DAPT inhibits titanium particle-induced osteolysis by suppressing the RANKL/Notch2 signaling pathway
Xiang Wei1,2, Baoting Fan3, Xuzhuo Chen3, Yutian Cheng4, Aobo Zhang1,2, Shiqi Yu5, Shanyong Zhang3 and Huaqiang Zhao1,2
1Shandong Province Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong Province, China
2Department of Oral and Maxillofacial Surgery, School of Stomatology, Shandong University, Jinan, Shandong Province, China
3Department of Oral and Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
4Department of Stomatology，Shandong Provincial Hospital affiliated to Shandong
First Medical University, Jinan, Shandong Province, China
5Shanghai Ninth People’s Hospital, School of Biomedical Engineering, Shanghai Jiaotong University, Shanghai, China
Address for correspondence:
Shanyong Zhang, Department of Oral and Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
Email: [email protected]
Huaqiang Zhao, Shandong Province Key Laboratory of Oral Tissue Regeneration,
School of Stomatology, Shandong University, Jinan, Shandong Province, China. Department of Oral and Maxillofacial Surgery, School of Stomatology, Shandong University, Jinan, Shandong Province, China
Email: [email protected]
Artificial prosthesis is wildly used in clinical medicine for degenerative disease such as osteoclast-related diseases. However, the material wear particles released from the surface of prostheses cause prosthetic loosening as a result of aseptic osteolysis in long-term use. Therefore, it is important to find an agent that inhibits the formation and function of osteoclast for therapeutic use. Notch signaling pathway plays a lot of roles in cell proliferation, differentiation and apoptosis. However, the role of Notch signaling pathway in osteoclastogenesis remains unclear. The aim of this study is to assess the effects of γ-secretase inhibitor DAPT on osteoclastogenesis via Notch signaling pathway in vitro and titanium particle-induced osteolysis in vivo. In animal experiments, the inhibitory effect of DAPT on titanium particle-induced osteolysis in a mouse calvaria model was demonstrated. Interestingly, few resorption pits were observed following administration of DAPT and almost no osteoclasts formed at high concentration of DAPT. In vitro experiments revealed the mechanism of the effects of DAPT on osteoclastogenesis. DAPT inhibited the formation and function of osteoclast by blocking RANKL-induced Notch2-NF-κB complex signaling pathway.
In conclusion, these results indicated that DAPT could prevent and cure titanium particle-induced prosthetic loosening and other osteoclast-related diseases.
DAPT, prosthetic loosening, osteoclast, osteolysis, Notch signaling pathway
In human body, bones are constantly rebuilt through resorption and synthesis processes, which depend on the balance of osteoblast and osteoclast activities1. Studies have shown that the temporomandibular joint disorder (TMD) is a common disease around the world, which affects the quality of life of patients greatly2. As the disease condition deteriorates, temporomandibular joint osteoarthritis (TMJ-OA), TMJ ankylosis and idiopathic condylar resorption frequently occur, which contribute to the degeneration of TMJ. Currently, severe TMD and tumor in TMJ site are usually treated by autogenous bone grafts from costicartilage, coronoid process and sternoclavicular joint3. However, the autogenous bone grafts are not suitable for long-term use due to the autogenous bone resorption. Thus, a new and more effective treatment is required. In the status quo, total joint arthroplasty (TJA) with prostheses is becoming a popular therapy4. Artificial prostheses made of titanium alloy have been found to be effective when they are used for a short period of time5. But, aseptic
loosening caused by periprosthetic osteolysis is a major complication that leads to arthroplasty failure in long-term use6,7. In previous studies, wear particles such as titanium particles released from the artificial prostheses were found to induce peripheral osteolysis by stimulating inflammatory cells, and destabilizing the balance between osteoblast and osteoclast activity8. Therefore, to inhibit the formation and function of osteoclast in differentiation process and resorption process could be an effective prevention and treatment strategy for aseptic loosening.
Osteoclasts are large multinucleated cells derived from the monocyte-macrophage lineage9,10, participating in bone remodeling and maintenance of homeostasis. Osteoclast is activated by RANK ligand (RANKL) that stimulates the receptor activator of the nuclear factor-κB (RANK), after which they trigger subsequent related signaling pathways11,12. RANKL is one of the tumor necrosis factor (TNF) receptor superfamily13, and its expression is regulated by IL-1, IL-6, TNF-α and macrophage-colony stimulating factor (M-CSF) secreted from macrophages, T lymphocytes and some other inflammatory cells. M-CSF regulates the function and survival of osteoclast precursors and promotes RANK expression in osteoclast precursors14,15. RANK-RANKL complex recruits TNF receptor-associated factor 6 (TRAF6) to the cell membrane16, which activates several signaling pathways such as NF-κB17,18, c-Fos19 and MAPK20, all of which participate in osteoclastogenesis. In
addition, nuclear factor of activated T cells cytoplasmic 1, NFATc1 is mainly activated by RANK-RANKL and it is served as a master transcription factor for osteoclast differentiation21.
Notch signaling pathway is a highly conserved signaling pathway and it plays an important part in different cell processes such as proliferation, differentiation and apoptosis22-24. The Notch-ligand complex causes proteolytic cleavage of Notch receptor by γ-secretase. The intracellular part of the receptor named Notch intracellular domain (NICD)25 translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcription activation of its target genes such as HES-1 genes and HES-5 genes26. It was reported that Notch signaling regulates osteoclastogenesis27. Other studies also reported that Notch2 expression increased by RANKL, and also Notch2 signaling induces NFATc1 expression by NICD28. Moreover, it was reported that Notch signals interact with other signaling molecules, such as NF-κB29, but all these findings are not well elaborated.
