Mol Cell Biochem (2015) 399:131–141 DOI 10.1007/s11010-014-2240-y

Type-2 cannabinoid receptor regulates proliferation, apoptosis, differentiation, and OPG/RANKL ratio of MC3T3-E1 cells exposed to Titanium particles Shang Qiu • Fengchao Zhao • Xianye Tang Fang Pei • Hongyan Dong • Liang Zhu • Kaijin Guo

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Received: 21 June 2014 / Accepted: 1 October 2014 / Published online: 8 October 2014 Ó Springer Science+Business Media New York 2014

Abstract The type-2 cannabinoid receptor (CB2) is expressed in osteoblasts and plays a role in bone metabolism through regulation on bone mass and bone turnover, but the functional importance of CB2 in osteoblasts under Titanium (Ti) stimulation is incompletely understood. This study aimed to investigate the CB2 expression in osteoblasts under Ti stimulation and the effects of CB2 activation on proliferation, apoptosis, differentiation, mineralization, OPG, and RANKL expression of MC3T3-E1 cells exposed to Ti particles. MC3T3-E1 cells were incubated in the presence of Ti particles with or without CB2specific agonist HU-308 and antagonist SR144528. Ti particles treatment obviously induced the CB2 expression in MC3T3-E1 cells, and reduced the cell survival in a doseand time-dependent manner (p \ 0.05). Addition of HU308 could dose-dependently alleviate the Ti-induced decrease of cell survival (p \ 0.05). The flow cytometry assay showed that comparing with the control group, the apoptosis rate and caspase-3 activity in the Ti group were significantly elevated (p \ 0.05), which could be alleviated by HU-308. Moreover, HU-308 effectively attenuated the decrease of cell mineralization capability, alkaline phosphates (ALP) and osteocalcin activity, and increase of OPG/RANKL ratio induced by Ti particles treatment (p \ 0.05). These effects were partially counteracted by combined treatment of CB2 antagonist SR144528 (p \ 0.05). In conclusion, CB2 activation has a favorable S. Qiu  F. Zhao  X. Tang  F. Pei  L. Zhu  K. Guo (&) Orthopaedic Department of Affiliated Hospital of Xuzhou Medical College, Jiangsu 223000, China e-mail: [email protected] H. Dong Institute of Neurobiology, Xuzhou Medical College, Jiangsu 223000, China

inhibitory effect on Ti-induced reactions in MC3T3-E1 cell through modulating proliferation, apoptosis, differentiation, and RANKL expression. These findings suggest that activation of CB2 might be an effective therapeutic strategy to promote bone formation and reduce bone dissolution. Keywords CB2  HU-308  Titanium  MC3T3-E1  Proliferation  Apoptosis  OPG  RANKL

Introduction Particulate wear particles from the interface between implant component and surrounding bone are thought to play a central role in osteolysis and aseptic loosening after total joint replacement [1]. Osteolysis is initiated by an aseptic inflammation response to phagocytosis of the implant wear debris, which disturbs osteoblast viability, function and stimulates osteoclastogenesis [2]. Titanium (Ti) particle can cause the imbalance of bone resorption and bone formation during the process of bone remodeling, and lead to failure of total joint replacement [3]. It is widely accepted that the OPG (osteoprotegerin)/ RANKL (receptor activator of NF-jB ligand) system plays critical roles in particle-induced osteolysis, and RANKL is an essential regulator to promote osteoclastogenesis [4]. RANKL binds to receptor activator of NF-jB (RANK) expressed on the surface of osteoclasts (OCs) and osteoclast precursors (OCPs) [5] and stimulates the differentiation and maturation of functional OCs [6]. OPG is a secreted decoying cytokine receptor, which can competitively bind to RANKL and suppress its biological effects on osteoclastogenesis [6]. The OPG/RANKL ratio has emerged as a critical parameter in the regulation of bone

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The Ti suspension was sonicated for 30 min to prevent aggregation before it was added to the cells.

