Oxidative stress-driven cellular heterogeneity in Knee Osteoarthritis: scRNA-seq pinpoints SPP1 and CYBA for diagnostic development

Knee osteoarthritis (KOA) is a progressive degenerative joint disease characterized by cartilage degeneration, synovial inflammation, and subchondral bone remodeling. Oxidative stress plays a critical role in KOA pathogenesis. This study aimed to identify oxidative stress–related hub genes by integrating single-cell RNA sequencing (scRNA-seq) with machine learning to uncover potential diagnostic and prognostic biomarkers for KOA. ScRNA-seq data from KOA cartilage tissues were obtained from the GEO database to characterize cellular heterogeneity and identify disease-associated chondrocyte subsets. CellChat was used to analyze intercellular communication, and Monocle2 was applied to reconstruct cell differentiation trajectories. Oxidative stress activity at the single-cell level was evaluated using AUCell, while high-dimensional weighted gene co-expression network analysis (hdWGCNA) was performed to identify oxidative stress–related gene modules. Candidate genes were screened using Venn analysis and further refined by LASSO regression, SVM-RFE, and XGBoost combined with SHAP analysis. Diagnostic performance was assessed, and biological relevance was explored through immune infiltration and pathway analyses. Experimental validation was conducted using qRT-PCR and western blot assays in H ₂ O ₂ -treated chondrocytes.

InfC and ProFC were detected specifically in OA cartilage and exhibited high oxidative stress activity. InfC was enriched in inflammatory and immune-related pathways, whereas ProFC was associated with cell cycle regulation and extracellular matrix remodeling. Integrated hdWGCNA and machine-learning analyses consistently identified CYBA and SPP 1 as candidate genes. The two-gene model achieved bootstrap-corrected AUCs of 0.937 and 0.825 in the training and validation cohorts, respectively. qRT-PCR and western blot analyses further showed increased CYBA and SPP1 expression in H ₂ O ₂ -treated chondrocytes.

Conclusion

InfC and ProFC may represent OA-associated chondrocyte states characterized by enhanced oxidative stress, while CYBA and SPP 1 may serve as candidate diagnostic biomarkers. Given the small cohort sizes and preliminary in vitro validation, these findings require confirmation in larger independent cohorts and functional studies.