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Biomaterials xxx (2014) 1e12

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Biomaterials

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The effect of metallic magnesium degradation products on osteoclast-induced osteolysis and attenuation of NF-kB and NFATc1 signaling

Zanjing Zhai a,1, Xinhua Qu a,1, Haowei Li a,1, Ke Yang b, Peng Wan b, Lili Tan b,Zhengxiao Ouyang a,c, Xuqiang Liu a, Bo Tian a, Fei Xiao a, Wengang Wang a, Chuan Jiang a,Tingting Tang a, Qiming Fan a, An Qin a,*, Kerong Dai a,d,*a Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine,Shanghai 200011, People’s Republic of Chinab Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, People’s Republic of ChinacDepartment of Orthopaedics, Hunan Provincial Tumor Hospital and Tumor Hospital of Xiangya School of Medicine, Central South University, Changsha,Hunan 410013, People’s Republic of Chinad Engineering Research Center of Digital Medicine and Clinical Translation, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200011, People’sRepublic of China

a r t i c l e i n f o

Article history:Received 20 February 2014Accepted 14 April 2014Available online xxx

Keywords:MagnesiumOsteoclastOsteolysisNF-kBNFATc1

* Corresponding authors. Shanghai Key LaboratoDepartment of Orthopaedics, Ninth People’s Hospital,School of Medicine, 639 Zhizaoju Road, Shanghai 2China.

E-mail addresses: [email protected] ([email protected], [email protected] (K. Dai).

1 Contributed equally.

http://dx.doi.org/10.1016/j.biomaterials.2014.04.0440142-9612/� 2014 Elsevier Ltd. All rights reserved.

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a b s t r a c t

Wear particle-induced aseptic prosthetic loosening is one of the most common reasons for total jointarthroplasty (TJA). Extensive bone destruction (osteolysis) by osteoclasts plays an important role in wearparticle-induced peri-implant loosening. Thus, strategies for inhibiting osteoclast function may havetherapeutic benefit for prosthetic loosening. Here, we mimicked the process of magnesium (Mg)degradation in vivo and obtained Mg leach liquor (MLL) by immersing pure Mg in culture medium. Forthe first time, we demonstrated that MLL suppresses osteoclast formation, polarization, and osteoclastbone resorption in vitro. An in vivo assay demonstrated that MLL attenuates wear particle-inducedosteolysis. Furthermore, we found that MLL significantly inhibits nuclear factor-kB (NF-kB) activationby retarding inhibitor-kB degradation and subsequent NF-kB nuclear translocation. We also found thatMLL attenuates the expression of NFATc1 at both the protein and mRNA levels. These results demonstratethat MLL has anti-osteoclast activity in vitro and prevents wear particle-induced osteolysis in vivo.Collectively, our study suggests that metallic magnesium, one of the orthopedic implants with superiorproperties, has significant potential for the treatment of osteolysis-related diseases caused by excessiveosteoclast formation and function.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Total joint arthroplasty (TJA) is the gold standard for treatmentof end-stage joint diseases such as osteoarthritis and rheumatoidarthritis [1]. However, aseptic loosening secondary to peri-prosthetic osteolysis remains a serious orthopedic problem [2].Currently, two approaches are mainly used to address with thisproblem. On the one hand, researchers are developing new

ry of Orthopaedic Implants,Shanghai Jiao Tong University00011, People’s Republic of

. Qin), [email protected],

al., The effect of metallic maBiomaterials (2014), http://d

materials that are more durable and resistant to reduce the wearrate [3,4]. However, given the limitations of currently availabletechnology and raw materials, the results have been far fromsatisfactory [5]. On the other hand, researchers are attempting toretard wear particle-induced osteolysis [6e8] by investigating theunderlying mechanisms. However, these mechanisms are complexand involve many different cytokines, chemokines, growth factors,and cell types. Wear particles activate macrophages, fibroblasts,foreign body giant cells, and T lymphocytes to release numerousproinflammatory cytokines and chemokines including tumor ne-crosis factor-a (TNF-a); interleukins-1, 6, 11, and 17 (IL-1, -6, -11,-17); prostaglandin E2 (PGE2); and macrophage-colony stimu-lating factor (M-CSF), all of which induce receptor activator ofnuclear factor-kB ligand (RANKL) expression by osteoblasts,marrow stromal cells, and activated T cells [9,10]. Increased RANKLlevels at the implant site enhance the differentiation and

gnesium degradation products on osteoclast-induced osteolysis andx.doi.org/10.1016/j.biomaterials.2014.04.044

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Z. Zhai et al. / Biomaterials xxx (2014) 1e122

activation of the already abundant pool of monocyte/macrophageprecursors into mature osteoclasts, thus shifting the local ho-meostasis to activated bone destruction [11,12]. Given that the latestage of wear particle-induced osteolysis enhances osteoclasticbone resorption, therapeutic targeting of osteoclast function pre-sents a logical method of treating or alleviating aseptic looseningafter total joint replacement. Indeed, many agents with anti-osteolytic activity have been studied over the last decade, suchas bisphosphonates [13], calcitonin [14], erythromycin [15], anddenosumab [16,17]. However, because the current methods ofpreventing and treating aseptic loosening are far from satisfactory,considerable scientific and public interest remains for investi-gating alternative agents or treatments for the prevention ofaseptic loosening in TJA.

Magnesium (Mg), which has long been used in orthopedic im-plants [18,19], is an exceptionally lightweight metal with superiorproperties [20]. Mg is essential for human metabolism and half ofthe total physiological Mg is stored in the bone tissue [19]. Previousstudies revealed the role of Mg in bone metabolism. For example,Mg-based structures accelerated hard callous formation [21,22] byenhancing osteoblast adhesion and stimulating new bone forma-tion [23]. Moreover, several studies revealed that Mg deficiency isnegatively associated with bone mass density [24]. Studies indifferent species also showed a positive effect of supplementingMgon bone density [25e27].