resorption. Ti could stimulate the differentiation of OCs from macrophage or OCPs by activating RANKL expression on osteoblasts and subsequently decreasing OPG/ RANKL ratio. Recently, there has been increasing interest on the role of cannabinoid receptors (CB) in regulating bone cell activity, bone remodeling, and bone mass [1, 7]. Ofek et al. have found that synthetic CB2-specific agonist HU-308 could stimulate proliferation of newborn mouse calvarial osteoblasts (NeMCO) cells and MC3T3-E1 osteoblastic cells, as well as promote activity of Tissue Non-specific Alkaline Phosphatase (TNSALP) and accumulation of extracellular mineral [8, 9]. HU-308 could also markedly reduced RANKL mRNA expression in bone marrow stromal cells undergoing osteoblastic differentiation, leaving OPG expression unaffected. Sophocleous et al. have reported that CB2 activation through HU-308 (10–30 nM, 10–12 days) could stimulate osteoblast differentiation and promote bone nodule (BN) formation in wild-type osteoblasts [10]. ALP levels failed to increase in CB2-/- cultures in response to Parathyroid Hormone (PTH), which means the PTH-induced ALP activity was blunted in osteoblasts derived from CB2-/- mice [10]. However, there is little information with regard to the effects of CB2 activation on osteoblasts exposed to Ti particles in terms of viability, apoptosis, differentiation, mineralization, and OPG/RANKL ratio.

Cell culture The osteoblastic cell line MC3T3-E1 purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) was incubated with Ti particles as an in vitro model. MC3T3-E1 cells were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in a-modified Eagle’s minimum essential medium (a-MEM, Invitrogen, Paisley, UK) supplemented with 10 % fetal bovine serum (Invitrogen, Paisley, UK), 100 lg/mL streptomycin (Invitrogen, Paisley, UK), and 100 U/mL penicillin (Invitrogen, Paisley, UK). After 24 h, unattached cells were removed, and the attached cells were cultured in osteogenic medium containing 50 lg/mL L-ascorbic acid, 10 nM dexamethasone, and 10 mM b-glycerophosphate (Saint Louis, MO, USA). The cells in osteogenic medium were pretreated with or without HU-308 (10-9, 10-8, and 10-7 M) for 6 h or SR144528 (1 lM) for 2 h, before further stimulation with Ti particles. The cells were maintained at 37 °C in a fully humidified atmosphere at 5 % CO2 in air with medium exchange every 3 days.

RT-PCR analysis and quantitative real-time PCR analysis Materials and methods MC3T3-E1 cells in 6-well plates, cocultured with or without Ti (2.5 mg/mL), HU-308 (10-9, 10-8 and 10-7 M), and SR144528 (1 lM), were harvested at 48 h after treatment. Ti particles and dead cells were removed by PBS washing. Total RNA was isolated using Trizol Reagent (Invitrogen, Paisley, UK) following the manufacturer’s instructions. RNA was then reverse transcribed to cDNA using TIANScript RT Kit (TIANGEN, Beijing, China). The forward and reverse primers for the selected genes are listed in Table 1. CB2, OPG, and RANKL mRNA expression were assessed by semi-quantitative PCR assays (RT-PCR). Amplification was performed using the Taq PCR MasterMix (TIANGEN, Beijing, China) with the

Preparation of Ti particles Commercially available pure Ti particles (average diameter 4.50 lm) were purchased from Johnson Matthey. Ti particles were sterilized by baking at 180 °C for 6 h followed by further treatment with 75 % ethanol for 48 h to remove the endotoxin [11]. The particle suspension was confirmed to be endotoxin-free by a commercial limulus assay kit (Chinese Horseshoe Crab Reagent Manufactory, China). Subsequently, the stock solution (10 mg/mL) prepared by suspending the particles in ME3T3-E1 growth medium was diluted to provide the needed concentrations of particles.

Table 1 The forward and reverse primers of RT-PCR used for amplify the selected genes Gene

