Single‐Cell Analysis of Cartilage‐Derived Cells Identifies Diverse Populations and a Cholesterol/ER‐Stress Chondrocyte Subset in Knee OA

This study aimed to elucidate the cellular and molecular mechanisms underlying knee osteoarthritis pathogenesis by analyzing chondrocyte populations and their spatial distribution using single-cell RNA sequencing (scRNA-seq). Knee cartilage cells underwent scRNA-seq. Cellular clusters were identified via gene expression profiles and annotated using gene ontology analyses. Correlations between cluster proportions and patient background characteristics were evaluated. Differentiation trajectories were evaluated using pseudotime analysis. Differentially expressed genes between osteoarthritis and non-osteoarthritis samples were identified, and their functional relevance was experimentally assessed. Cartilage stratification analysis was also performed. A total of 104,951 cells from 12 patients (9 osteoarthritis, 3 non-osteoarthritis) were analyzed. Initially, 11 clusters were identified; three non-chondrocytic clusters (mesenchymal stromal cell-like cells, blood cells, fibroblasts) were identified and excluded from chondrocyte analysis. Eight chondrocyte subpopulations were annotated, including a novel stress-responsive metabolic chondrocytes (MetabCs) associated with serum total cholesterol levels and linked to cholesterol metabolism and endoplasmic reticulum stress pathways. Correlation and trajectory analyses revealed two paths: one toward fibrocartilage chondrocytes (FCs) associated with aging and another toward MetabCs associated with osteoarthritis progression. Novel osteoarthritis-related genes identified included chondroprotective HILPDA and destructive AKR1C2. Stratification analysis indicated that worn cartilage chondrocytes exhibited a hybrid phenotype, shifting toward upper-zone features while retaining deep-zone characteristics. In conclusion, a novel chondrocyte cluster was identified as implicated in osteoarthritis pathogenesis, where elevated serum cholesterol levels potentially increase endoplasmic reticulum stress response, promoting MetabC formation. Chondrocytes altered their phenotype in response to spatial distribution changes caused by cartilage thinning.