However, previous studies provided only a partial perspectiveon the effects of Mg on bone, and research involving the directeffect of Mg on osteoclasts is lacking. Therefore, in the presentstudy, several concentrations of Mg leach liquor (MLL) at differentpH values were tested to investigate the role of Mg in osteoclastsin vitro and in wearing particle-induced osteolysis in vivo.

2. Materials and methods

2.1. Reagents

Alpha-minimum essential medium (MEM), fetal bovine serum (FBS), andpenicillin were purchased from Gibco BRL (Gaithersburg, MD, USA). Soluble mouserecombinant macrophage-colony stimulating factor (M-CSF) and RANKL were pur-chased from R&D Systems (USA). Tartrate-resistant acid phosphatase (TRAP) stain-ing solution was obtained from SigmaeAldrich (St Louis, MO, USA). The CellCounting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Japan).Primary antibodies targeting b-actin, phospho-IkBa, IkBa, MMP-9, c-Src, and NFATc1were purchased from Cell Signaling Technology (CST; Danvers, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for detecting mouse IL-6, IL-1b, TNF-a andRANKL were purchased from R&D Systems (USA).

2.2. Preparation of biomaterial extracts

MLL was prepared according to methods described by the International Orga-nization for Standardization (ISO 10993-12). Briefly, the ratio of pure Mg to culturemedium was 100 mg/mL. Mg was incubated at 37 �C for the indicated duration incomplete a-MEM (Hyclone; USA). The extracts were analyzed using inductivelycoupled plasma optical emission spectroscopy (ICP-OES; VISTAPRO; Agilent; USA) todetermine the elemental concentrations of Mg. Changes in the pH values of theextracts were detected using a pH test-meter (Denver UB-7; YaoHua Co.; China).Samples were sterilized by gamma irradiation at 25 KGY before use. In the cytologyassay, the three samples used in this study were named Mg-1, Mg-2, and Mg-3 andwere collected at the indicated time points. More specifically, Mg-1 was collected at6 h with pH 7.7 � 0.05 and Mg2þ 2 � 0.05 (mM); Mg-2 was collected at 12 h with pH8.0� 0.04 andMg2þ10.0� 0.187 (mM); Mg-3was collected at 24 hwith pH 8.4� 0.07and Mg2þ 16 � 0.193 (mM).

In addition, we adjusted the pH values of Mg-1 (pH ¼ 7.7 � 0.05,Mg2þ ¼ 2 � 0.05 mM) and Mg-2 (pH ¼ 8.0 � 0.04, Mg2þ ¼ 10.0 � 0.187 mM) to thephysiological level (pH ¼ 7.4) while the Mg ion concentration remained, andrenamed as Mg-L (pH ¼ 7.4 � 0.05, Mg2þ ¼ 2 � 0.069 mM) and MgeH(pH ¼ 7.4 � 0.05, Mg2þ ¼ 10.0 � 0.17 mM).

2.3. Cytotoxicity assay

The proliferative effect of MLL on bone marrow macrophages (BMMs) wasdetermined by using the CCK-8 kit (Dojindo Molecular Technology; Japan). Cellswere plated in 96-well plates at 5 � 103 cells/well in triplicate. After 24 h, the cellswere treated with Mg-1, Mg-2, and Mg-3 for the indicated duration. Then, 10 mL of

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CCK-8 was added to each well and the plates were incubated at 37 �C for 2 h. Theoptical density (OD) was measured using an ELX800 absorbance microplate reader(Bio-Tek; USA) at 450 nm (650 nm reference). Cell viability was calculated relative tothe control ([experimental group OD � blank OD]/[control group OD � blank OD]).

2.4. Apoptosis assay

The apoptotic effect of MLL on BMMs was determined by using the VybrantApoptosis Assay Kit #2 (Invitrogen Life Technologies, USA). Cells were treated withMLL samples for 48 h and then washed twice with cold phosphate-buffered saline(PBS) and pelleted; the supernatants were discarded and the cells were resuspendedin 1� annexin-binding buffer. Early apoptosis was detected by staining with AlexaFluor 488 annexin V and propidium iodide using the Vybrant Apoptosis Assay Kit #2(Invitrogen Life Technologies, USA). Fluorescence-activated cell sorting (FACS) wasperformed using a FACScan flow cytometer (Becton Dickinson; Sunnyvale, CA). Datawere acquired using CELL Quest software.

2.5. Colony formation assay

RAW264.7 cells derived from murine macrophages were seeded in triplicate in48-well plates at 3 � 103 cells/well and cultured for 4 d in the presence of MLLsamples. After 4 d, the cells were fixed and stained with 40 , 6-diamidino-2-phenylindole (DAPI; Sigma). Colonies or areas with �50 nuclei were counted.

2.6. In vitro osteoclastogenesis assay

In vitro osteoclastogenesis assays were performed to examine the effects of MLLon osteoclast differentiation. BMM cells were prepared as previously described[28,29]. Briefly, cells extracted from the femurs and tibiae of a 6-week-old C57/BL6mouse were incubated in complete cell culture medium containing 30 ng/mLM-CSFin a T-75cm2

flask for proliferation. When the medium was changed, the cells werewashed to deplete residual stromal cells. After reaching 90% confluence, the cellswere washed with PBS three times and trypsinized for 30 min to harvest BMMs.Adherent cells on dish bottoms were classified as BMMs; these BMMs were platedon 96-well plates at a density of 8 � 103 cells/well in triplicate and incubated in ahumidified incubator containing 5% CO2 at 37 �C for 24 h. Then, the cells weretreated with various MLL samples containing M-CSF (30 ng/mL) and RANKL (50 ng/mL).