Forward primer

CB2

50 -CGCCGGAAGCCCTCATACC-30

OPG RANKL GAPDH

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0

5 -AGCTGCAGTACGTCAAGCAGGA-3 0

Reverse primer 50 -CCTCATTCGGGCCATTCCTG-30 0

50 -TTTGCAAACTGTATTTCGCTCTGG-30 0

5 -ATCGTTGGATCACAGCACATCAG-3 50 -GGATGTCGGTGGCATTAATAGTGAG-30

50 -GGATGTCGGTGGCATTAATAGTGAG-30 50 -TGGTGAAGACGCCAGTGGA-30

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following conditions: 30 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 55 °C, 1 min at 70 °C. Correct size of PCR products was confirmed by electrophoresis separation and sequence analysis. An equal volume of each PCR sample was analyzed by 2 % agarose gel electrophoresis containing ethidium bromide (0.5 lg/mL). Expression of OPG and RANKL were quantified using real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR) analysis with SYBR Premix Ex Taq (TaKaRa, Dalian, China) on LightCycler detector (Roche, Basel, Switzerland). Amplification was performed as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 1 min at 60 °C. OPG and RANKL expression were normalized on expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Western blotting analysis MC3T3-E1 cells were harvested at 48 h after treatment, and Ti particles and dead cells were removed by PBS washing. Then the culture medium was discarded, cells were washed with PBS solution, and the mixture of RIPA and PMSF (100:1) was added to prepare cell suspension. After lysis at 0 °C for 30 min, the supernatants (total protein) were collected with 16,0009g centrifuging at 4 °C for 15 min. Protein concentrations were determined by BCA protein assay. Equal amount of protein samples was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred from the gel onto a nitrocellulose membrane. The membrane was blocked with 3 % BSA (BioSharp, Hefei, China) for 3 h at room temperature, followed by incubation with primary antibody (anti-CB2, 1:500, Santa Cruz, TX, USA) at 4 °C overnight, then incubated with IRDye-labeled monkey anti-goat secondary antibody (1:500, LI-COR, Nebraska, USA) at 37 °C for 2 h. Finally, the blots were visualized and analyzed by Odyssey infrared laser imaging system (GENE, California, USA). GAPDH was used as the internal inference, and each experiment was repeated at least three times. Immunofluorescence assay After treatment, MC3T3-E1 cells in 24-well plates were fixed with 4 % paraformaldehyde/PBS for 10 min and then permeabilized in 0.3 % Triton X-100 for 5 min. After washing with PBS solution, cells were blocked with 3 % normal donkey serum albumin in PBS for 30 min and incubated with 1:100 dilution of primary goat anti-CB2 antibody (Santa Cruz, TX, USA) at 4 °C overnight. After washing three times with PBS, cells were incubated with the secondary antibody donkey anti-goat IgG (VICMED,

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Xuzhou, China) labeled with rhodamine (TRITC) at room temperature for 1 h. Fluorescence staining was viewed under a fluorescence microscope (Olympus, Tokyo, Japan) with an excitation wavelength at 552 nm and emission wavelength at 570 nm. Assessment of cell viability/proliferation MC3T3-E1 cells were cultured in 96-well plates at a density of 4,000 cells/well, and collected at 1, 2, 3, 4, 5, 6, and 7 day after treatment. Ti particles and dead cells were removed by PBS washing. Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) is a quantitative measure for cell viability and proliferation through detecting a watersoluble formazan dye from WST-8 tetrazolium salt. After treatment, 10 lL of CCK-8 reagent in 90 lL fresh a-MEM per well was added to each plate well, and then incubated at 37 °C for 1 h. The optical density (OD) was measured at 450 nm wavelength using a microplate reader (GENE, California, USA). Survival rate was calculated following manufacturer’s specification. Experiments were conducted with three replicates and repeated at least three times.

DAPI staining MC3T3-E1 cells were maintained in 24-well plates at a density of 1 9 104 cells per well, incubated with or without Ti (2.5 mg/mL), HU-308 (10-9, 10-8 and 10-7 M), and SR144528 (1 lM), and harvested at 48 h after treatment. Cells were washed with PBS saline, fixed with 4 % paraformaldehyde for 15 min, and then incubated with 40 ,60 diamidino-2-phenylindole (DAPI) solution (10 lg/mL) for 10 min at room temperature. After washing with PBS three times, apoptotic morphological changes in the nuclear chromatin of cells were examined under a fluorescence microscope (Olympus, Tokyo, Japan). Flow cytometric assessment of apoptosis MC3T3-E1 cells were plated in 6-well plates (three replicates for each cell sample), and treated as described above. Apoptosis was analyzed through detecting the redistribution of phosphatidylserine in the plasma membrane by Annexin V-FITC Apoptosis Detection kit (KeyGEN BioTHCH, Nanjing, China). After treatment, cells were harvested with 0.25 % trypsin (without EDTA), and washed twice with PBS solution. Each cell sample was suspended with 500 lL Binding Buffer, mixed with 5 lL Annexin V-FITC and 5 lL propidium iodide (PI) for 30 min at 37 °C in dark. The stained cells were analyzed directly by flow cytometry (BD FACS Calibur, New Jersey, USA) using the Cell Quest Software (BD, New Jersey, USA).

