Filamin A regulates monocyte migration through Rho small GTPases during osteoclastogenesis. J Bone Miner Res 2010; 25:1077–91. 21. Chen EH, Grote E, Mohler W, et al. Cell-cell fusion. FEBS Lett 2007;581: 2181–93. 22. Kukita T, Wada N, Kukita A, et al. RANKL-induced DC-STAMP is essential for osteoclastogenesis. J Exp Med 2004;200:941–6. 23. Yagi M, Miyamoto T, Sawatani Y, et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med 2005;202:345–51. 24. Helming L, Gordon S. Molecular mediators of macrophage fusion. Trends Cell Biol 2009;19:514–22. 25. Miyamoto T. Regulators of osteoclast differentiation and cell-cell fusion. Keio J Med 2011;60:101–5. 26. Ishii M, Saeki Y. Osteoclast cell fusion: mechanisms and molecules. Mod Rheumatol 2008;18:220–7. 27. Hung CJ, Kao CT, Chen YJ, et al. Antiosteoclastogenic activity of silicate-based materials antagonizing receptor activator for nuclear factor kappaB ligandinduced osteoclast differentiation of murine marcophages. J Endod 2013;39: 1557–61. 28. Takayanagi H, Kim S, Koga T, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 2002;3:889–901. 29. Matsumoto M, Kogawa M, Wada S, et al. Essential role of p38 mitogen-activated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1. J Biol Chem 2004;279:45969–79. 30. Kim K, Lee SH, Ha Kim J, et al. NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP). Mol Endocrinol 2008;22:176–85. 31. Wiens M, Wang X, Schroder HC, et al. The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells. Biomaterials 2010;31:7716–25. 32. Schroder HC, Wang XH, Wiens M, et al. Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation. J Cell Biochem 2012;113:3197–206. 33. Caudrillier A, Hurtel-Lemaire AS, Wattel A, et al. Strontium ranelate decreases receptor activator of nuclear factor-KappaB ligand-induced osteoclastic differentiation in vitro: involvement of the calcium-sensing receptor. Mol Pharmacol 2010;78: 569–76. 34. Zhai Z, Qu X, Li H, et al. The effect of metallic magnesium degradation products on osteoclast-induced osteolysis and attenuation of NF-kappaB and NFATc1 signaling. Biomaterials 2014;35:6299–310. 35. Candeiro GT, Correia FC, Duarte MA, et al. Evaluation of radiopacity, pH, release of calcium ions, and flow of a bioceramic root canal sealer. J Endod 2012;38: 842–5.
Bioaggregate Inhibits Osteoclast Differentiation
7