2.7. TRAP activity

After 5 d of culture, the osteoclasts were fixed using 4% paraformaldehyde (PFA)in PBS for 10min and the substrates were rinsed three times with PBS. Samples werestained for TRAP activity, an enzymatic marker of osteoclasts, using an acid phos-phatase kit (SigmaeAldrich) according to the manufacturer’s protocol withoutcounter-staining. Images were obtained using a Nikon SMZ 1500 stereoscopic zoommicroscope (Nikon Instruments Inc.; Melville, NY). We quantified the total area ofTRAP-positive regions and the total number of osteoclasts on five randomly selectedfields of view for each sample.

2.8. F-actin ring immunofluorescence

For F-actin ring immunofluorescent staining, MLL pre-treated osteoclasts werefixed with 4% PFA for 15 min at room temperature (RT) and permeabilized for 5 minwith 0.1% v/v Triton X-100. Cells were incubated with rhodamine-conjugatedphalloidin (1:100; Invitrogen Life Technologies, USA) diluted in 0.2% w/v bovineserum albumin (BSA)-PBS for 1 h at RT and then washed extensively with 0.2% w/vBSA-PBS and PBS. Cells were then incubated with Hoechst 3342 dye (1:5000;Invitrogen Life Technologies, USA) for visualization of nuclei, washed with PBS, andmounted with ProLong Gold anti-fade mounting medium (Invitrogen Life Tech-nologies, USA). Fluorescence detection was performed on a NIKON A1Si spectraldetector confocal system equippedwith 20� (dry) lenses. Fluorescence images werecollected using NISeC Elements software and analyzed using Image J software(National Institutes of Health).

2.9. Resorption pit assay

For the bone resorption assay, BMMs were seeded on bone slices in 96-wellplates at a density of 8 � 103 cells/well with three replicates and stimulated withM-CSF (30 ng/mL) and RANKL (50 ng/mL). Four days later, cells were treated withMLL samples for 48 h post-culture. Cells were then fixed with 2.5% glutaraldehyde.Bone slices were imaged by using scanning electron microscopy (SEM; FEI Quanta250) with 200� magnification at 10 kV. Three view fields were randomly selectedfor each bone slice for further analysis. Pit areas were quantified using Image Jsoftware (National Institutes of Health). Similar independent experiments wererepeated at least three times.

2.10. Titanium particle-induced osteolysis mouse model

A wear particle-induced osteolysis model was generated as previously reported[28]. In brief, titanium particles were washed continuously in 100% ethanol for 48 hto remove adherent endotoxins, followed by resuspension in sterile PBS solution at aconcentration of 300 mg/mL. Twenty 8-week-old C57BL/J6 mice divided into four


Fig. 1. Preparation, characterization, and cytotoxicity analysis of magnesium leach liquor (MLL) samples. (A) Magnesium concentration was determined by inductively coupledplasma optical emission spectroscopy after the indicated immersion time. (B) The pH value of the extracts was measured by using a pH test-meter. (C) The cell viability of MLL-stimulated bone marrow macrophage (BMM) cells treated with Cell Counting Kit-8 (CCK-8) was measured over time by using various MLL samples. (D) MLL-treated BMMs wereincubated for 48 h, and then apoptosis was assessed using flow cytometry. (E) The apoptosis ratio was calculated in various samples. (F) MLL-treated RAW264.7 cells were fixed andstained with 40-6-diamidino-2-phenylindole (DAPI). (G) DAPI-positive colonies with �50 cells were counted. (H) Area of colonies with �50 cells was determined.

Table 1Immersion time and characterization of magnesium leach liquor (MLL) samples.Values are expressed as the mean � standard error of the mean.

Mg-1 Mg-2 Mg-3 Mg-L Mg-H

Immersion time (h) 6 12 24 6 12pH value 7.7 � 0.05 8.0 � 0.04 8.4 � 0.07 7.4 � 0.05 7.4 � 0.05Mg2þ(mM) 2 � 0.05 10 � 0.187 16 � 0.193 2 � 0.069 10 � 0.17

Z. Zhai et al. / Biomaterials xxx (2014) 1e12 3

groups of five mice each were used. Titanium particles (30 mg) were embeddedunder the periosteum of the mice around the middle suture to induce bonedestruction (three groups). One group of mice was used as a sham control group. Allprocedures were conducted in accordance with the official guidelines for animalcare of the Shanghai Jiao Tong University School of Medicine (Shanghai, China;Animal Ethics Approval #201040). Two days after implantation of titanium particles,MLL or PBS was injected into the periosteum every other day for 14 d before theanimals were sacrificed and further analysis was performed. No adverse effects ormortality occurred.

Please cite this article in press as: Zhai Z, et al., The effect of metallic magnesium degradation products on osteoclast-induced osteolysis andattenuation of NF-kB and NFATc1 signaling, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.044

Fig. 2. Magnesium leach liquor (MLL) inhibited RANKL-induced osteoclast formation in a pH- and magnesium concentration-dependent manner. (A) Bone marrow macrophages(BMMs) were treated with Mg-1 or Mg-2 samples followed by M-CSF (30 ng/mL) and RANKL (50 ng/mL) stimulation for 5 d. Cells were then fixed with 4% paraformaldehyde (PFA)and subjected to tartrate-resistant acid phosphatase (TRAP) staining. (B) TRAP-positive multinuclear cells were counted. (C) BMMs were treated with samples at different pH valuesfollowed by M-CSF (30 ng/mL) and RANKL (50 ng/mL) stimulation for 5 d. Cells were then fixed with 4% PFA and subjected to TRAP staining. (D) TRAP-positive multinuclear cellswere counted. (E) BMMs were treated with samples with different Mg concentrations followed by M-CSF (30 ng/mL) and RANKL (50 ng/mL) stimulation for 5 d. Cells were thenfixed with 4% PFA and subjected to TRAP staining. (F) TRAP-positive multinuclear cells were counted. All experiments were performed at least three times, and significance wasdetermined using the Student-Newman-Keul’s test (*P < 0.05; **P < 0.01).