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Measurement of caspase-3 activity Measurement of the activity of caspase-3 was conducted by the caspase-3 activity kit (Beyotime Institute of Biotechnology, Nanjing, China). Lysates of MC3T3-E1 cells on 96-well microtitre plates were collected at 48 h after respective treatment. Cells were homogenized in 100 lL reaction buffer (1 % NP-40, 20 mM Tris–HCl, 137 mM Nad and 10 % glycerol) containing 10 lL caspase-3 substrate (Ac-DEVD-pNA) (2 mM) and 10 lL cell lysate per sample, and incubated at 37 °C for 4 h. The absorbance was measured with an ELISA reader at 405 nm wavelength. The activity of caspase-3 was expressed as values of enzyme activity compared with the control. Experiments were conducted with three replicates and repeated at least three times. ELISA assay MC3T3-E1 cells were seeded in 6-well plates at a density of 1 9 106 cells/well and were allowed to attach for 24 h. The cells were cocultured with or without Ti (2.5 mg/mL), HU-308 (10-9, 10-8 and 10-7 M), and SR144528 (1 lM) for 48 h. At the end of the treatment, Ti particles and dead cells were removed by PBS washing. ALP, OCN, and Col I content in cell lysates were measured using ELISA Assay Kits (RapidBio Lab, California, USA) according to manufacturer’s instructions. The standard curve and samples were analyzed as described by the manufacturer. The absorbance was measured at 450 nm. Experiments were conducted with three replicates and repeated at least three times.

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Bonferroni’s correction for post hoc t test. A p values \0.05 were considered statistically significant.

Results Expression of CB2 in MC3T3-E1 cells exposed to Ti particles Expression of CB2 in MC3T3-E1 cells was assessed by RT-PCR, western blotting, and immunofluorescence assay. Results of RT-PCR and western blotting indicated that expression of CB2 mRNA and protein in MC3T3-E1 cells exposed to Ti particles markedly increased after 48 h of treatment (Fig. 1a, b), as compared to the control group. Immunofluorescence analysis was used to elucidate the expression and subcellular localization of CB2 protein. As shown in Fig. 1c, the red fluorescent intensity of protein CB2 in the Ti-treated MC3T3-E1 cells was higher than that in the control cells, although there were some morphological changes (denoting apoptosis) in cells treated with Ti particles.

Effects of Ti on the cell viability and proliferation of MC3T3-E1 cells Cells were incubated with different concentrations of Ti particles (0, 0.625, 1.25, 2.5, 5, and 10 mg/mL) for different times (1, 2, 3, 4, 5, 6, and 7 days). CCK-8 assay was

Mineralization assay MC3T3-E1 cells were cultured in 24-well plates and treated with or without Ti (2.5 mg/mL), HU-308 (10-9, 10-8 and 10-7 M), and SR144528 (1 lM) for 21 days. Then cells were collected after washing Ti particles and dead cells with PBS. The mineralization of the extracellular matrix in the MC3T3-E1 cells was evaluated by Alizarin Red S staining (Genmed, Boston, USA) following manufacturer’s instructions. Alizarin Red S staining was viewed under a microscope (Olympus, Tokyo, Japan) and counted with Leica Qwin image analysis system (Leica, Solms, German). Statistical analysis Statistical analyses were performed using SPSS for Windows version 13.0. Data were presented as mean ± SD from three independent experiments. All data were subjected to Analysis of Variance (ANOVA) with

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Fig. 1 Expression of CB2 in MC3T3-E1 cells exposed to Ti particles. a CB2 mRNA expression in MC3T3-E1 cells exposed to Ti particles for 48 h was detected using RT-PCR analysis; b CB2 protein expression in MC3T3-E1 cells exposed to Ti particles for 48 h was detected using western blotting analysis; c CB2 protein expression in MC3T3-E1 cells detected using immunofluorescence assay. The results are representative of three independent experiments. Images are representative of 3–6 wells per condition. Scale = 20 lm. Cont control group, Ti Ti group; GAPDH was used as the internal reference

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used to measure the cell viability and proliferation. As shown in Fig. 2a, Ti particles could reduce the cell viability in a dose- and time-dependent manner. Ti concentration below 1.25 mg/mL caused no obvious decrease of cell viability, compared with the control group cells (p [ 0.05). However, high concentration of Ti (2.5, 5, and 10 mg/mL) treatment resulted in significant decrease of cell survival (p \ 0.05). In the 5 and 10 mg/mL groups, the viable cells after Ti treatment for 5–7 days reduced to less than 10 % of the control group. Generally, the viability of the low concentration group (\1.25 mg/mL) did not vary much at early stages of 7 days culturing. Thus, 2.5 mg/mL of Ti concentration (approximate 50 % viability at 2 day) was selected in the following experiments.

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that CB2 activation can protect osteoblasts through alleviating the decrease of viable cells induced by Ti particles.

