Integrated transcriptomic and epigenomic profiling of human cartilage reveals coordinated changes in fibronectin expression, splicing, and receptor signaling during osteoarthritis progression

Osteoarthritis (OA) is defined by progressive loss of articular cartilage, but how chondrocytes change as the disease advances is unclear because most studies have focused on end-stage tissue. We profiled human knee articular cartilage across the full range of macroscopic OA grade, from young healthy to aged severe OA donors, by bulk RNA sequencing of 92 knees, single-nucleus RNA sequencing of 108,600 chondrocyte nuclei from 30 donors and single-nucleus multiome sequencing of 24 of these donors, treating grade as a continuous variable. With increasing grade, cartilage increased extracellular matrix (ECM) synthesis and remodeling genes, lost glucocorticoid-responsive, lipid-metabolic and antioxidant programs, and changed the splicing of a small set of ECM transcripts, led by the extra domain B (EDB) exon of fibronectin (FN1). Nine chondrocyte clusters were present in every disease stage and formed a continuum ordered by cartilage zone and hypertrophic differentiation, with PRG4+ superficial and COL10A1+ hypertrophic chondrocytes as the only distinct cell types at its ends and a pre-hypertrophic state between them. With grade, all clusters shared a response in which ECM production rose and homeostatic programs fell, and they differed in which of its components was strongest: ECM production in the superficial cells, the secretory pathway in the hypertrophic and CYTL1+ cells, and inflammatory signaling in the ANXA1+ and CRISPLD2+ cells. Inferred communication between chondrocytes was remodeled with stage. FN1 was induced in every cluster in excess of its fibril-assembly machinery, together with the fibronectin-cleaving protease HTRA1, and FN1 signaling shifted in receptor use from integrin α5β1 toward CD44 and, less robustly, αvβ8, accompanied by a rise in TGF-β/SMAD response genes. In four early-OA donors, tenascin-C was already largely induced and the FN1 receptor shift from α5β1 toward CD44 was partly present, whereas the rise of FN1 outside superficial chondrocytes, of FGF1 and of developmental and ossification programs came with progression. Chromatin accessibility changed in the same direction across clusters, with regions linked to FN1 opening and regions linked to ITGA5 closing. OA progression in human cartilage therefore appears to be a change of chondrocyte state that differs in emphasis along a zonal and hypertrophic continuum and includes changes in fibronectin regulation, production, processing, and receptor engagement.