2.11. Micro-computed tomography (micro-CT) scanning

A high-resolution micro-CT scanner (Skyscan 1176; Skyscan; Aartselaar,Belgium) was used for qualitative and quantitative analyses of osteolysis in mousecalvariae at a resolution of 9 mm using the following settings: X-ray voltage, 50 kV;electric current, 500 mA; rotation step, 0.7�. To reduce metal artifacts, the wearparticles were removed before scanning. After reconstruction, a square region ofinterest (ROI) around the midline suture was chosen for further qualitative andquantitative analysis, with the bone volume against tissue volume (BV/TV), bonemineral density (BMD), bone mineral content (BMC), and percentage of totalporosity of each sample measured according to previous studies [30].

2.12. Histological and histomorphometric analysis

After micro-CT analysis, the samples were decalcified in 10% ethyl-enediaminetetraacetic acid (EDTA; pH 7.4) at 4 �C for 6e8 weeks, followed byparaffin embedding. For analysis of the middle suture osteolysis area, non-osseoustissue adjacent to and continuous with the midline suture was taken as theosteolysis area and analyzed by using Image Pro-Plus 5.0 (Media Cybernetics; USA).Five consecutive hematoxylin and eosin (H&E)-stained sections were evaluated. Inaddition, TRAP staining was performed and TRAP-positive multinucleated osteo-clasts were counted in each sample.

2.13. Calvaria culture

Calvaria were removed en bloc under sterile conditions from five animals pergroup and randomly assigned for culture. Each calvaria was placed into one well of a12-well plate and cultured with 1 mL of serum- and phenol-free Dulbecco’s modi-fied Eagle medium (DMEM) containing glutamine (Invitrogen; Paisley, UK) and 1%penicillin and streptomycin at 37 �C in the presence of 5% CO2 for 24 h. Twenty-fourhours later, the culture medium was collected and stored at �80�C for assaying IL-


1b, IL-6, TNF-a, and RANKL secretion. Then, calvariaewere calcinated and their ashesweighed to normalize the cytokine production values.

2.14. ELISA For IL-1b, IL-6, TNF-a, and RANKL detection

ELISA was conducted using Mix-NMatch ELISArray� kits (SABiosciences; Fred-erick, MD) coatedwith a panel of target-specific capture antibodies (TNF-a, IL-1b, IL-6, and RANKL) to determine relative cytokine levels in the organ culture superna-tant. The detection limits of the assay were 1.6 pg/mL for IL-6, 3 pg/mL for IL-1b,5.1 pg/mL for TNF-a, and 5 pg/mL for RANKL. Cytokine levels lower than thedetection limit were considered to be 0 pg/mL. Absorbance was measured using anELX800 absorbance microplate reader (Bio-Tek; USA) at 490 nm and 540 nm per themanufacturer’s instructions.

2.15. NF-kB and NFATc1 luciferase reporter gene activity assay

The effects of MLL on RANKL-induced NF-kB activation were measured usingRAW264.7 cells that had been stably transfected with an NF-kB or NFATc1 luciferasereporter construct, as previously described [31]. Briefly, cells were seeded into 48-well plates and maintained in the cell culture media for 24 h. Cells were then pre-treated with or without the MLL samples for 1 h, followed by addition of RANKL(50 ng/mL) for 8 h or 24 h. Luciferase activity was measured using the PromegaLuciferase Assay System (Promega;Madison,WI, USA) and normalized to the controlactivity level.

2.16. Confocal microscopy for NF-kB localization

RAW264.7 cells were plated at a density of 1 � 104 cells/well in 6-well platescontaining sterile cover slips and grown at 37 �C for 24 h. The medium was thenreplaced with serum-free medium, and the cells were allowed to grow for another24 h before treatment. Cells were treated with MLL for 4 h, followed by stimulationwith RANKL (50 ng/mL) for 20 min. After treatment, cells were washed twice with


Fig. 3. Magnesium leach liquor (MLL) inhibited RANKL-induced F-actin ring formation and osteoclastic bone resorption. (A) Bone marrow macrophages (BMMs) were incubatedwith M-CSF (30 ng/mL) and RANKL (50 ng/mL), followed by treatment with or without MLL samples. Cells were fixed and stained for F-actin. (B) BMM-derived pre-osteoclasts werestimulated with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 3 d. Later, cells were cultured in the presence of the indicated MLL samples and with M-CSF (30 ng/mL) and RANKL(50 ng/mL) for another 48 h. Scanning electron microscopy (SEM) images of bone resorption pits are shown. (C) Osteoclasts having actin rings were counted. (D) Resorption pit areaswere measured using Image J and are presented graphically. All experiments were performed at least three times, and significance was determined using the Student-Newman-Keul’s test (*P < 0.05; **P < 0.01).