Effects of CB2 activation on apoptosis and caspase-3 activity of MC3T3-E1 cells exposed to Ti

The effects of CB2 activation and inhibition on proliferation of MC3T3-E1 cells exposed to Ti were assessed by CCK-8 assay (Fig. 2b). Compared with the control group, the CB2-specific agonist HU-308 (10-7 M) treatment alone seemed to have no obvious effects on the cell viability. However, addition of HU-308 (10-9, 10-8 and 10-7 M) could dose-dependently alleviate the Ti-induced decrease of cell survival rate. The cell survival rates in the Ti ? HU-308 groups (10-8 and 10-7 M) were significantly higher than that of Ti-treated group (p \ 0.05). Further, addition of SR144528 (CB2-specific blocker) could block the alleviating effect of HU-308 on cell survival under Ti treatment (p \ 0.05). These results indicate

To explore the protective effects of HU-308 in Ti-induced MC3T3-E1 cells, we measured the cell apoptosis by DAPI staining (Fig. 3) and Annexin-V-FITC/PI methods (Fig. 4a). Results of DAPI staining showed that in the control group (Fig. 3a) and HU-308 group (Fig. 3b), there were scarcely any condensed apoptotic cell nuclei. However, in the Ti group (2.5 mg/mL), almost half of the nucleus showed apoptotic morphological changes. Addition of HU-308 caused apoptotic nuclei gradually reduced with the increase of HU-308 concentration (Fig. 3d–f). In the Ti ? HU-308 ? SR144528 group (Fig. 3g), the amount of apoptotic nuclear changes increased significantly, compared with the corresponding Ti ? HU-308 group (Fig. 3f). Results of flow cytometric assay showed that compared with the control group, the apoptosis rate in the Ti group (at 48 h) significantly elevated from 1.38 to 18.14 % (Fig. 4a, p \ 0.05). With the increase of HU-308 concentration, the apoptosis rate of Ti ? HU-308 group gradually decreased (Fig. 4a). In the Ti ? HU-308 (10-8 and 10-7 M) group, the apoptosis rate reduced to 5.94 and 2.14 %, respectively. However, addition of SR144528 blocked the decrease of apoptosis rate, which is significantly higher than that of Ti ? HU-308 (10-7 M) group (Fig. 4a, p \ 0.05).

Fig. 2 The effects of Ti and CB2 activation on survival of MC3T3E1 cells. a Effects of Ti particles on the proliferation of MC3T3-E1 cells. MC3T3-E1 cells were exposed to increasing doses of Ti (0.625–10 mg/mL) for 1, 2, 3, 4, 5, 6, and 7 days, and cell viability was assessed using the CCK-8; 9p \ 0.05 vs the control group; b effects of CB2 activation and inhibition on Ti-induced decrease of

survival of MC3T3-E1 cells. The cells were incubated with or without HU-308 (10-9, 10-8 and 10-7 M) and Ti (2.5 mg/mL) for 48 h. In some experiments, the cells were treated with SR144528 (1 lM). The data were presented as mean ± SD from three independent experiments. 9p \ 0.05 vs the control group; #p \ 0.05 vs the Ti-treated group; rp \ 0.05 vs the Ti ? HU-308-7 group

Effects of CB2 activation on proliferation of MC3T3E1 cells exposed to Ti particles

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Fig. 3 Effects of CB2 activation and inhibition on Ti-induced apoptosis of MC3T3-E1 cells assessed by DAPI staining. a In the control group, almost all cells had uniformly stained nuclei. b Cells were treated with HU-308 (10-7 M). c Ti (2.5 mg/mL) for 48 h induced typical morphological changes of apoptosis (i.e. nuclei fragmentation with condensed chromatin). d–f Cells were treated with

HU-308 (10-9, 10-8 and 10-7 M) and Ti (2.5 mg/mL), respectively, for 48 h. The apoptosis cells decreased compared to the Ti treatment group in various degree. g Cells were treated with HU-308 (10-7 M), Ti (2.5 mg/mL) and SR144528 (1 lM) for 48 h, the apoptosis cells increased compared to group (f). Images are representative of 3–6 wells per condition

Fig. 4 Effects of CB2 activation on Ti-induced apoptosis and caspase-3 activity. a Effects of CB2 activation and inhibition on Tiinduced apoptosis of MC3T3-E1 cells assessed by flow cytometric. b Effects of CB2 activation and inhibition on Ti-induced caspase-3 activation in MC3T3-E1 cells. The cells were incubated with or

without HU-308 (10-9, 10-8 and 10-7 M) and Ti (2.5 mg/mL) for 48 h. In some experiments, the cells were treated with SR144528 (1 lM). The data were presented as mean ± SD from three independent experiments. 9p \ 0.05 vs the control group; #p \ 0.05 vs the Ti-treated group; rp \ 0.05 vs the Ti ? HU-308-7 group