DAPT (N-[N-(3,5-difluorohenacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester, GSI-IX), a γ-secretase inhibitor, can inhibit the Notch signaling pathway. It was reported that DAPT could alleviate pathological lesions and strengthen anti-inflammatory responses after cerebral ischemia/reperfusion (I/R) injury30. It was also reported that DAPT significantly reduced cancer stem cells population and tumor
self-renewal ability31. However, it is still unknown whether DAPT can prevent and cure particle-induced osteolysis via suppression of Notch signaling pathway. As far as we know, few studies have explored the role of Notch signaling pathway in osteoclast differentiation and there is no research exploring the effects of DAPT on titanium particle-induced osteolysis for animal and clinical trials.
Therefore, this study aims to 1) examine the potential therapeutic effects of DAPT on particle-induced osteolysis; 2) explore the effects of DAPT on the formation and function of osteoclast; 3) demonstrate the underlying molecular mechanism of DAPT in osteoclastogenesis.
2 MATERIALS AND METHODS
DAPT (GSI-IX) purchased from Selleck Chemicals (Houston, TX, USA), was dissolved in Dimethylsulfoxide) to prepare 10 mM solution. Alpha modification of Eagle medium (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Sydney, Australia). Recombinant M-CSF and RANKL were obtained from R&D (R&D Systems, Minneapolis, MN, USA). Tartrate-resistant acid
phosphatase (TRAP) staining solution was purchased from Sigma-Aldrich. SYBR○RE Premix Ex TaqTM II were obtained from TaKaRa Biotechnology (Otsu, Shiga, Japan).
Antibodies used in Western blot were purchased from Cell Signaling Technology (CST, USA).
2.2 Cells isolation and culture
Primary bone marrow-derived macrophages (BMMs) were extracted from the femurs and tibias bone marrow of three to five week-old C57/BL6 mice according to the method described by Qin et al32. Cells were cultured in complete α-MEM media. Floating cells were cleared after 24 hrs. The adherent cells were incubated for 3-5 days until BMMs reached the desired confluence.
2.3 Cell viability assay
The cytotoxic effect of DAPT was assessed with cell counting kit-8 (CCK-8) under the manufacturer’s instructions. BMMs were seeded on 96-well plates (1×104 cells/well) in complete α-MEM (10% FBS) in triplicate. Subsequently, BMMs were added with different concentration gradients of DAPT (0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320 μM) for 48, 72, and 96 hrs. Then the cells in each well were treated with CCK-8 buffer (Dojindo Molecular Technology, Japan) according to the instructions. The 450 nm absorbance wavelength (630 nm reference wavelength) was measured using a microplate reader (Infinite 200 pro, Tecan).
2.4 Osteoclastogenesis assay in vitro
BMMs were seeded on a 96-well plate (8×103 cells/well) in complete α-MEM in triplicate with 30 ng/ml M-CSF, 50 ng/ml RANKL and different concentrations of DAPT (0, 2.5, 5, and 10 μM). The cell media was renewed every two days. When the mature osteoclasts formed, the cells in each well were washed with PBS twice, and they were fixed by 4% paraformaldehyde for 30 mins. Osteoclasts with more than 3 nuclei were considered as positive for TRAP, and these cells were counted by Image J software.
2.5 Bone resorption assay
BMMs were seeded on a 96-well plate in complete α-MEM in triplicate with bone slice at the bottom of plate (Osteo Assay Stripwell Plate, Corning, USA) at a density of 8×103 cells/well. Different concentrations of DAPT (0, 2.5, 5, and 10 μM) were added with M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 5-7 days. Then, the osteoclasts were removed, and bone slices were scanned under Scanning Electron Microscope (SEM, Hitachi S-4800). The bone resorption area was measured by Image J software.
2.6 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay.
BMMs were seeded on 6-well plate (1×105 cells/well) in complete α-MEM with 30 ng/ml M-CSF, 50 ng/ml RANKL and different concentrations of DAPT (0, 2.5, 5, and 10 μM) for 5 days. Total RNA was extracted when the mature osteoclasts formed, using Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the protocol. Related cDNA was synthesized from 1 μg of total RNA. Subsequently,
RT-qPCR was acted on LightCycler96 (Roche, USA) using the SYBR○RE Premix Ex
TaqTM II. A total volume of 20 μl mixed by 10 μl of SYBR○RE Premix Ex TaqTM II, 7.2 μl ddH2O, 2 μl cDNA and 0.4 μl of each primer composed for each gene. Cycling condition comprised of denaturation and amplification with PCR primers for NFATc1, TRAP, c-Fos, CtsK, CTR and DC-STAMP, and all reactions were run in triplicate. GAPDH was seen as housekeeping gene. To measure the mRNA expression level of each gene, the comparative 2-ΔΔCT method was used. The mouse primer sequence of NFATc1, TRAP, c-Fos, CtsK, CTR, DC-STAMP and GAPDH were as follows: NFATc1 forward 5’-CCGTTGCTTCCAGAAAATAACA-3’ and reverse 5’-TGTGG GATGTGAACTCGGAA-3’; TRAP forward 5’-CTGGAGTGCACGATGCCAGCG
ACA-3’ and reverse 5’-TCCGTGCTCGGCGATGGACCAGA-3’; c-Fos forward 5’-CCAGTCAAGAGCATCAGCAA-3’ and reverse 5’-AAGTAGTGCAGCCCGGA
GTA -3’; CtsK forward 5’-CTTCCAATACGTGCAGCAGCAGA-3’ and reverse 5’- TCTTCAGGGCTTTCTCGTTC-3’; CTR forward 5’-TGCAGACAACTCTTGGTTG
G-3’ and reverse 5’-TCGGTTTCTTCTCCTCTGGA-3’; DC-STAMP forward 5’-AAAACCCTTGGGCTGTTCTT-3’ and reverse 5’- AATCAGGACGACTCCTTG
G-3’; and GAPDH forward 5’-ACCCAGAAGACTGTGGATGG-3’ and reverse 5’- CACATTGGGGGTAGGAACAC-3’.