PBS and fixed onto the cover slips by incubation in 4% PFA for 30 min. Cells werethen washed with PBS three times and permeabilized in 0.1% Triton X-100 for30 min at RT. Cover slips were blocked in 3% BSA for 1 h at RT. Antibodies targetingthe NF-kB p65 subunit (1:100) were added to the 1% BSA solution and incubatedfor 12 h at 4 �C. For nuclear staining, DAPI (Sigma; St. Louis, MO, USA) was added ata final concentration of 0.1 mg/mL and incubated for 10 min in the dark. The coverslips were then washed three times with PBS. Nuclear translocation of p65 wasimaged using a NIKON A1Si spectral detector confocal system (Nikon; Tokyo,Japan).

2.17. Western blot analysis

RAW264.7 cells were seeded at 5 � 105 cells/well into 6-well plates and treatedwith or withoutMLL samples and RANKL (50 ng/mL) for the indicated duration. Cellswere lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 50 mM

TriseHCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM

sodium vanadate, 1% deoxycholate, and protease inhibitor cocktail. The lysate wascentrifuged at 12,000 �g for 10 min, and the protein in the supernatant wascollected. Protein concentrations were measured by performing the bicinchoninic(BCA) assay. Each protein lysate (30 mg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 8e10% gels, and proteinswere then transferred to polyvinylidene difluoride membranes (Millipore; Bedford,MA, USA). Nonspecific interactions were blocked with 5% skim milk for 1 h, andmembranes were then probedwith the specific primary antibodies overnight at 4 �Cas indicated. Membranes were incubated with the appropriate secondary antibodiesconjugated with IRDye 800CW (molecular weight, 1166 Da), and antibody reactivitywas detected by exposure in an Odyssey infrared imaging system (LI-COR).


2.18. Quantitative polymerase chain reaction (PCR) analysis

For real-time PCR, 10 � 104 BMMs were seeded in each well of a 24-well plateand cultured in complete medium containing a-MEM, 10% FBS, 100 U/mL penicillin,M-CSF (30 ng/mL), and RANKL (50 ng/mL). Cells were then treated with or withoutMLL samples for the indicated durations. Total RNA was prepared using the RNeasyMini kit (Qiagen; Valencia, CA, USA) according to the manufacturer’s instructions,and cDNA was synthesized from 1 mg of total RNA using reverse transcriptase(TaKaRa Biotechnology; Otsu, Japan). Real-time PCR was performed using the SYBRPremix Ex Tag kit (TaKaRa Biotechnology) and an ABI 7500 Sequencing DetectionSystem (Applied Biosystems; Foster City, CA, USA). The detector was programmedwith the following PCR conditions: 40 cycles of denaturation at 95 �C for 5 s andamplification at 60 �C for 34 s. All reactions were run in triplicate and werenormalized to expression levels of b-actin. The following primer sets were used aspreviously described [32,33]: mouse b-actin: forward, 50-TCTGCTGGAAGGTGGA-CAGT-30 and reverse, 50-CCTCTATGCCAACACAGTGC-30; mouse NFATc1: forward, 50-CCGTTGCTTCCAGAAAATAACA-30 and reverse, 50-TGTGGGATGTGAACTCGGAA-30;mouse TRAP: forward, 50-CTGGAGTGCACGATGCCAGCGACA-30 and reverse, 50-TCCGTGCTCGGCGATGGACCAGA-30; mouse cathepsin K: forward, 50-CTTCCAA-TACGTGCAGCAGA-30 and reverse, 50-TCTTCAGGGCTTTCTCGTTC-30; mouse CTR:forward, 50-TGCAGACAACTCTTGGTTGG-30 and reverse, 50-TCGGTTTCTTCTCCTCTGGA-30 .

2.19. Statistical analysis

Values are presented as the mean � standard deviation (SD). Values were ob-tained from three or more experiments. Statistical analyses were performed using a


Fig. 4. Histomorphometric analysis of calvaria in mice treated with magnesium leach liquor (MLL) for 14 d after surgery. (A) Representative microtomography images of the calvariain each group. (B) Bone mineral content (BMC), bone mineral density (BMD), bone volume to tissue volume (BV/TV), and percentage of porosity of the whole calvaria (% totalporosity). (*P < 0.05 and **P < 0.01 compared to the vehicle control).


one-way analysis of variance (ANOVA) followed by the Student-Newman-Keul’s test.P < 0.05 was considered significant.

3. Results

3.1. Mg Concentration and pH value of the extracts

TheMg concentration in the extracts was determined during theimmersion period. As shown in Fig. 1A, the concentration of Mgions changed over time. Specifically, the Mg concentration peakedat 2 d and remained constant thereafter. As shown in Fig. 1B, the pHvalue of the MLL exhibited a similar tendency. The Mg concentra-tion, immersion time, and pH of the MLL samples used in thefollowing studies are shown in Table 1.

3.2. Effects of Mg samples on cytotoxicity

Cell viability assays were performed to examine the potentialcytotoxicity of MLL in BMMs. Our CCK-8 proliferation assay


showed that Mg-1 and Mg-2 did not affect cell proliferation overthe whole culture duration (Fig. 1C). By contrast, Mg-3 markedlysuppressed cell proliferation even after 1 d of treatment (Fig. 1C).In addition, Mg-1 and Mg-2 had no pro-apoptotic effect on BMMs.However, Mg-3 induced apoptosis in almost 30% of the cells(Fig. 1DeE). Furthermore, the RAW264.7 colony assay showed thatnone of the Mg extracts had an effect on colony formation.However, the colony area in Mg-3-treated cells was relativelylower than that of the control group (Fig. 1FeH). Based on theseresults, the non-cytotoxic samples (Mg-1, Mg-2) were used insubsequent experiments.