Similarly, Ti could induce significant increase of caspase-3 activity compared with the control group (Fig. 4b, p \ 0.05). Co-incubation of HU-308 resulted in dosedependant decrease of caspase-3 activity. The caspase-3 activity of Ti ? HU-308 groups (10-8 and 10-7 M) was markedly lower than that of Ti-treated group (Fig. 4b, p \ 0.05). Addition of SR144528 into the Ti ? HU-308 (10-7 M) group led to increase of caspase-3 activity (Fig. 4b, p \ 0.05). These results implicated that Ti-

induced apoptosis occurs through the activation of caspase3, and CB2 activation appeared to participate in preventing Ti-induced apoptosis pathway.

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CB2 activation regulates ALP, Col I, and OCN expression in Ti-treated MC3T3-E1 cells The Effects of CB2 activation on Ti-induced decrease of ALP, Col I, and OCN expression in MC3T3-E1 cells were

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in the Ti ? HU-308 seemed to have no obvious increasing trend (p [ 0.05). These results indicated that CB2 activation can promote ALP and OCN expression, and protect osteoblast differentiation under Ti stimulation. CB2 activation promotes matrix mineralization in MC3T3-E1 cells exposed to Ti Mineral staining with alizarin red S was conducted to evaluate the effects of CB2 activation on Ti-induced degradation of matrix mineralization in MC3T3-E1 cells (Fig. 6). Compared with the control group, HU-308 (10-7 M) group exhibited accelerated accumulation of mineralized nodules, as evidenced by increased mineralized nodules numbers, sizes, as well as nodule areas (from 20.14 to 27.02 %, p \ 0.05). The Ti-treated group, however, showed less deposited mineralized nodules. In the Ti ? HU-308 (10-9, 10-8, and 10-7 M) groups, the nodule area (%) increased in a dose-dependant manner, compared with the Ti-treated group (p \ 0.05). Addition of SR144528 resulted in decrease of nodule area (%) as compared with the Ti ? HU-308 group (p \ 0.05); indicating CB2 activation could promote the matrix mineralization capability of MC3T3-E1 cells, and alleviate the degradation of matrix mineralization capability induced by Ti.

CB2 activation regulates OPG and RANKL gene expression in Ti-treated MC3T3-E1 cells

Fig. 5 Effects of CB2 activation and inhibition on Ti-induced decrease of ALP, Col, OCN activity in MC3T3-E1 cells. The cells were incubated with or without HU-308 (10-9, 10-8, and 10-7 M) and Ti (2.5 mg/mL) for 7 days. In some experiments, the cells were treated with SR144528 (1 lM). The ALP, Col, and OCN activity were examined with ELISA assay. The data were presented as mean ± SD from three independent experiments. 9p \ 0.05 vs the control group; #p \ 0.05 vs the Ti-treated group; rp \ 0.05 vs the Ti ? HU-308-7 group

analyzed by ELISA assay. As shown in Fig. 5, the ALP, Col I, and OCN expressions in the HU-308 (10-7 M) group significantly elevated compared with the control group (p \ 0.05). The ALP and OCN expression of Ti ? HU-308 (10-9, 10-8 and 10-7 M) groups were higher than that of Ti-treated group in a dose-dependant manner (p \ 0.05). Compared with the Ti-treated group, the Col I expressions

Results of RT-PCR (Fig. 7a) showed that the mRNA level of OPG among various groups had no obvious difference; the RANKL mRNA level of Ti-treated group was markedly higher than that of the control group; RANKL expressions of Ti ? HU-308 (10-9, 10-8, and 10-7 M) groups were lower than that of Ti-treated group, which seemed to be inversely related with the concentration of HU-308. Results of quantitative real-time PCR analysis (Fig. 7b) were in coincidence with the RT-PCR results (Fig. 7a). The OPG expression in Ti-treated group, Ti ? HU-308, and Ti ? HU-308 ? SR144528 groups showed no significant difference compared with the control group (p [ 0.05). Ti induced high expression of RANKL and alleviated by HU308 dose-dependently, which was partially inversed by addition of SR144528 (p \ 0.05). Accordingly, the OPG/ RANKL ratio significantly decreased in the Ti-treated group compared with the control group, which was alleviated by HU-308 and partially inversed in by SR144528. These results suggested that CB2 activation can suppress the expression of RANKL, therefore effectively attenuate reduce of OPG/RANKL ratio induced by Ti.

