Recombinant anxa1

The hFLS from normal healthy human synovial tissues purchased from Cell Applications (Cell Applications) were cultured according to the supplier’s recommendations. Cultured cells were stimulated with UHMWPE debris at a density of 0.1 mg/cm³ for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂ using an inverted culture system^{3 (link)}. In separate experiment, cultured hFLS cells were stimulated with recombinant human TNF-α (Peprotech) in the presence or absence recombinant AnxA1 (R&D Systems).

Human fetal osteoblasts purchased from Cell Applications (San Diego, CA, USA) were cultured in osteoblast growth medium (Cell Applications). Cells were differentiated in osteoblast differentiation medium (Cell Applications) for 14 days and cultures were regularly replenished with fresh media every 3 days^{40 (link)}. Cells were stimulated with either recombinant human TNF-α (Peprotech) for 48 h in the presence or absence recombinant AnxA1 (R&D Systems).

Human monocytes were cultured in a growth medium supplemented with recombinant macrophage colony-stimulating factor RANKL plus 100 ng/mL recombinant AnxA1 (R&D systems). We next analyzed the transcriptional profiling of stimulated cells for 8 days by RNA sequencing. An average of 69 million reads (paired-end reads of 101 bp) per sample were mapped by the STAR software and read count was determined using the RSEM software. Significant differences were calculated using DESeq2 R package (https://www.r-project.org/). Analyses were performed using the Database for Annotation Visualization and Integrated Discovery online tools (DAVID: david.abcc.ncifcrf.gov). Heat map was used to visualize the differences in fold changes in each enriched GO term (http://biit.cs.ut.ee/clustvis/). The RNA-seq data are publicly available at the Gene Expression Omnibus (GEO) database (http: www.ncbi.nlm.nih.gov/geo/) under the accession numbers (GSE183145) and (GSE171542)^{38 (link)}.