Bo, Z.; Xu, H.; Liang, Y.

Osteoarthritis cartilage has heterogeneous chondrocyte states, yet their transitions remain unresolved from public single-cell data. We retrospectively reanalyzed a public knee cartilage single-cell RNA-seq dataset GSE255460 from 8 osteoarthritis and 3 non-osteoarthritis donors totaling 19 samples. After sample-wise quality control and doublet removal we performed batch-corrected clustering, chondrocyte subclustering with marker-based annotation, and trajectory inference using Slingshot. Regulatory chondrocytes were tested for osteoarthritis versus control differential expression, followed by Gene Ontology and KEGG enrichment with Benjamini-Hochberg false discovery rate <0.05, and protein-protein interaction hub screening. We retained 27,036 cells. Chondrocytes exhibited branching continuous states; regulatory cells localized near the main manifold and adjacent to inferred branches, suggesting a transition-adjacent state. In regulatory cells, osteoarthritis-upregulated genes were enriched for collagen-containing extracellular matrix organization, endoplasmic reticulum secretory/proteostasis, cell-matrix adhesion including focal adhesion, and TGFbeta/SMAD signaling. Protein-protein interaction analysis identified five high-connectivity hubs: COL5A1, COL5A2, COL6A1, COL1A2, and COL3A1. Our findings support a transition-adjacent regulatory program in OA with coordinated extracellular matrix remodeling and secretory/adhesion/TGFbeta signatures, nominating collagen hubs for validation.