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138 Fig. 6 Effects of CB2 activation and inhibition on Tiinduced decrease of matrix mineralization of MC3T3-E1 cells. Mineral staining with alizarin red S was used to determine the mineralization capability of MC3T3-E1 cells. Cells were treated as described above. a Representative photomicrographs from each group. b The nodule area (%) of each group. Scale = 200 lm. The data were presented as mean ± SD from three independent experiments. 9 p \ 0.05 vs the control group; # p \ 0.05 vs the Ti-treated group; rp \ 0.05 vs the Ti ? HU-308-7 group

Fig. 7 Effects of CB2 activation and inhibition on OPG and RANKL gene expression in MC3T3-E1 cells exposed to Ti particles. The cells were incubated with or without HU-308 (10-9, 10-8, and 10-7 M) and Ti (2.5 mg/ mL) for 48 h. In some experiments, the cells were treated with SR144528 (1 lM). a OPG, RANKL, GAPDH gene expression analyzed by RTPCR analysis; b, c OPG, RANKL gene expression evaluated by quantitative realtime PCR analysis; d ratio of OPG/RANKL. The data were presented as mean ± SD from three independent experiments in MC3T3-E1 cells. 9p \ 0.05 vs the control group; #p \ 0.05 vs the Ti-treated group; r p \ 0.05 vs the Ti ? HU308-7 group

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Discussion It is reported that OCs are capable of phagocytosing the implant-derived biomaterial wear particles in periprosthetic tissues, and OCs can remain fully functional, hormoneresponsive, bone-resorbing cells after particle-phagocytosis [12]. Presence of wear debris particles in periprosthetic zones could mediate the osteolytic effects through inducing OC differentiation and/or activation, but diminishing osteoblast function, which is involved with various exogenous cytokines, growth factors, and prostaglandin E2 in mesenchymal stem/osteoprogenitor cells (MSCs) [13, 14]. Ti particle is a kind of common wear debris in clinical practice based on its inert and highly biocompatible properties [7, 15]. And Ti particle was chosen in this study to be incubated with osteoblasts to mimic the situation of injuring osteoblast in vivo. It has been reported that CB2 protein is present in trabecular osteoblasts [16], as well as in OCs [10]. Ti-particle stimulation could dramatically increase the CB2 expression (mRNA and protein) in mice pouch membranes [17]. Here we discovered that Ti particle could markedly enhance CB2 expression (mRNA and protein) in MC3T3-E1 preosteoblast cell line. Recently, endocannabinoids system including endogenous ligands and their receptors (CB1 and CB2) has been discovered in the skeleton [10]. CB2 has aroused increasing interest and concerns in the regulation of bone metabolism [10], although the psychoactive effect mediated by activation of CB1 receptors is still lacking [18]. Ofek et al. have reported that osteoblastic and osteoclastic CB2 activation contributes to the maintenance of normal bone mass via two mechanisms: direct stimulation of osteoblasts and suppression of OCs by inhibiting RANKL-induced osteoclastogenesis [8]. CB2 activation is mitogenic to osteoblasts in culture and inhibits activity and formation of OCs in osteoporotic post-menopausal women [19]. CB2-specific agonists could attenuate and rescue ovariectomy-induced bone loss, while CB2 null mice show a marked age-related bone loss [10, 20]. Lozano-Ondoua et al. also have observed that sustained administration of the CB2 agonist resulted in inhibition of bone loss in the sarcoma cancer model [21]. Numerous reports have further confirmed the Ofek’s conclusion of CB2 activation to maintain bone mass by genetic and pharmacological methods in many pathological conditions. However, there is little information with regard to the role of CB2 in osteoblast in terms of cell proliferation, differentiation, expression of OPG and RANKL. The effect of CB2 activation on osteoblast exposed to Ti particles is still unknown. Now we hypothesize that CB2 activation may have positive effects on particle-mediated osteolysis and protect osteoblast viability and function against Ti particles exposure through inhibiting Ti-induced downregulation of OPG/RANKL ratio.