2.7 Western blot analysis
BMMs cells were seeded on 6-well plates (5×105 cells/well) in complete α-MEM. When the cells reached the desired confluence, serum-free α-MEM with or without DAPT (10 μM) was replaced. After the pretreatment for 8 hrs, the cells were treated with RANKL for a certain period of time. After being washed twice by 1xPBS, total proteins were extracted from cultured cells on ice with the use of radioimmunoprecipitation assay (RIPA) lysis buffer. After centrifugation at 12000x g for 15 mins, the protein-containing supernatant was gathered, and the concentrations were measured using NanoDrop Lite Spectrophotometer assay. Equal amount of protein from each sample was added in 10% SDS-PAGE gels. After electrophoresis, proteins were transferred to PVDF membranes (Bedford, MA, USA). Membranes were blocked by skimmed milk that is dissolved in TBS-Tween, and then incubated
with primary antibodies overnight at 4℃. Washed by PBS three times, the membranes were treated with horseradish peroxidase-conjugated secondary antibodies. Antigen-antibody reaction was exposed to Tanon 4600 system. With the use of Image
J software, the band intensity was quantified by grey analysis and normalized to
2.8 Molecular Modeling
Homology model of p65 was built using the protein structure (PDB code: 1MY5) as a template. The three-dimensional coordinate for DAPT was established with the use of ChembioDraw Ultra and ChemBio3D Ultra software. DAPT was designed to dock to the possible binding pocket of p65 using AutoDock and AutoDock Vina33. Default parameters were set in the docking process. The possible binding sites were predicted by PyMOL software.
2.9 Animal model of titanium particle-induced calvarial osteolysis
A mouse calvarial osteolysis model was established to explore whether DAPT could inhibit osteolysis in vivo34. Animal Care and Experiment Committee of Shanghai Jiaotong University School of Medicine approved all the procedures of animal experiments. The guidelines for the Ethical Conduct in the Care and Use of
Nonhuman Animals in Research were strictly observed. Twenty healthy 8-week-old C57/BL6 mice were randomly assigned to four groups: sham operation group (Sham), titanium particles group (Vehicle), titanium particles with injection of low-dose DAPT (0.5 mg/kg/day) group (Low) and high-dose DAPT (2 mg/kg/day) group (High). To remove endotoxin, commercial pure titanium particles (Alfa Aesar, China) were
baked at 180℃ for 6 hrs and soaked in 70% ethanol for 48 hrs35. After anesthesia, the calvarial periosteum of mice was separated from the calvaria by sharp dissection. 20 mg pretreated titanium particles were then embedded on the surface of the calvaria
around the middle suture32. During the following 14 days, mice in the experimental group were injected intraperitoneally with two different concentrations of DAPT (0.5 and 2 mg/kg/day) respectively. The sham group and vehicle group were injected with PBS every day. The mice were finally killed after two weeks, and the calvaria were fixed into paraformaldehyde after excision operation for further study.
2.10 Micro-CT scanning
The prepared calvaria were scanned by Micro-computed tomography (Micro-CT) (Skyscan 1076; Aartselaar, Belgium) in high-resolution with 50 kV X-ray voltage, 500 mA electric current and 0.7° rotation step. After three-dimensional reconstruction of Micro-CT, the area surrounding the midline suture of calvaria was chosen as a
square region of interest (ROI). Quantitatively, the bone volume against tissue volume (BV/TV), number of porosity, percentage of porosity, trabecular thickness, trabecular separation and trabecular number were measured for each sample36. Soaked in 10% EDTA for 2 weeks after scanning by Micro-CT, decalcified calvaria were made for further study.
2.11 Histomorphometric and histological analysis
After decalcification, the calvaria were embedded in paraffin for hematoxylin & eosin staining (H&E) and TRAP staining. Then the specimens were observed under a microscope, and TRAP-positive multinucleated cells were counted.