3.3. Effects of Mg samples on osteoclastogenesis

To investigate the effect of MLL on osteoclastogenesis, BMMswere treated with Mg-1 or Mg-2 during osteoclast formation. Asshown in Fig. 2A, the control group formed numerous TRAP-positive multinucleated osteoclasts. By contrast, the formation ofosteoclasts was inhibited after MLL treatment, as demonstrated by


Fig. 5. Histological staining of calvaria sections. (A) Representative hematoxylin and eosin (H&E)- and tartrate-resistant acid phosphatase (TRAP)-stained histological slices. (BeC)Histomorphometric analysis of the erosion area (percentage of infiltrated fibrotic area against total tissue area) was performed, and the number of TRAP-positive multinucleatedosteoclasts on the bone surface was measured (*P < 0.05, **P < 0.01 compared to the vehicle control).


the decreased number of osteoclasts. In particular, the Mg-2 groupcontained approximately 90% fewer osteoclasts than the controlgroup (Fig. 2B). To examine the effects of alkalinity and Mg onosteoclastogenesis, we first selected three NaOH-adjusted culturemedia with the same pH values as the MLL groups (7.4, 7.7, or 8.0).As shown in Fig. 2C, osteoclastogenesis was inhibited at pH valueshigher than 7.4 in both the NaOH and MLL groups. However, theMLL groups had a greater inhibitory effect on osteoclastogenesisthan the NaOH groups (Fig. 2D). Thus, to further clarify the directrole of Mg on osteoclast formation, we adjusted the pH values ofMg-1 and Mg-2 to the physiological level (7.4) while maintainingthe Mg ion concentration, and renamed as Mg-L and Mg-H(Table 1). As shown in Fig. 2E, MLL suppressed osteoclastogenesisin an Mg concentration-dependent manner. Culture media with ahigh Mg concentration had a significant inhibitory effect on oste-oclast formation. Collectively, these results demonstrate that boththe Mg and pH values in the solution contribute to the inhibitoryeffects of MLL on osteoclastogenesis.

3.4. Effects of Mg samples on F-actin ring formation

To further examine the effects of MLL on osteoclastogenesis, weexamined whether MLL affected RANKL-induced osteoclast actinring formation, which is a prerequisite for osteoclast bone resorp-tion and is the most obvious characteristic of mature osteoclastsduring osteoclastogenesis [34]. As expected, characteristic podo-somal condensation and F-actin ring formation were observed inuntreated control (Ctrl) osteoclasts as visualized by phalloidin-Alexa Fluor 647 staining and confocal microscopy (Fig. 3A). How-ever, the size and number of actin ring structures were significantly


decreased when the cells were incubated with Mg-1 or Mg-2(Fig. 3B), suggesting that MLL suppressed actin ring formation.Mg-1 and Mg-2 produced drastic alterations of F-actin ring for-mation and morphology, where F-actin tended to aggregate assmall pleomorphic rings that appeared largely unstructured andoften varied in both size and number (Fig. 3A and C).

3.5. Effects of Mg samples on osteoclastic bone resorption

Because the formation of a well-polarized F-actin ring is anessential prerequisite for efficient bone resorption by osteoclasts,we next explored the effect of MLL on bone resorption. BMM-derived pre-osteoclasts stimulated with M-CSF and RANKL for 3 dwere cultured on devitalized bovine bone discs, and the effect ofMLL on osteoclast bone resorptive function was assessed. SEMrevealed that both Mg-1 and Mg-2 effectively inhibited osteoclast-mediated bone resorption (w30% and 50% suppression, respec-tively) (Fig. 3B and D).

3.6. Effects of Mg samples on wear particles-induced osteolysis

Having established that MLL indeed inhibited osteoclast for-mation and bone resorption in vitro, we next explored its potentialprotective effects under pathological osteolysis conditions. Weutilized a titanium particle-induced mouse calvarial osteolysismodel to directly evaluate the effects of MLL against localizedparticle-induced osteolysis, in which 30 mg of titanium wear par-ticles was embedded under the periosteum at the middle suture ofthe calvaria in 8-week-old C57BL/J6 mice treated without or withMLL. Importantly, no mortality was observed during or after


Fig. 6. Magnesium leach liquor (MLL) inhibited the local expression of (A) TNF-a, (B) IL-1b, and (D) RANKL while had no obvious effect on (C) IL-6 expression. Cytokine levels in theculture mediumwere determined after 24 h by conducting an enzyme-linked immunosorbent assay (ELISA; n ¼ 5 per group). Results are presented as the mean � standard error ofthe mean (*P < 0.05, **P < 0.01 compared to the vehicle control).


particle implantation, and the mice retained normal activitythroughout the duration of the experiment. After 14 d, the micewere sacrificed and the degree of particle-induced osteolysis wasassessed using high-resolution micro-CT and histological evalua-tion. As expected, implantation of titanium wear particles inducedsevere osteolysis, as evidenced by the extensive surface erosion onthe calvaria (vehicle; PBS injection) when compared to the negativecontrol (sham; no titanium particles) (Fig. 4A). By contrast, treat-ment with Mg-1 and/or Mg-2 significantly reduced wear particle-induced bone destruction, particularly in the Mg-2 group(Fig. 4A). Quantitative analysis of bone parameters furtherconfirmed that Mg-1 or Mg-2 significantly increased the BMC,BMD, and BV/TV (Fig. 4B), and decreased the total bone porosity ofthe calvaria (Fig. 4B).

Histological assessment and histomorphometric analysisfurther confirmed the attenuation of wear particle-induced boneerosion by MLL (Fig. 5A). Wear particle injection resulted in thepresence of multiple osteoclasts along the eroded bone surface, asrevealed by staining for the osteoclast marker enzyme TRAP(Fig. 5A; black arrowheads). Consistent with the micro-CT quanti-tation, histomorphometric analysis demonstrated that both Mg-1and Mg-2 significantly reduced the bone erosion induced by thetitanium particles. In addition, the number of osteoclasts wasdecreased, although the difference was not statistically significantrelative to the Mg-1-treated group (Fig. 5B and C). Collectively,these data imply that MLL primarily disrupted osteoclast resorptionfunction, rather than osteoclast formation, in vivo (Fig. 5B and C),thus indicating that MLL may be an effective anti-resorptive agentfor the treatment of particle-induced osteolysis.


