ECM remodeling features in reparative chondrocytes during knee osteoarthritis

Knee osteoarthritis (KOA) is characterized by progressive cartilage degeneration and disruption of extracellular matrix (ECM) homeostasis. Chondrocytes are not a homogeneous or static population during disease progression but exhibit pronounced functional heterogeneity. Although single-cell transcriptomic studies have identified multiple chondrocyte states in osteoarthritic cartilage, how these states dynamically relate to ECM remodeling and disease progression remains incompletely understood.

We integrated multiple publicly available single-cell RNA sequencing datasets of human knee cartilage to construct a unified cellular atlas and systematically compared chondrocyte states between control and KOA samples. Differential expression analysis, functional enrichment, pseudotime trajectory inference, and cell-cell communication analysis were applied to characterize ECM-related chondrocyte states and their dynamic transitions. Key signaling cues identified from single-cell analyses were further evaluated using in vitro cultured human articular chondrocytes.

We observed a marked expansion of the previously described reparative chondrocyte population (RepC) in KOA cartilage. Rather than reflecting a simple increase in cell proportion, KOA-associated RepC exhibited enhanced ECM remodeling programs characterized by collagen reorganization and strengthened ECM-cell interactions. Pseudotime analysis positioned RepC downstream of proliferation chondrocytes and near a major branching region toward either regulator chondrocytes with further extension toward fibrocartilage chondrocytes or effector chondrocytes. In KOA, RepC was preferentially represented at mid-to-late pseudotime stages within the reconstructed trajectory framework. Cell-cell communication analysis suggested that RepC showed prominent inferred ECM-related interactions, particularly involving collagen and FN1-integrin pathways. Consistently, FN1 or TGF-β1 stimulation in vitro induced expression of multiple RepC-associated genes and enhanced SMAD2/3 phosphorylation, recapitulating key features of the RepC state observed in single-cell analyses.

These findings highlight ECM remodeling features of reparative chondrocytes during KOA and support a state-centric view in which disproportionate representation of reparative states within the pseudotime trajectory framework is associated with maladaptive ECM remodeling in KOA.