2.12 Data analysis
Data are displayed as means ± standard deviation (SD). The data of results were analyzed using the SPSS software. P<0.05 showed a statistically significant difference between different groups. 3 RESULTS 3.1 Effect of DAPT cytotoxicity in vitro To determine the possibility that osteoclastogenesis is affected by the cytotoxicity of DAPT, CCK-8 cell viability assays were conducted. As shown in Figure 1, DAPT exerted cytotoxic effect on BMMs at the concentration higher than 80 μM. The IC50 of DAPT was 316.2, 281.8, and 288.4 μM after 48, 72, and 96 hrs. To sum up, these results indicated that the maximum concentration (10 μM) used in this study did not cause any cytotoxicity. Therefore, the effects of DAPT on osteoclastogenesis are considered to be non-cytotoxic. 3.2 Effect of DAPT on osteoclastogenesis To investigate the effects of DAPT on osteoclastogenesis, BMMs were seeded on 96-well plates in complete α-MEM with 30 ng/ml M-CSF and 50 ng/ml RANKL. Different concentrations of DAPT (0, 2.5, 5, and 10 μM) were added for 5-7 days. As shown in Figure 2a, numerous multinucleated TRAP-positive osteoclasts were observed in the control group. However, the number of TRAP-positive cells declined after treatment with DAPT in a dose-dependent manner. Image J software was adopted to quantitatively analyze the results. Compared with the control group, DAPT significantly decreased the number of mature osteoclasts and almost not any multinucleated osteoclasts formed at 10 μM of DAPT (Figure 2b,c). On the whole, these results showed that DAPT inhibited osteoclast differentiation in vitro. 3.3 Effect of DAPT on osteoclast-induced bone resorption Since osteoclast differentiation can be inhibited when they were exposed to DAPT, an assumption was put forward that the function of osteoclast could also be compromised after treatment with DAPT. Therefore, BMMs were seeded on bone slices with or without different concentrations of DAPT (0, 2.5, 5, and 10 μM). SEM scanning of bone slices was performed when the mature osteoclasts formed. As shown in Figure 3a, in the control group, a large scale of bone resorption area was observed. Conversely, the bone resorption area in DAPT group was observably smaller than that in the control group. Image J software was utilized to further analyze the results. As shown in Figure 3b, about 40% and 15% of resorbed bone areas were measured in 2.5 μM and 5 μM DAPT treatment group respectively, and almost not any bone resorption area was observed in 10 μM DAPT treatment group. Overall, these data indicated that DAPT restrained the function of osteoclast in vitro in a dose-dependent manner. 3.4 Effect of DAPT on RANKL-induced gene expression Since the formation and function of the osteoclast were inhibited by DAPT, we tested whether DAPT could suppress the expression of osteoclast-specific genes by RT-qPCR after BMMs being treated with different concentrations of DAPT (0, 2.5, 5, and 10 μM). As shown in Figure 4, the expression level of osteoclast-specific genes, which was upregulated during RANKL-induced osteoclast differentiation10, dramatically declined after DAPT treatment compared to the expression level in the control group. Particularly, the expression level of the key transcription factor, NFATc1 gene attenuated owing to the presence of DAPT, and other osteoclast-specific genes displayed a similar trend in a dose-dependent manner. In general, these results proved that DAPT suppressed the osteoclast-specific genes. 3.5 Effect of DAPT on NFATc1 and RANKL-induced Notch signaling pathway Previous studies revealed that NFATc1 plays a crucial part in osteoclastogenesis21. Therefore, the effects of DAPT on RANKL-induced NFATc1 were researched. As mentioned before, the data shown in Figure 4 suggested that the transcriptional level of NFATc1 increased following the stimulation of RANKL, but DAPT inhibited the transcription in a dose-dependent manner. To further verify the inhibitory effect of DAPT on NFATc1, the protein levels were measured by western blot analysis. As shown in Figure 5a, NFATc1 protein level increased following RANKL stimulation, but it decreased with the addition of DAPT. Grey analysis confirmed this trend (Figure 5b), which suggested that RANKL-induced NFATc1 expression was inhibited by DAPT. Notch signaling pathway plays a significant role in osteoclastogenesis27. Therefore, the underlying molecular mechanism of Notch signaling pathway was further investigated by western blot analysis. As shown in Figure 5a,b, NICD2 was highly expressed on the stimulation of RANKL. However, the high expression of NICD2 was significantly suppressed with DAPT treatment. Similarly, the level of HES-1 protein, one of the downstream proteins of Notch, was also apparently suppressed by DAPT treatment. Overall, these results indicated that DAPT inhibited Notch signaling pathway during osteoclastogenesis. 3.6 Effect of DAPT on RANKL-induced NF-κB signaling pathway There are several studies which revealed that RANKL-induced NF-κB signaling pathway is attached with great importance for the activation of NFATc1 during osteoclastogenesis37. Therefore, IκBα and p65, which are the key proteins in NF-κB signaling pathway, were examined by western bolt analysis. As shown in Figure 5c,d, the amount of IκBα protein remained unchanged after treatment with or without DAPT. However, p65 and p-p65 protein levels dramatically reduced owing to the presence of DAPT. These data suggested that p65 was indispensable for Notch2-mediated activation of osteoclast but without affecting IκBα, which indicated that p65 could link the Notch and NF-κB signaling pathways. 