Trypsin solution

Chitosan (200–600 mPa·s; 0.5% in 0.5% acetic acid at 20 °C, Tokyo Kasei Kogyo, Tokyo, Japan), genipin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), acetic acid (FUJIFILM Wako Pure Chemical Corporation, Japan), disodium hydrogen phosphate (Na₂HPO₄; FUJIFILM Wako Pure Chemical Corporation, Japan), sodium dihydrogen phosphate (NaH₂PO₄; FUJIFILM Wako Pure Chemical Corporation, Japan), Giemsa Stain Solution (Merck, Darmstadt, Germany), 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation, Japan), Dulbecco’s PBS (PBS; Sigma-Aldrich, St. Louis, MO, USA), polyetherurethane (PU) film containing 0.1% zinc diethyldithiocarbamate (ZDEC) (ZDEC-PU; Hatano Research Institute, Hadano, Japan), PU film containing 0.25% zinc dibuthyldithiocarbamate (ZDBC) (ZDBC-PU; Hatano Research Institute, Hadano, Japan), high-density polyethylene sheet (HDPE; Hatano Research Institute, Hadano, Japan), Chinese hamster lung fibroblast V79 cells (V79 cells; RIKEN RCB0008 and Japan Health Sciences Foundation JCRB0603), Eagle’s minimum essential medium (MEM; Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS; Corning Cellgro, Manassas, VA, USA), 0.5% and 0.25% trypsin solution (FUJIFILM Wako Pure Chemical Corporation, Japan and Thermo Scientific, Carlsbad, CA, USA, respectively), and CELL COUNTING KIT-8 (Dojindo Laboratories, Kumamoto, Japan) were used in this study.

Cells and homogenized tissues were lysed using TRIzol Reagent (Invitrogen) for RNA extraction. To obtain RNA from calvarial bones following implantation of wear particles, frozen samples were crushed using tissue homogenizer (Power Masher II, Nippi, Tokyo, Japan) in liquid nitrogen. Cells (1 × 10⁶) from the granulomatous tissue around UHMWPE particles were obtained on day 7 and then digested by trypsin solution (Wako) for 10 min in 37°C-water bath. Thereafter, chloroform (Wako) was added for phase separation and the DNA-free RNA was purified from the aqueous layer using RNeasy Plus Mini kit columns (Qiagen, Hilden, Germany). Purified RNA (0.5 μg) was reverse transcribed using the GoScriptTM reverse transcriptase kit (Promega, Madison, USA) in order to carry out a qRT-PCR analysis. The cDNAs were assayed using the SYBR^® Premix Ex Taq™ II (Takara, Shiga, Japan) and gene-specific primers listed in Supplementary Table (13 (link)). Specific primers were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify 60–100 bp of the genes. The gene expression was calculated by the ^2−ΔΔCt method after normalizing to the expression of the housekeeping genes, including GAPDH and β-actin. Amplification efficiencies of the target and reference genes ranged between 90 and 110%.