3.7. Effects of Mg samples on cytokine production

To further explore the mechanisms underlying the effect of MLLon osteolytic diseases, wear particle-induced cytokine productionwas measured. The presence of wear particles induced a significantincrease in IL-1b and TNF-a levels in the vehicle groups (Fig. 6A andB). However, particle implantation did not significantly affect theIL-6 level relative to the sham control group (Fig. 6C). The levels ofIL-1b and TNF-a were lower in the Mg-1-treated group than in thevehicle control group, although the difference was not significant,whereas Mg-2 significantly suppressed the expression of both IL-1band TNF-a. In addition, significant differences in RANKL releasewere found between the vehicle and sham groups. Furthermore,local RANKL production appeared to be greatly inhibited by MLLfollowing particle implantation (Fig. 6D).

3.8. Suppression of Mg samples on NF-kB activation

RANKL-induced NF-kB activation is essential for osteoclast dif-ferentiation and function. To determine whether MLL inhibits NF-kB-mediated osteoclastogenesis, we investigated NF-kB activationusing three different approaches in the presence of MLL. First, usingwestern blot assays, we confirmed that MLL inhibited RANKL-induced phosphorylation and degradation of the inhibitory sub-unit of NF-kB (IkBa) (Fig. 7A). To confirm the western blot data, weexamined the effect of MLL on NF-kB activity using a luciferase re-porter gene assay. As shown in Fig. 7B, the transcriptional activity ofNF-kBwas sharply increasedwhen the cellswere exposed to RANKL.This increase in NF-kB activity was inhibited by both Mg-1 and Mg-


Fig. 7. Magnesium leach liquor (MLL) inhibited RANKL-induced activation of the NF-kB and NFTAc1 signaling pathways. (A) RAW264.7 cells were seeded at 5 � 105 cells/well in 6-well plates and pretreated with or without Mg-2 for 4 h prior to RANKL stimulation (50 ng/mL) for 10 min. Cells were lysed for western blotting with specific antibodies againstphospho-IkBa, IkBa, and actin. (B) RAW264.7 cells that had been stably transfected with an NF-kB luciferase reporter construct were seeded in 48-well plates and maintained in thecell culture medium for 24 h. The cells were then pretreated with or without Mg-2 for 1 h, followed by incubation with RANKL (50 ng/mL) for 8 h. NF-kB luciferase activity wasmeasured. (C) RAW264.7 cells were plated at a density of 1�104 cells in 6-well plates and treated with Mg-2 for 4 h, followed by stimulation with RANKL (50 ng/mL) for 20 min. Thelocalization of p65 was visualized using immunofluorescence analysis. (D) RAW264.7 cells that had been stably transfected with an NFATc1 luciferase reporter construct wereseeded in 48-well plates and maintained in the cell culture medium for 24 h. The cells were then pretreated with or without Mg-2 for 1 h, followed by incubation with RANKL(50 ng/mL) for 8 h. NFATc1 luciferase activity was measured. (E) RAW264.7 cells were cultured with RANKL (50 ng/mL), and Mg-2 for 0, 1, or 3 d. Cells were then lysed forimmunoblot analysis with antibodies against NFATc1, MMP9, c-Src, and actin. (F) Fold changes in NFATc1, MMP9, and c-Src levels were normalized to the expression of actin.


2. Finally, we also performed immunofluorescence staining of theNF-kB subunit p65 with or without MLL treatment. As shown inFig. 7C, almost all p65was translocated into the nucleus after 20minof incubation with RANKL. However, nuclear translocation of p65was blocked when the cells were incubated with MLL. These resultsindicate that MLL inhibits RANKL-mediated NF-kB activation.

3.9. Suppression of Mg samples on NFATc1 expression

NFATc1 is a well-known master regulator of osteoclastogenesisand function. To determine whether MLL regulates the expressionof NFATc1, we next assessed the effects of MLL on RANKL-inducedNFATc1 expression. As shown in Fig. 7D, NFATc1 transcriptionalactivity increased when the cells were exposed to RANKL. MLL


abolished this activity when added at the beginning of the assay. Toconfirm that MLL suppressed the expression of NFATc1, we exam-ined the expression of NFATc1 at both the protein and mRNA levels.NFATc1 expression increased when cells were exposed to RANKL,andMLL abrogated the RANKL-induced increases at both themRNAand protein levels (Fig. 7E and F), suggesting that MLL suppressesRANKL-induced NFATc1 expression.

3.10. Effects of Mg samples on osteoclastogenesis-related geneexpression

Osteoclast differentiation is mediated by the expression of alarge number of genes such as those encoding MMP9, NFATc1,TRAP, c-Src, CTR, and cathepsin K, most of which are target genes of


Fig. 8. Magnesium leach liquor (MLL) suppressed RANKL-induced expression of osteoclast-specific genes. Bone marrow macrophages (BMMs) were cultured with macrophage-colony stimulating factor (M-CSF; 30 ng/mL) and RANKL (50 ng/mL) with or without Mg-2 for 0, 1, 2, 3, or 4 d. Osteoclast-specific gene expression (TRAP, CTR, Cts k, andNFATc1) was analyzed by real-time polymerase chain reaction (PCR) and the results were normalized to the expression of actin. All experiments were performed at least three times(*P < 0.05; **P < 0.01).