3.7 Interaction between DAPT and p65 protein Computational molecular docking was performed to predict the possible binding sites of p65 with DAPT. As shown in Figure 6a,b, DAPT had some reasonable connections and also it docked appropriately to p65 protein. In detail, DAPT interacted with Arg-201 and Ser-203 of p65, which resulted in the failure of phosphorylation of p65. Therefore, the NF-κB signaling pathway was blocked under the effects of DAPT, which was consistent with the trend of western blot analysis. 3.8 Effect of DAPT on titanium particle-induced osteolysis It has been proven that DAPT is able to inhibit the formation and function of osteoclast in vitro previously. However, whether DAPT has effects on titanium particle-induced osteolysis in vivo is unknown. Therefore, to assess the potential effects of DAPT on particle-induced osteolysis, a murine calvarial osteolysis model was established. After embedding 20 mg pretreated titanium particles on the periosteum, therapeutic does of DAPT were injected for 2 weeks. As shown in Figure 7a, compared with the smooth calvarial surface in the sham group, three-dimensional reconstruction of Micro-CT scanning showed that a number of osteolysis pits were observed on the calvaria as a result of the stimulation of titanium particles in the vehicle group. Remarkably, the number of osteolysis pits decreased in a dose-dependent manner in experimental group, where the high-dose DAPT (2 mg/kg/day) dramatically attenuated the bone resorption pits while the low-dose DAPT (0.5 mg/kg/day) mildly suppressed the osteolysis on calvarial surface. For quantitative analysis, compared with the sham control group, many porosity pits were counted in the vehicle group, accompanied with a lower value of BV/TV than that of the sham group. On the contrary, the number of porosity pits in the ROI gradually decreased while the BV/TV value increased after injection of DAPT (Figure 7b-d). Furthermore, because of the osteolysis, the trabecular thickness and trabecular separation increased in the vehicle group. However, these two values remained within the normal range in high-dose DAPT group (Figure 7e,f). Meanwhile, the number of trabecular declined in the vehicle group but increased following the treatment with DAPT (Figure 7g). Further histological staining and histomorphometric analysis confirmed the protective effect of DAPT on titanium particle-induced osteolysis. As shown in Figure 8a, substantial inflammatory infiltration such as macrophages and TRAP-positive osteoclasts were observed on the surface of calvaria in the vehicle group. In contrast, the bone destruction area reduced in DAPT-treated group, and so was the number of inflammatory cells and osteoclasts (Figure 8b). Collectively, these data indicated that DAPT inhibited the osteoclastogenesis in vivo. 4 DISCUSSION TMJ-OA is a common joint disease resulting in chronic degeneration of bones and soft tissue38. Clinical evidence shows that TMJ-OA occurs in 8-16% of population39. As the disease progresses, ankylosis and condylar resorption occur. In recent years, TJA has been found to be an effective therapy to cure severe degeneration of TMJ structure, even tumors in TMJ site. However, periprosthetic osteolysis and subsequent aseptic loosening are factors causing failure of joint prostheses40. Since there are more younger patients (30 to 35 years of age), the prostheses are required to have a long lifetime. The wear titanium particle-induced aseptic loosening is caused by activation of osteoclast, and thus osteoclast is regarded as the target to treat titanium particle-induced osteolysis. The bisphosphonates were the first medication to treat osteoclast-related diseases. However, serious adverse effects of bisphosphonates such as gastrointestinal toxicity and osteonecrosis limited the use of it41. Estrogen, the primary female sex hormone, was also employed as a therapeutic medication to treat osteoporosis. But severe adverse reaction such as breast cancer restricted its application42. Therefore, we postulated that osteoclast inhibitors could be effective treatments for wear particle-induced osteolysis. DAPT, a γ-secretase inhibitor, was recently reported to significantly enhance tumor immunity43. Also, it has anti-inflammation effects after cerebral I/R injury30. However, whether DAPT could inhibit the formation and function of osteoclast is unknown, and the mechanism underlying the effects of DAPT on osteoclastogenesis remains unclear. In our study, DAPT was confirmed to have inhibitory effects on osteoclastogenesis in vitro. The administration of DAPT suppressed TRAP-positive osteoclasts and few osteoclasts were stained at the concentration of 10 μM without cytotoxicity. Also, the results of bone slices test showed that almost not any bone resorption pits were observed, which indicated that DAPT could restrain the function of osteoclast. What’s more, three-dimensional reconstruction images of calvarial bone confirmed that DAPT significantly attenuated osteolysis in vivo. Further studies were performed to explore the possible molecular mechanism of the inhibitory effects. It was found that the mRNA expression level of osteoclast-related genes decreased following the treatment with DAPT. Several previous studies reported that RANKL-induced ERK, JNK pathway played a crucial role in the formation of osteoclast44,45, but few studies discussed the Notch signaling pathway. Notch signaling is highly conserved and it plays an essential role in varieties of cell processes. However, the impact of Notch signaling in the field of osteoclastogenesis remains controversial. The results of western blot revealed that NICD2 and HES1 increased during the process of osteoclast differentiation after RANKL stimulation. However, DAPT inhibited the RANKL-induced Notch2 signaling pathway, which indicated that Notch2 plays a