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In the present study, we found that cell proliferation decreased and cell apoptosis rate increased in caspase-3 pathway when MC3T3-E1 cells were treated with Ti particles. CB2-selective agonist HU-308 could significantly increase the cell proliferation and obviously reduce cell apoptosis rate in MC3T3-E1 cells exposed to Ti; however, these effects could be distinctly restrained by CB2 selective antagonist. Ti has been reported to reduce osteoblast viability by inducing apoptosis and increase the caspase-3 activity of osteoblasts [22, 23]. However, Ti particles significantly stimulated the proliferation of BMSC-derived osteoblastic cells at both high (5 mg/mL) and low (0.625 mg/mL) particle concentrations [24]. These seemingly contradictory results may be caused by the different types of cells and experimental concentrations. In this study, we first confirmed a proper particle concentration that could affect osteoblast proliferation and differentiation without severely compromising cell viability. The particle concentration of 2.5 mg/mL that did not induce complete cell death but affected cellular function remarkably was chosen to be used in the following experiments. Our results mainly accorded with reports by Kwon [23], who reported that osteoblast proliferation was significantly inhibited by Ti particle concentrations of more than 0.06 wt % (0.625 mg/mL), but we failed to observe severely compromising cell viability at low concentration (0.625 and 1.25 mg/mL). HU-308 was reported to stimulate proliferation of NeMCO cells or osteoblast [8, 9, 25]. Similarly, we found that CB2 activation could effectively alleviate Ti particle-induced downregulation of osteoblast viability and upregulation of apoptosis. Apoptosis rates in Ti group and Ti plus HU-308 (10-9 M) group were 18.14 % and 17.57 %, respectively; but survival rates were 50.72 % and 65.89 % (p \ 0.05), respectively. It is a possible that CB2 activation stimulates osteoblast proliferation/survival rates not only by inhibiting cell apoptosis but also through decreasing other types of cell death, such as necrosis and cell senescence. However, Sophocleous et al. failed to detect any effects on osteoblast proliferation using wildtype mice with 10–30 nM concentrations of HU-308 for 10–12 days (either during the osteogenic cultures or when primary calvarial osteoblasts were cultured under normal conditions) and found that HU-308 significantly increased osteoblast numbers when combined with ovariectomy [10]. In our study, we found that caspase-3 activity increased clearly in the Ti group, which was inhibited remarkably by HU-308 and partially inversed by SR144528. These results implicated that Ti particle-induced osteoblasts apoptosis might take place through the caspase-3 pathway, which may be blocked by CB2 activation through suppressing caspase-3 activity. Apart from osteoblast viability, osteoblast differentiation and mineralization also play key roles in bone

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formation. ALP, OCN, and Col I are important markers of osteoblast differentiation and function, and BN formation can reflect osteoblastic differentiation and mineralization [26–28]. Metallic or polymeric particles have been reported to impair the ability of mature osteoblastic cells to synthesize ALP [24], collagen I and mineralize matrix [29, 30]. In the present study, we found that CB2 activation increased expression of ALP and OCN protein and matrix mineralization (compared to Ti alone) when MC3T3-E1 cells were treated with Ti particles. However, these effects could be restrained by CB2 selective antagonist SR144528. Activation of CB2 stimulates osteoblast-differentiated functions as ALP activity, matrix mineralization, and collagen accumulation [8, 31]. Osteoblasts derived from CB2-/- mice had a reduced capacity to form BNs in vitro and impaired PTH-induced ALP activity [10]. The CB2selective agonist HU-308 also stimulated BN formation in wild-type osteoblasts but had no effect in CB2 null osteoblasts [10]. Taken together, CB2 activation may play a crucial role in protecting osteoblast against Ti exposure by enhancing osteoblast viability and promoting osteoblastic differentiation. Osteoblasts-released OPG and RANKL are key components involved in bone remodeling [32]. OPG has been identified as a naturally decoy receptor for RANKL to prevent its interaction with RANK, thus inhibit OC differentiation [33]. It is widely accepted that decrease of OPG/RANKL ratio causes a stimulation of OCs and eventually enhancement of bone resorption [34, 35]. Recently, Mine et al. revealed that Ti particles had adverse effects on the expressions of RANKL and OPG in osteoblastic cells, leading to decrease of OPG/RANKL ratio [36]. HU-308 could markedly reduced RANKL mRNA in bone marrow stromal cells undergoing osteoblastic differentiation [8]. In this study, we have also observed HU-308 could clearly reverse Ti particle-induced increase of RANKL and decrease of OPG/RANKL ratio in a dosedependent manner, indicating that CB2 activation could inhibit osteoclastogenesis by alleviating Ti-induced decrease of OPG/RANKL ratio. Taken together, Ti particles stimulate the expression of CB2 in osteoblasts and CB2 activation could protect osteoblasts in terms of viability, differentiation, mineralization, and OPG/RANKL ratio when osteoblasts were treated with Ti particles. These findings implicated that CB2 activation may have potential value for prevention and treatment of wear particle-induced osteolysis. Acknowledgments The study was supported by Xuzhou Science and Technology Project (XM13B080). Conflict of interest of interest.

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The authors declare that there are no conflicts

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