Effect of ProRoot MTA® and Biodentine® on osteoclastic differentiation and activity of mouse bone marrow macrophages

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.


Introduction
Root resorption, a complication of Dentistry, may occur after dental trauma, surgical procedure, orthodontic treatment, or bleaching. 1 A large area of damaged root surface and continual resorption process may result in the loss of teeth. As resorption is mediated by osteoclasts/odontoclasts, reducing osteoclastic activity is a key to inhibit the progression of root resorption. Several studies have reported a method to suppress the progression of root resorption. In cases of avulsion, or surgical procedure of intentional replantation, the application of fluoride, dexamethasone, or other solutions is recommended. 2,3 Osteoclasts (OCLs) are multinucleated giant cells resulted from a hematopoietic monocyte or macrophage lineage. 4 Odontoclasts are multinucleated giant cells involved in the resorption of hard dental structures and are similar to OCLs regarding cellular origins, characteristics, and function including the dissolution of mineralized hard tissues. 5,6 In this respect, anti-resorptive drugs have been used for patients with osteoporosis, e.g. bisphosphonate was used to inhibit root resorption. 7,8 Bisphosphonates are a family of popular anti-resorptive agents.
A family member, alendronate (ALD), exhibited increased anti-resorptive capacity upon the addition of nitrogen into the side chain. 9 In addition to ALD, nitrogen-containing bisphosphonates also include zolendronate, risedronate, ibandronate, and others. 10 Local treatment with ALD has been shown to prevent root resorption by inhibiting macrophages. 11,9 Calcium silicate-containing cements (CSCs) are widely applied in endodontic treatments, not only as a retrograde filling material, but also for pulp capping material, apexification, and re-vascularization procedures. 12,13 Recent studies have reported the calcium silicate-containing mineral trioxide aggregate (MTA) inhibits OCL differentiation and bone resorption, and possesses good biocompatibility and bioactivities to promote healing mechanisms. 14,15,11 Biodentine ® (BIOD; Septodont, Saint-Maur-des-Fossés, France), a novel biocompatible repair and dentine-like components to MTA have been used since 2011. 16 BIOD is considered to be an alternative to MTA with comparable outcomes in various clinical applications. 17,16 This investigation aimed to assess the differentiation effects of ProRoot MTA ® (PMTA) and BIOD on OCLs originated from murine bone marrow macrophages (BMMs), and compare these effects with those of ALD.
As few researches have examined the anti-resorptive activity of BIOD, the results of this study may support the application of CSCs as a choice of treatment to prevent root resorption.

Agent preparation
The two CSCs used in this investigation, BIOD and PMTA (PMTA; Dentsply, Tulsa, OK, USA), and 10 -8 mol/L ALD (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were prepared as described in previous studies. 18,19 Briefly, BIOD and PMTA were mixed according to their respective manufacturer's instructions and packed into sterilized plastic molds (5 mm inner diameter, 2 mm thickness). Once set, discs were removed from the molds and sterilized with ethylene oxide gas. Two discs of each material were then placed into 4 mL of α-Minimum Essential Medium (α-MEM; HyClone, Logan, UT, USA) containing 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) in a glass vial, and incubated at 37°C for 72 h.
After incubation, conditioned media were collected and passed through a 0.22 μm filter.

Tartrate-resistant acid phosphatase (TRAP) staining
BMMs were seeded in 96-well plates at a density of

Cytotoxicity assay
Viability of OCLs after exposure to agents for 48 and 72 h was assessed. WST assays showed the cytotoxicity of agents did not affect cell viability

Resorption pit assay
To determine whether CSC treatment affected the activity of OCLs, the resorption area was examined.
After 3 days incubation on the dentin disc, mature OCLs showed stained pits with purple color (Figure 5).
CSCs and ALD inhibited the function of mature OCLs, as indicated by fewer pits on the discs.   In accordance with a previous study, 19 neither CSCs nor cements containing antiosteoporotic drugs were cytotoxic to BMMs and OCLs, as evaluated by WST assay. Given that local treatment of ALD can compromise the revascularization of the alveolus around the replanted teeth after avulsion, 9 nontoxic concentrations (≤10 −8 mol L -1 ) were applied to determine the effects of ALD on osteoclastogenesis using RAW cells. However, BMMs used in this study are known to be more sensitive than RAW cells, yielding higher cytotoxicity compared with the results of previous studies, even without statistical differences.
In addition, previous studies have reported that BIOD and PMTA have similar properties concerning effects on cell viability. 31,32 This agrees with the findings of this study, which showed BIOD and PMTA had comparable effects on osteoblast cell biocompatibility.
In this study, OCLs derived from BMMs were confirmed to express active TRAP enzyme, a key marker involved in osteoclastic bone resorption. These previous studies. 16,9,19,38 BIOD also inhibited the mRNA expression of osteoclastic markers, but to a lesser extent than PMTA or ALD (P<0.05, Figure 4).
To further delineate the immediate effects of signaling pathways for RANKL/LPS-induced osteoclastogenesis, phosphorylation of IκBα and ERK1/2 was assessed. RANKL interacts with the OCL surface receptor RANK, which recruits tumor necrosis factor receptor-associated factor 6 (TRAF6) to further activate various downstream signaling pathways such as nuclear factor-κB (NF-κB) and three mitogen activated protein kinases (MAPKs) including p38 MAPK, ERK1/2, and c-jun N-terminal kinase. 39 NF-κB is inactive in the cytosol because it is bound to IκBα, but becomes active once IκBα has been phosphorylated.
According to these results, phosphorylation of ERK1/2 was significantly inhibited by CSCs or ALD, suggesting CSCs targeted the MAPK cascade ( Figure   6). NF-κB signaling is also downregulated, although phosphorylation of IκBα seemed to be less inhibited compared with ERK1/2. These results suggest downregulation of MAPKs and NF-κB signaling is a mechanism involved in the antiosteoclastogenic activity of CSCs and ALD.

Conclusions
Within the limitations of this study, we conclude