crucial part in the process of osteoclastogenesis. Activation of Notch2 subsequently contributes to the stimulation of NFATc1, which is the master regulator of osteoclastogenesis37. In contrast, DAPT significantly decreased the expression of NFATc1 by blocking Notch cleavage and liberation of NICD2, thereby, so as to the downstream product HES1. It is worthwhile mentioning that we also found that DAPT inhibited NF-κB subunit p65 without affecting IκBα, which indicated that p65 might be vital in Notch2-mediated activation of NFATc1. Overall, we inferred that Notch2-NF-κB complex regulated the response to RANKL stimulation. Generally speaking, in this study, we found DAPT was able to prevent and cure titanium particle-induced bone destruction. In clinical practice, we assume that DAPT could effectively treat the aseptic prostheses loosening, so as to extend the duration of prostheses. Hence, for more patients, especially young patients who had TJA due to severe degeneration of TMJ, their physiological structure and function of TMJ can be better recovered. And also, optimal occlusion can be restored so that their quality of life could be improved through prostheses. What’s more, for patients who suffer from tumors in TMJ site, such as joint synovial chondroma, the risk of aseptic prostheses loosening could reduce after TJA. Therefore, DAPT has great potential clinical significance in the treatment of aseptic loosening, the main complication of TJA. However, there are some limitations in this study. Firstly, because osteoclast and osteoblast are in a dynamic balance, the effects of DAPT on osteoblast are unclear and require further investigation. Secondly, the titanium particles around the implant in human patients are continuously liberated in a low concentration rather than being embedded on the surface of calvarial bone in a relatively high concentration, as reported by Marius Von Knoch46. In this study, an acute pathological osteolysis model induced by titanium particles was established due to the limited time and cost, which is slightly different from the real clinical situation. Thus, we proposed that better animal models, such as beagle and goat with implantation of customized TMJ prosthesis, are required to imitate the environment of human TMJ in the future investigations. Thirdly, previous studies reported that metal implants undergo corrosion in different ways in human body47. For instance, metal ions released from implants activated T-lymphocytes that produce substantial inflammatory cytokines including TNF-α, which triggers some immune response and osteoclastogenesis48,49. In our study, we did not explore the side effects of metal ions, so the detailed investigations on this aspect are anticipated in future studies. 5 CONCLUSION The presented study confirmed that DAPT could inhibit osteoclastogenesis in vitro and titanium particle-induced osteolysis in vivo via blockade of Notch2 and NF-κB signaling pathway. Apparently, local injection of optimal concentration of DAPT could be regarded as a potential preventive or therapeutic treatment for periprosthetic loosening after TJA operation and even other osteolysis-related diseases in the future. ACKNOWLEDGMENTS This study was supported by National Natural Science Foundation of China (Grant No. 81671010); Science and Technology Commission of Shanghai Municipality (Grant No. 16441908800); Shanghai Hospital Development Center (Grant No. 16CR3104B). CONFLICT OF INTEREST The authors declare that they have no conflict of interests. AUTHOR CONTRIBUTIONS SYZ and BTF designed most of the experiments. XW, XZC and ABZ were responsible for carrying out experiments. XW and SQY carried out the cell experiments. XW and XZC performed surgery on animals. HQZ analyzed the data. XW wrote the manuscript. YTC reviewed and edited the manuscript. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. REFERENCES 1. Kim HS, Suh KS, Sul D, Kim BJ, Lee SK, Jung WW. The inhibitory effect and the molecular mechanism of glabridin on RANKL-induced osteoclastogenesis in RAW264.7 cells. Int J Mol Med 2012;29(2):169-77. 2. Abrahamsson AK, Kristensen M, Arvidsson LZ, Kvien TK, Larheim TA, Haugen IK. Frequency of temporomandibular joint osteoarthritis and related symptoms in a hand osteoarthritis cohort. Osteoarthritis and Cartilage 2017;25(5):654-657. 3. Khadka A, Hu J. Autogenous grafts for condylar reconstruction in treatment of TMJ ankylosis: current concepts and considerations for the future. Int J Oral Maxillofac Surg 2012;41(1):94-102. 4. Teeny SM, York SC, Mesko JW, Rea RE. Long-term follow-up care recommendations after total hip and knee arthroplasty - Results of the American Association of Hip and Knee Surgeons member survey. Journal of Arthroplasty 2003;18(8):954-962. 5. Vanloon JP, Debont LGM, Boering G. Evaluation of Temporomandibular-Joint Prostheses - Review of the Literature from 1946 to 1994 and Implications for Future Prosthesis Designs. Journal of Oral and Maxillofacial Surgery 1995;53(9):984-996. 6. Abu-Amer Y, Darwech I, Clohisy JC. Aseptic loosening of total joint replacements: mechanisms underlying osteolysis and potential therapies. Arthritis Research & Therapy 2007;9. 7. Harris WH. Wear and periprosthetic osteolysis - The problem. Clinical Orthopaedics and Related Research 2001(393):66-70. 8. Dumbleton JH, Manley MT, Edidin AA. A literature review of the association between wear rate and osteolysis in total hip arthroplasty. Journal of Arthroplasty 2002;17(5):649-661. 9. Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007;40(2):251-264. 10. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423(6937):337-342. 11. Purdue PE, Koulouvaris P, Potter HG, Nestor BJ, Sculco TP. The cellular and molecular biology of periprosthetic osteolysis. Clin Orthop Relat Res 2007;454:251-61. 