Materials & Methods
The NCBI Gene Expression Omnibus (GEO) is a public data repository that stores gene expression profiles, raw series, and platform records ( https://www.ncbi.nlm.nih.Gov/geo/ ). The subchondral bone dataset, GSE51588 , included 10 normal and 40 OA samples. The platform used was GPL13497 . The number of datasets for subchondral bone samples was limited. Only GSE51588 was available for human samples, and we chose OA-Normal (10:40) for validation. The 40 OA samples included medial tibia samples (MT, significant degeneration, lesioned) and lateral tibia samples (LT, minimal degeneration, preserved). The second dataset for subchondral bone samples in GEO datasets was GSE30322 , but it consisted of Sprague Dawley (SD) rats for OA-Normal (15:15). Additionally, GSE110606 contained human chondrocytes for OA-Normal (1:1), while GSE114007 included human cartilage samples for OA-Normal (20:18). Lastly, GSE55457 and GSE55457 comprised synovial tissue samples, including OA, Normal, and rheumatoid arthritis subgroups. We chose OA-Normal samples from each dataset for validation (10:10). The OA samples were used for further training analyses ( Table 1 ). The data matrix series were downloaded, and the limma package in R software was used to identify differentially expressed genes (DEGs) ( Ritchie et al., 2015 ). P <0.05 and —logFC2—>1 were the cutoff criteria.

The Database for Annotation, Visualization, and Integrated Discovery (DAVID; https://david.ncifcrf.gov/ ) provides a comprehensive set of functional annotation tools and helps investigators understand the biological meaning behind large lists of genes. Gene Ontology (GO) annotation covers gene cell components (CCs), molecular functions (MFs), and biological processes (BPs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis extracts pathway information from molecular interaction networks. In the present study, the DAVID online tool was used for GO and KEGG pathway enrichment analyses of the upregulated and downregulated genes. R language was used for data visualization, and P <0.05 was considered to indicate significant GO and KEGG terms.

The construction of protein-protein interaction (PPI) networks is critical for understanding cell biology and interpreting genomic data. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://www.string-db.org/ ) is an online biological database and website designed to construct PPI networks in molecular biology. A PPI network was constructed using the STRING database to understand the DEGs more in-depth. The DEGs (species: Homo sapiens ) were mapped to the STRING database (PPI score > 0.4), and those with a combined score > 0.99 were identified via Cytoscape software 3.7.1 ( http://www.cytoscape.org/ ) to visualize the PPI network. The top 10 hub genes were identified via the maximal clique centrality (MCC) method.

This study was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (approval number: ky2020-078), and we obtained written informed consent from the participants. Six GSE datasets containing data from the joint tissues of OA patients were used for validation to screen coexpressed genes in multiple tissues. The DEGs in the associated datasets, including GSE51588 (OA-Normal for subchondral bone samples), GSE30322 (subchondral bones of SD rat OA models), GSE110606 and GSE114007 (chondrocyte and cartilage samples), and GSE55235 and GSE55457 (synovial tissues), were used in this study to screen for coexpressed genes that aligned with the expression profile of the OA subchondral bone samples ( Table 1 ). Differentially expressed genes (—logFC2—>1, P <0.05) were considered for further validation. A Venn diagram was constructed to display the results.

The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (approval number: ky2020-078), and all of the patients provided informed consent. Total knee placement samples were collected. Ethical approval and consent for the use of resected tissue were obtained. Subchondral bone samples were obtained and stored in liquid nitrogen until analysis. Cartilage and synovium samples were cut into small pieces (∼1 mm 3 ) and transferred into 0.2% collagenase II and 0.4% collagenase I solutions, respectively. The samples were then incubated for 4 h at 37  °C. Then, α -MEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin was added, and the samples were incubated at 37 °C with 5% CO 2 until they reached >80% confluence. We discarded the cell debris three days later and passaged the cells three times to eradicate residual contaminating macrophages. The resulting monocultures of synovial fibroblasts (SFs) were used for experiments until passage 8 ( Pérez-García et al., 2016 ). Passage 2 chondrocytes were used for experiments. SF and chondrocytes were cultured in 6-well plates, and 10 ng/ml IL-1 β was added to each well for 48 h for further analyses.

Total RNA from SF, chondrocytes, and subchondral bone was obtained using a reagent (ES Science Technique, Shanghai, China). RNA was reverse-transcribed using the ES Science cDNA Reverse Transcription Kit (ES Science Technique, Shanghai, China), and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed with the ES Science SYBR Green Kit (ES Science Technique, Shanghai, China). The primers used are listed in Table 2 . Per the manufacturer’s instructions, 1 µg of total RNA was added to 2 µL of DNA enzyme and diluted with ddH 2 O to a final volume of 16 µL. The mixture was incubated at 25 °C for 5 min to remove genomic DNA. Then, 4 µL of 5 × RT mix (10 U/µL) was added to 16 µL of total RNA treated with DNA enzyme for reverse-transcription at 42 °C for 15 min. A qPCR system (20 µL) was prepared as follows: 10 µL of 2x Super SYBR Green qPCR Master Mix (0.05 U/µL), 2 µL of template, 0.4 µL each of forward (10 µmol/L) and reverse (10 µmol/L) primer, and ddH 2 O to obtain a final volume of 7.2 µL. qRT–PCR was performed as follows: one cycle of 95 °C for 5 min, 40 cycles of 95 °C for 10 s, and 40 cycles of 60 °C for 30 s. For relative quantification, we normalized the target gene expression to that of the housekeeping gene (GAPDH). The results are presented as the relative expression with respect to the untreated condition using the formula 2 −ΔΔCt . The experiment was repeated at least three times. The data were analyzed with GraphPad Prism 7.0 (GraphPad Software, Boston, MA, USA). Differences were assessed using Student’s two-tailed t test, and P < 0.05 was considered to indicate significance.

xCell is a novel and robust method based on single sample gene set enrichment analysis (ssGSEA) that estimates the abundance scores of 64 cell types in the microenvironment. There are two approaches to running xCell: one is the online website tool ( https://xCell.ucsf.edu/ ), and the other is the xCell package in R software. We estimated the abundance scores of 64 cell types in lesioned and preserved subchondral bone samples using the online tool. P <0.05 was considered to indicate significance. The immune cell scores were also calculated for each sample. The relationships among immune cells were calculated using the Pearson coefficient. The “ggplot2″package was used to construct violin diagrams to visualize the differences in immune cell infiltration. A flow chart depicting the study process for the OA gene expression analysis is shown in Figure 1 .