Results
Differential gene expression analysis was performed on OA and normal subchondral bone samples. There were 1,010 DEGs in the GSE51588 MT-LT cohort, 423 upregulated and 587 downregulated. The most upregulated genes were STMN2 (logFC = 5.52924, P < 0.05) and POSTN (logFC = 3.984087, P < 0.05), and the most downregulated genes were LEP (logFC = −4.41131, P < 0.05) and APOB (logFC = −4.00156, P < 0.05). The distribution of all DEGs according to the two dimensions of −log10 ( P value) and logFC is represented by a volcano map ( Figure 2A ). The DEGs were evaluated using a heatmap ( Figure 2B ). The detailed logFC and P values of the DEGs are shown in the supplementary data.

GO annotation and KEGG pathway enrichment analyses were performed to better understand the functional significance of the DEGs. Functional enrichment analysis of the upregulated DEGs revealed that the top five biological processes were skeletal system development, sister chromatid cohesion, ossification, mitotic nuclear division, and extracellular matrix organization ( Figure 3A ). The main cellular components and molecular functions involved included spindle microtubules, proteinaceous extracellular matrix, platelet-derived growth factor binding, and metalloendopeptidase activity ( Figs. 3B – 3C ). KEGG pathway analysis of the upregulated DEGs revealed that the top five terms were associated with the Wnt signaling pathway, proteoglycans in cancer, protein digestion and absorption, the PI3K-Akt signaling pathway, and the Hippo signaling pathway ( Figure 3D ).

The functional enrichment analysis of the downregulated DEGs revealed that the top five BP terms were associated with xenobiotic metabolic process, triglyceride biosynthetic process, positive regulation of inflammatory response, positive regulation of fat cell differentiation, and positive regulation of B-cell activation ( Figure 4A ). The main cellular components and molecular functions were associated with the receptor complex, plasma membrane, transporter activity, and retinol dehydrogenase activity ( Figs. 4B – 4C ). KEGG pathway analysis of the downregulated DEGs revealed that the top terms were associated with tyrosine metabolism, regulation of lipolysis in adipocytes, the PPAR signaling pathway, neuroactive ligand–receptor interaction, and the AMPK signaling pathway ( Figure 4D ).

Using the STRING database, we constructed a putative PPI network map for the relationships between the DEGs, which was visualized with Cytoscape. Finally, a total of 1,010 DEGs were mapped to 5,766 nodes in the network with a combined score >0.4. Network interactions with a score >0.99, including 116 nodes, were visualized with Cytoscape software ( Figure 5A ). The top 10 hub genes identified by the MCC included JUN, TNF, IL-1 β , LEB, CXCL8, and FN1 ( Figure 5B ).

Six multiple-tissue datasets, including subchondral bone, cartilage, and synovium samples, were used for validation. Two coexpressed genes, LRRC15 and Col11A1, were screened out. A Venn diagram was constructed to visualize the results ( Figure 6 ). The LRRC15 and Col11A1 genes were upregulated in all datasets. To confirm the results of our bioinformatics analysis, we conducted qRT–PCR to validate the expression of the genes coexpressed with chondrocyte, fibroblast-like synovial (FLS), and subchondral bone samples. LRRC15 and Col11A1 expression levels were higher in the OA group than in the normal group ( Figs. 7A – 7C ).

The immune response in subchondral bones is a significant factor in OA development. Multiple immune cells participate in the maturation and activation of osteoblasts through highly complex mechanisms ( Luo et al., 2023 ). We analyzed immune infiltrating cells using the xCell algorithm to assess differential immune cell infiltration between OA and normal tissues. A correlation heatmap of the 64 types of immune cells revealed that ly endothelial cells, mv endothelial cells, and endothelial cells were significantly positively correlated. CD4+ T cells and CD4+ Tem cells also demonstrated a positive correlation. ( Fig. S1A ). A heatmap was constructed of the abundance scores of the immune cells in each sample ( Fig. S1B ).

A violin plot of the differential immune cell infiltration levels revealed that fewer adipocytes and endothelial cells infiltrated in the lesioned samples versus the preserved samples, whereas MSCs, myocytes, plasma cells, Th2 cells, ly endothelial cells, and mv endothelial cells infiltrated more ( Figs. 8A – 8H ).