Reagents and cell preparation
TUS (purity >98%, Fig. 3. A) was purchased from Dalian Meilun Biotechnology (Liaoning, China) and dissolved in alpha modification of minimum essential medium (α-MEM) at the concentration of 0.2 M as storage solution stocking at 4℃. The α-MEM medium was obtained from Gibco-BRL (Gaithersburg, MD, USA). The fetal bovine serum (FBS), penicillin /streptomycin, soluble mouse recombinant M-CSF and RANKL were acquired from R&D Systems (Minneapolis, MN, USA). The cell counting kit (CCK-8) was obtained from Dojindo Molecular Technology (Japan) The DMSO and TRAP staining kit was obtained from Sigma-Aldrich (St. Louis, MO, USA).
RAW264.7 cells was purchased from American Type Culture Collection (Rockville, MD, USA) and were incubated in α-MEM supplemented with 10% FBS and 1% penicillin/streptomycin, namely complete α-MEM. 4 to 6 week-old C57BL/6 mice were acquired from Shanghai Laboratory Animal Company (SLACCAS, Shanghai, China). Primary BMMs were separated from the whole bone marrow of murine femurs and tibias and cultured in complete α-MEM with the addition of 30 ng/ml M-CSF. All cells we used in this study were incubated in the incubator with constant high humidity, 37 ℃ and 5% CO2 atmosphere [26]. Non-adherent cells were removed before each passage.
Cell viability
The cytotoxic effects of TUS on RAW264.7 and BMMs cells were assessed using a CCK-8 assay according to the instruction book. RAW264.7 or BMMs cells were plated in 96-well plates at a density of 3 × 103 cells/well for adhesion overnight. Cells were then treated with diverse concentrations of TUS (0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 or 100 uM) for 48 h or 96 h. A 10 μl volume of CCK-8 buffer was added to each well, and cells were incubated at 37 ℃ for an additional 1 h. Then, the optical density (OD) was detected at a wavelength of 450 nm (650 nm reference) on an ELX800 absorbance microplate reader (Bio-Tek, USA). Cell viability was calculated relative to control using the following formula: (experimental group OD - zeroing OD)/(control group OD - zeroing OD) [27].
Osteoclast formation assay
To investigated the effect of TUS on osteoclast formation in vitro, RAW264.7 or BMMs cells were seeded in 96-well plates respectively at a density of 2.5×103 cells/well and then cultured for adhesion overnight. After confirming the healthy condition of the cells, they were cultured with complete α-MEM with 50 ng/mL of RANKL (30 ng/mL of M-CSF for BMMs cells solely) and different non-cytotoxic concentrations of TUS (0, 6.25, 12.5, 25 uM), until osteoclast formation was clearly observed 5 days later. The osteoclasts were fixed with 4% paraformaldehyde for 20min and then stained for TRAP. TRAP-positive cells with more than five nuclei were counted as osteoclasts.
Bone resorption pit evaluation
RAW264.7 cells were plated onto the surface of sterile bovine bone slices in 96-well plates at a density of 3×103 cells/well and then cultured complete α-MEM containing 50 ng/mL of RANKL (30 ng/mL of M-CSF for BMMs cells solely) and different concentrations of TUS (0, 6.25, 12.5, 25 uM). After confirming osteoclast formation, the cells were treated with complete culture medium supplemented with 30 ng/mL of M-CSF, 50 ng/mL of RANKL, and different TUS concentrations for another 48 h. Finally, bone resorption effect on the bovine bone slices was evaluated using scanning electron microscope (SEM; FEI Quanta 250).
RNA extraction and quantitative PCR analysis
Quantitative PCR was applied to measure the expression of osteoclast specific genes. We seeded BMMs in 24-well plates at a density of 1×105 cells/well and treated them with complete α-MEM, 50 ng/ml RANKL and 30 ng/ml M-CSF. Cells were administered with different concentrations of TUS (0, 6.25, 12.5 or 25 uM). Then, we extracted RNA with the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the instruction book. Next, we synthesize cDNA from 1 mg of total RNA using reverse transcriptase kit (TaKaRa Biotechnology, Otsu, Japan). The SYBR Premix Ex Tag kit (TaKaRa Biotechnology) and an ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA, USA) was used in qPCR. PCR conditions were 40 cycles of denaturation at 95 °C for 5s and amplification at 60 °C for 34s [26]. The reactions were conducted in triplicate. The mice primer sequence set is listed in Table 1.