12. Ren W, Wu B, Peng X, Hua J, Hao HN, Wooley PH. Implant wear induces inflammation, but not osteoclastic bone resorption, in RANK(-/-) mice. J Orthop Res 2006;24(8):1575-86. 13. Raggatt LJ, Partridge NC. Cellular and Molecular Mechanisms of Bone Remodeling. Journal of Biological Chemistry 2010;285(33):25103-25108. 14. Arai F, Miyamoto T, Ohneda O, Inada T, Sudo T, Brasel K, Miyata T, Anderson DM, Suda T. Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappa B (RANK) receptors. Journal of Experimental Medicine 1999;190(12):1741-1754. 15. Fuller K, Owens JM, Jagger CJ, Wilson A, Moss R, Chambers TJ. Macrophage-Colony-Stimulating Factor Stimulates Survival and Chemotactic Behavior in Isolated Osteoclasts. Journal of Experimental Medicine 1993;178(5):1733-1744. 16. Naito A, Azuma S, Tanaka S, Miyazaki T, Takaki S, Takatsu K, Nakao K, Nakamura K, Katsuki M, Yamamoto T and others. Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 1999;4(6):353-62. 17. Franzoso G, Carlson L, Xing L, Poljak L, Shores EW, Brown KD, Leonardi A, Tran T, Boyce BF, Siebenlist U. Requirement for NF-kappaB in osteoclast and B-cell development. Genes Dev 1997;11(24):3482-96. 18. Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R. Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nat Med 1997;3(11):1285-9. 19. Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA, Wagner EF. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 1994;266(5184):443-8. 20. Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, Hata K, Yamashita K, Hiraga T, Watanabe T and others. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest 2004;114(4):475-84. 21. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, Saiura A, Isobe M, Yokochi T, Inoue J and others. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 2002;3(6):889-901. 22. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: Cell fate control and signal integration in development. Science 1999;284(5415):770-776. 23. Lai EC. Notch signaling: control of cell communication and cell fate. Development 2004;131(5):965-73. 24. Ashley JW, Ahn J, Hankenson KD. Notch signaling promotes osteoclast maturation and resorptive activity. J Cell Biochem 2015;116(11):2598-609. 25. Bray SJ. Notch signalling: a simple pathway becomes complex. Nature Reviews Molecular Cell Biology 2006;7(9):678-689. 26. Han H, Tanigaki K, Yamamoto N, Kuroda K, Yoshimoto M, Nakahata T, Ikuta K, Honjo T. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. International Immunology 2002;14(6):637-645. 27. Duan L, Ren YJ. Role of notch signaling in osteoimmunology-from the standpoint of osteoclast differentiation. European Journal of Orthodontics 2013;35(2):175-182. 28. Fukushima H, Nakao A, Okamoto F, Shin M, Kajiya H, Sakano S, Bigas A, Jimi E, Okabe K. The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis. Mol Cell Biol 2008;28(20):6402-12. 29. Vacca A, Felli MP, Palermo R, Di Mario G, Calce A, Di Giovine M, Frati L, Gulino A, Screpanti I. Notch3 and pre-TCR interaction unveils distinct NF-kappaB pathways in T-cell development and leukemia. EMBO J 2006;25(5):1000-8. 30. Wang JJ, Zhu JD, Zhang XH, Long TT, Ge G, Yu Y. Neuroprotective effect of Notch pathway inhibitor DAPT against focal cerebral ischemia/reperfusion 3 hours before model establishment. Neural Regen Res 2019;14(3):452-461. 31. Zhao ZL, Zhang L, Huang CF, Ma SR, Bu LL, Liu JF, Yu GT, Liu B, Gutkind JS, Kulkarni AB and others. NOTCH1 inhibition enhances the efficacy of conventional chemotherapeutic agents by targeting head neck cancer stem cell. Sci Rep 2016;6:24704. 32. Qin A, Cheng TS, Lin Z, Cao L, Chim SM, Pavlos NJ, Xu J, Zheng MH, Dai KR. Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption. PLoS One 2012;7(4):e34132. 33. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010;31(2):455-61. 34. Jin S, Park JY, Hong JM, Kim TH, Shin HI, Park EK, Kim SY. Inhibitory effect of (-)-epigallocatechin gallate on titanium particle-induced TNF-alpha release and in vivo osteolysis. Exp Mol Med 2011;43(7):411-8. 35. Liu F, Zhu Z, Mao Y, Liu M, Tang T, Qiu S. Inhibition of titanium particle-induced osteoclastogenesis through inactivation of NFATc1 by VIVIT peptide. Biomaterials 2009;30(9):1756-62. 36. Wedemeyer C, Xu J, Neuerburg C, Landgraeber S, Malyar NM, von Knoch F, Gosheger G, von Knoch M, Loer F, Saxler G. Particle-induced osteolysis in three-dimensional micro-computed tomography. Calcif Tissue Int 2007;81(5):394-402. 37. Asagiri M, Sato K, Usami T, Ochi S, Nishina H, Yoshida H, Morita I, Wagner EF, Mak TW, Serfling E and others. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J Exp Med 2005;202(9):1261-9. 38. Kalladka M, Quek S, Heir G, Eliav E, Mupparapu M, Viswanath A. Temporomandibular joint osteoarthritis: diagnosis and long-term conservative management: a topic review. J Indian Prosthodont Soc 2014;14(1):6-15. 39. Mejersjo C. Therapeutic and prognostic considerations in TMJ osteoarthrosis: a literature review and a long-term study in 11 subjects. Cranio 1987;5(1):69-78. 40. Greenfield EM, Bi Y, Ragab AA, Goldberg VM, Van De Motter RR. The role of osteoclast differentiation in aseptic loosening. J Orthop Res 2002;20(1):1-8. 41. Marx RE. The deception and fallacies of sponsored randomized prospective double-blinded clinical trials: the bisphosphonate research example. Int J Oral Maxillofac Implants 2014;29(1):e37-44. 42. Maeda SS, Lazaretti-Castro M. An overview on the treatment of postmenopausal osteoporosis. Arq Bras Endocrinol Metabol 2014;58(2):162-71. 43. Mao L, Zhao ZL, Yu GT, Wu L, Deng WW, Li YC, Liu JF, Bu LL, Liu B, Kulkarni AB and others. gamma-Secretase inhibitor reduces immunosuppressive cells and enhances tumour immunity in head and neck squamous cell carcinoma. Int J Cancer 2018;142(5):999-1009. 