Single-Cell Resolution Mapping Reveals Cell Death-Associated Stress Programs and Osteogenic-Chondrogenic Differentiation Imbalance in Subchondral Bone Mesenchymal Stem Cells of Knee Osteoarthritis.

BACKGROUND: KOA is a degenerative joint disorder characterized by progressive cartilage degradation and subchondral bone remodeling. Subchondral bone MSCs orchestrate tissue homeostasis through osteogenic and chondrogenic differentiation, yet the mechanisms underlying their lineage imbalance in OA pathogenesis remain poorly understood.

METHODS: We reanalyzed publicly available human tibial-plateau subchondral-bone single-cell RNA-sequencing and bulk-expression datasets using library-aware integration, donor-paired regional pseudobulk summaries, diffusion pseudotime, candidate cell-cell interaction analysis, and weighted gene co-expression analysis. Quantitative RT-PCR in four ATCC cell lines (NHAC, SW1353, MG-63, and U2OS) assessed ferroptosis-related gene expression (GPX4, SLC7A11, ACSL4, and TFRC). Immunofluorescence with COL2A1 co-staining and Western blotting assessed protein-level changes across four experimental groups (Control, OA, OA + OE-GPX4, and OA + si-GPX4).

RESULTS: After quality control, 25,885 of 31,090 cells were retained and assigned to 12 marker-supported display labels. The single-cell analysis defined eight molecular program modules, whereas the bulk analysis identified six data-derived coexpression blocks. Donor-paired regional analyses characterized observed composition and expression differences, and diffusion pseudotime organized 749 mesenchymal/perivascular stromal cells without establishing lineage conversion. Bulk OA-versus-Normal analysis identified 1,805 differentially expressed genes, and the exploratory cross-platform comparison showed no global monotonic correspondence (Spearman rho = 0.009, p = 0.282). Quantitative RT-PCR showed lower GPX4 and SLC7A11 and higher ACSL4 and TFRC in OA-like chondrocytes, whereas osteosarcoma lines showed higher GPX4, SLC7A11, and TFRC. Immunofluorescence and Western blotting showed corresponding GPX4, SLC7A11, and ACSL4 changes across the four experimental groups, with COL2A1 co-staining providing a matrix-associated readout.

CONCLUSIONS: Integrated single-cell and bulk analyses identified regional transcriptional patterns, stromal-state ordering, and stress-associated programs in subchondral bone. In vitro assays supported ferroptosis-related protein changes in OA-like chondrocytes. These findings provide candidate mechanisms for further functional testing but do not establish lineage conversion or causal relationships.