Mmscs

Mouse MSCs (mMSCs; Cyagen, Guangzhou, China) were cultured in MSC basal media, including MSC growth supplement (MCGS), L-glutamine, penicillin, and streptomycin at 37 °C and 5% CO₂. For differentiation experiments, cells were cultured with mMSC Differentiation Basal Medium-osteogenic containing β-glycerophosphate, dexamethasone, ascorbic acid, MCGS, L-glutamine, penicillin, and streptomycin. After confluence, the cells were treated with 10⁻¹⁰ M or 10⁻⁸ M icariin (Sigma, St. Louis, MO, USA) with/without 10 μM Wnt/β-catenin inhibitor ICG-001 (Tocris Bioscience, Bristol, UK), starting 4 h before treatment with 0.1 mg/mL Ti particles. The concentration of Ti particles used for incubation was similar to that of wear debris retrieved from periprosthetic tissues36 (link). In addition, 10⁻¹⁰ M and 10⁻⁸ M icariin significantly enhanced osteoblast differentiation without cytotoxicity as previously described10 (link).

mMSCs and MLE-12 cells were used in the present study. mMSCs, obtained from Cyagen Biosciences Inc. (Guangzhou, China), were isolated from the bone marrow of C57BL/6 mice. The cells were verified as mesenchymal stem cells according to the identification of cell surface phenotypes (CD34+, CD44+, CD29+, SCA-1+ and CD117−) and the multipotent differentiation potential along the adipogenic, osteogenic, and chondrogenic lineages offered by the supplier [10] (link). MLE-12 cells, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and mMSCs were cultured in Dulbecco's modified Eagle media/nutrient F-12 (DMEM/F12) 1∶1 mixture (Thermo Scientific Hyclone, Beijing, China) supplemented with 2% (for MLE-12 cells) or 10% (for mMSCs) fetal bovine serum (FBS; Wisent Inc., St-Bruno, Quebec, Canada), 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Scientific Hyclone) in a humidified 5% CO₂ incubator at 37°C. The culture media was changed every 3 days, and the cells were passaged when they reached 90% confluency.

MMSCs (Cyagen Biosciences Inc., Guangzhou, China) cultured in MSC basal media supplemented with MSC growth supplement (MCGS), 1% l-glutamine and 1% penicillin–streptomycin in a 5% CO₂ incubator at 37 °C were treated with or without 20 or 40 μM RSV before incubation with 2 mg PMMA particles. MMSCs cultured in medium served as control. After 72 h, the expression levels of KCNQ1OT1, β-catenin, Runx2, Osterix and OCN were determined using qRT-PCR and western blotting. The ALP activity was also measured. In addition, ARS staining was used to examine the differentiation of MMSCs after 21 days.
For further analyzing the biological functions of RSV in osteolysis, MMSCs treated with 40 µM RSV were incubated with or without ICG-001 (10 µM), a selective Wnt/β-catenin inhibitor (Tocris Bioscience, Bristol, UK) for 4 h prior to addition of 2 mg PMMA particles. In addition, MMSCs in media (namely control group) and MMSCs only treated with 2 mg PMMA particles (namely PMMA group) were used as negative controls. Three days later, ALP activity and the expression levels of Runx2, Osterix and OCN at mRNA levels were determined. The differentiation of mMSC was investigated using ARS staining after 21 days.