44. Li M, Wang W, Geng LI, Qin Y, Dong W, Zhang X, Qin AN, Zhang M. Inhibition of RANKL-induced osteoclastogenesis through the suppression of the ERK signaling pathway by astragaloside IV and attenuation of titanium-particle-induced osteolysis. International Journal of Molecular Medicine 2015;36(5):1335-1344. 45. Liu X, Qu X, Wu C, Zhai Z, Tian B, Li H, Ouyang Z, Xu X, Wang W, Fan Q and others. The effect of enoxacin on osteoclastogenesis and reduction of titanium particle-induced osteolysis via suppression of JNK signaling pathway. Biomaterials 2014;35(22):5721-30. 46. von Knoch M, Jewison DE, Sibonga JD, Sprecher C, Morrey BF, Loer F, Berry DJ, Scully SP. The effectiveness of polyethylene versus titanium particles in inducing osteolysis in vivo. J Orthop Res 2004;22(2):237-43. 47. Okazaki Y, Gotoh E. Comparison of metal release from various metallic biomaterials in vitro. Biomaterials 2005;26(1):11-21. 48. Hallab NJ, Caicedo M, Finnegan A, Jacobs JJ. Th1 type lymphocyte reactivity to metals in patients with total hip arthroplasty. J Orthop Surg Res 2008;3:6. 49. Taki N, Tatro JM, Lowe R, Goldberg VM, Greenfield EM. Comparison of the roles of IL-1, IL-6, and TNFalpha in cell culture and murine models of aseptic loosening. Bone 2007;40(5):1276-83. FIGURE LEGENDS FIGURE 1 Cytotoxic assays of DAPT treatment. BMMs were cultured in complete α-MEM containing the indicated concentrations of DAPT for 48, 72, and 96 hrs respectively. The cell viability, relative to control, was measured by the CCK-8 assay. The inhibition rate of BMMs at 48, 72, and 96 hrs was calculated by GraphPad Prism software, and the IC50 was 316.2, 281.8, and 288.4 μM respectively. **P<0.01, ***P<0.001 and ****P<0.0001, compared to 0 μM treatment (control). BMMs, bone marrow-derived macrophages; CCK-8, cell-counting kit-8; IC50, half-maximal inhibitory concentration FIGURE 2 DAPT inhibited RANKL-induced osteoclastogenesis in vitro. (a) BMMs were cultured in complete α-MEM with different concentrations of DAPT followed by 30 ng/ml M-CSF and 50 ng/ml RANKL for 5-7 days. Cells were subsequently fixed with 4% paraformaldehyde, and subjected to TRAP staining. (b) The number of TRAP-positive cells was counted. (c) The area of mature osteoclasts was measured by Image J. *P<0.05, ***P<0.001 and ****P<0.0001, compared to 0 μM treatment (control). RANKL, receptor activator of the nuclear factor-κB ligand; BMMs, bone marrow-derived macrophages; M-CSF, macrophage colony-stimulating factor; TRAP, tartrate-resistant acid phosphatase FIGURE 3 DAPT inhibited osteoclast bone resorption. (a) BMMs were seeded on bone slices with different concentrations of DAPT followed by 30 ng/ml M-CSF and 50 ng/ml RANKL in complete α-MEM for 5-7 days. (b) Bone resorption area was measured by Image J software. ***P<0.001 and ****P<0.0001, compared to 0 μM treatment (control). BMMs, bone marrow-derived macrophages; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of the nuclear factor-κB ligand FIGURE 4 DAPT inhibited RANKL-induced expression of osteoclast-specific genes. BMMs were treated with M-CSF (30 ng/ml), RANKL (50 ng/ml) and different concentrations of DAPT in complete α-MEM for 5-7 days. TRAP, NFATc1, c-Fos, CtsK, CTR, DC-STAMP expression levels were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RNA expression levels were normalized to the expression of GAPDH. **P<0.01, ***P<0.001 and ****P<0.0001, compared to 0 μM treatment (control). BMMs, bone marrow-derived macrophages; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of the nuclear factor-κB ligand FIGURE 5 DAPT suppressed RANKL-induced NAFTc1, Notch2 and NF-κB signaling. (a) Suppression of RANKL-induced NFATc1, NICD2 and HES-1 by DAPT treatment (10 μM). (b) The band intensity corresponding to NFATc1, NICD2 and HES-1 was quantified and normalized to β-actin by Image J software, and is presented by column using GraphPad Prism software. (c) The amount of p65 and p-p65 expressed highly in control group, but it significantly reduced by DAPT treatment (10 μM). IκBα was not affected by exposure to DAPT. (d) The band intensity corresponding to IκBα, p65 and p-p65 was quantified and normalized to β-actin by Image J software, and is presented by column using GraphPad Prism software. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001, compared to 0 μM treatment (control). RANKL, receptor activator of the nuclear factor-κB ligand FIGURE 6 DAPT interacted with p65. (a) Chemical structure of DAPT with a molecular formula of C23H26F2N2O4 and a molecular weight of 432.46 g/mol. (b) Molecular docking model predicted by PyMOL software showed the possible interaction binding pocket between DAPT and p65 FIGURE 7 DAPT inhibited titanium particle-induced mouse calvarial osteolysis. (a) Three-dimensional reconstructed images of representative Micro-CT from each group. (b) Bone volume against tissue volume (BV/TV). (c) The number of porosity on the surface of calvaria in each group. (d) The percentage of total porosity in each group. (e) The trabecular thickness in each group. (f) The trabecular separation in each group. (g) The trabecular number in each group. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 FIGURE 8 DAPT inhibited titanium particle-induced mouse calvarial osteolysis assessed by histological staining and histomorphometric analysis. (a) H&E and TRAP staining were performed, and the middle suture of the calvaria in each group were shown. (b) The number of TRAP positive cells in each group. **P<0.01, ***P<0.001. H&E, hematoxylin & eosin staining; TRAP, tartrate-resistant acid phosphatase Accepted Article Accepted Article Accepted Article Accepted Article Accepted Article Accepted Article Accepted Article Accepted Article