NFATc1 [35]. Given our findings that MLL suppressed the NF-kBpathway and NFATc1 expression, we next investigated whetherMLL also regulates osteoclastogenesis-related marker geneexpression. Our results indicate that similar to NFATc1, MLL alsoinhibits RANKL-induced protein expression of c-Src and MMP-9(Fig. 7E) and mRNA expression of TRAP, cathepsin K, and CTR dur-ing osteoclast formation (Fig. 8).

4. Discussion

Periprosthetic osteolysis and subsequent aseptic looseningremain two of the most common complications limiting the long-term durability of TJA and are the major reasons for complexjoint revision arthroplasty operations [1,36]. Extensive efforts havebeen made to improve the implant design and biomaterials inaddition to the sterilization and surgical techniques. However,these approaches are unlikely to eliminate particle generation frombearing surfaces, which stimulates a cascade of adverse biologicalreactions causing osteolysis and loosening, thereby leading tofailure of the total joint replacement [37]. Although the precisemechanism remains unclear, wear debris-induced persistentinflammation, osteoclast formation, and osteoclastic bone resorp-tion are important events in periprosthetic osteolysis [38,39].

Mg is an exceptionally lightweight metal with superior prop-erties [20]. Although several studies have found that Mg deficiencyis negatively associated with BMD [40] because of reduced osteo-blastic activity [41], retarded bone differentiation, and matrixcalcification [42], the role of Mg in osteoclast formation remainsunclear. In the present study, for the first time, we demonstratedthat MLL inhibits osteoclast formation through the effects of bothalkalinity and Mg ion concentration. In addition, MLL also impaired


F-actin ring formation and thus suppressed resorptive activityin vitro. In vivo, MLL significantly reduced titanium particle-inducedosteolysis, which was consistent with the decline in osteoclast ac-tivity and cytokine levels (e.g., TNF-a and IL-1b). Meanwhile, in ourexperiment titanium remained stable and had no pharmacologicalinteraction with Mg. Our results show that MLL inhibits the phos-phorylation and degradation of the NF-kB inhibitory subunit IkBaand suppresses the nuclear translocation and activation of the p65subunit of NF-kB, which was further supported by the observedreduction of NF-kB activity. The inhibition of the NF-kB signalingpathway in osteoclasts is consistent with previous studies [43]. Inaddition, NFATc1 is well-known as a master transcription factorthat is closely regulated by NF-kB activity [44,45]. Here, we showedthat MLL inhibits NFATc1 transcriptional activity and expression atboth the protein and mRNA levels. Since the activation and nucleartranslocation of NFATc1 are mediated by a specific phosphatase,calcineurin, which is activated by the RANKL-mediated calcium-calmodulin signaling pathway, thus the natural calcium antagonistand potent L-type calcium channel inhibitor effects of Mg [46] mayunderlie the inhibitory effect of MLL on NFATc1 that we observed.NFATc1 regulates the expression of genes associatedwith osteoclastdifferentiation and function, such as the TRAP, MMP9, CTR,cathepsin K, and c-Src genes. In this study, we examined theexpression of NFATc1-regulated genes, such as TRAP, CTR, andcathepsin K, and found that they were all down-regulated by MLL,suggesting that MLL affects not only the expression of NFATc1 butalso the expression of its downstream genes.

In summary, for the first time, our results demonstrate that MLLsuppresses osteoclastogenesis and osteoclast activity in vitro andin vivo. Moreover, we found that the inhibitory effects of MLL mayoccur through: (1) blockade of the NF-kB and NFATc1 pathways


Fig. 9. Schematic diagram of the mechanism by which magnesium leach liquor (MLL) inhibits osteoclast differentiation and function. The RANKL-RANK-activated NF-kB signalingpathway induces NFATc1 gene expression, and Ca2þ-dependent calcineurin signaling plays a critical role in NFATc1 auto-amplification. Both of these pathways regulate theexpression of osteoclastogenesis-related genes such as TRAP, CTR, MMP9, NFATc1, c-Src, and cathepsin K. MLL blocks the NF-kB signaling pathway and may also prevent Ca2þ-dependent calcineurin signaling, subsequently suppressing NFATc1 expression and auto-amplification. Finally, MLL blocks osteoclast differentiation and bone resorption.


(Fig. 9), (2) the alkalinity provided byMLL, and (3) down-regulationof wearing particle-induced inflammatory molecules, includingTNF-a, IL-1b, and RANKL.

5. Conclusion

MLL derived from pure Mg incubated in culture mediuminhibited osteoclastogenesis and titanium particle-inducedosteolysis though inhibition of local bone resorption and inflam-matory cytokine release. These results suggest that metallic mag-nesium has significant potential for the treatment of osteolysis-related diseases caused by excessive osteoclast formation andfunction.

Acknowledgments

This study was supported by Key National Basic Research Pro-gram of China (Grant No. 2012CB619101), Major Basic Research ofScience and Technology Commission of Shanghai Municipality(Grant No. 11DJ1400303), a major basic research grant from theScience and Technology Commission of Shanghai Municipality(Grant No. 11DJ1400303), National Natural Science Foundation ofChina (Grant No. 81190133), a scientific research grant from theNational Natural Science Foundation for the Youth of China (GrantNo. 81201364), a scientific research grant for Youth of Shanghai(Grant No. ZZjdyx 2097), a scientific research grant from 985project–stem cell and regenerative medicine centre and an inno-vative research grant from the Shanghai Municipal Education


Commission (Grant No. 13YZ031), Chinese Academy of Sciences(No. XDA01030502), Doctoral Innovation Foundation fromShanghai Jiaotong University School of Medicine (BXJ201330).

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