NF-κB luciferase reporter gene assay
To examine whether TUS affected NF-κB gene expression, RAW264.7 cells were stably transfected with a p-NF-κB-TA-Luc luciferase reporter construct. Concisely, cells were plated in a 24-well plate at a density of 1 × 105 cells/well. 24 hours later, cells were pre-treated with different concentrations of TUS (0, 6.25, 12.5, 25 uM) for 1 h, prior to incubation with 50 ng/mL RANKL for another 8 h. Cells were then lysed and luciferase activity was measured using the Promega Luciferase Assay System according to the instruction book [15].
Western blotting analysis
We seeded RAW264.7 cells in 6-well plates at a density of 5 × 105 cells/well. When the cells were confluent, they were pre-treated with or without 25 uM TUS for 4 h. Cells were then incubated with 50 ng/mL RANKL for 0, 5, 10, 20, 30, or 60 min. Total proteins were extracted from cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Well Biology, Changsha, China) with protease inhibitor cocktail. Next, the lysates were centrifuged at 12,000 g for 15 min, and the supernatants that contained the proteins were collected. Protein concentrations were measured with bicinchoninic acid assay (Well Biology).
We resolved 30 mg of each protein lysate using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels, and transferred products to polyvinylidene difluoride (PVDF) membranes. Following transfer, membranes were blocked with 5% skimmed milk powder in TBS-Tween (TBS: 0.05 M Tris, 0.15 M NaCl, pH 7.5, and 0.2% Tween-20) for 1 h, and then incubated with primary antibodies overnight at 4 ℃ [15]. Membranes were then rinsed with TBS-Tween and incubated with the corresponding secondary antibodies conjugated with IRDye 800CW (molecular weight 1162 Da) for 2 h. Bands were detected through the Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NE, USA). Quantitative analysis of bands’ intensity was performed by image J software.
Titanium particle-induced calvarial osteolysis model
Protocols for this study is censored by the Medical Ethics Committee of the Second Xiangya Hospital, Central South University and all experiment procedures were performed in accord with the related recommendations of guiding principles.
We established a murine calvarial osteolysis model to determine the preventative effects of TUS on osteolysis in vivo. In short, 16 healthy 8-week-old C57BL/6 mice (weight: 21.47 ± 1.22 g) were bred in specific pathogen-free (SPF) plastic-isolator cages assigned randomly into 3 groups: sham PBS control (sham), titanium (Ti) particles with PBS (vehicle), and Ti particles with 10 mg/kg/day of TUS (TUS group). To remove endotoxins adherent on Ti particles, commercial pure Ti particles were sterilized by baking at 180 ℃ for 6 h, followed by treatment of 70% ethanol for 2 days. The murines were anesthetized, and the cranial periosteum was separated from the calvarium by sharp dissection. Then, 30 mg of Ti particles were planted under the periosteum at the middle suture of the calvarium [15]. Mice in TUS group were injected intraperitoneally with TUS at 10 mg/kg/day for 8 weeks. Mice in the sham and vehicle groups received PBS daily. At the end of the experiment, the mice were sacrificed, and the calvaria were excised and fixed in 4% paraformaldehyde for micro-computed tomography (CT) analysis.
Micro CT scanning
The fixed calvaria were observed using a high-resolution micro-CT system (μCT50, Scanco, Switzerland). The scanning protocol was set at an isometric resolution at 8.3 mm, and X-ray energy settings of 80 kV and 80 mA. After reconstruction, a square ROI around the midline suture was selected for further qualitative and quantitative analysis. BV/TV, the number of porosity and percentage of total porosity of each sample were analyzed.
Histomorphometric analysis
After micro-CT scanning, the calvaria samples were decalcified in 10% ethylene diamine tetraacetic acid (EDTA) for 3 weeks, and then embedded in paraffin. Histological sections were preconditioned for TRAP and hematoxylin and eosin (H&E) staining. The specimens were then examined and photographed under a high quality microscope. The number of TRAP-positive multinucleated osteoclasts was counted in each sample.
Statistical analysis
The data were expressed as means ± SD. Results were analyzed using Student’s t-test using the SPSS 13.0 software (SPSS Inc., USA). P < 0.05 indicated a significant difference between groups.