Critical Requirement of Senescence-Associated CCN3 Expression in CD44-Positive Stem Cells for Osteoarthritis Progression

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage breakdown, synovial inflammation, and subchondral bone remodeling. Previous studies have shown that cellular communication network factor 3 (CCN3) expression increases with age in cartilage, and its overexpression promotes OA-like changes by inducing senescence-associated secretory phenotypes. This study aimed to investigate the effect of <i>Ccn3</i> knockout (KO) on OA development using a murine OA model. Destabilization of the medial meniscus (DMM) surgery was performed in wild-type (WT) and <i>Ccn3</i>-KO mice. Histological scoring and staining were used to assess cartilage degeneration and proteoglycan loss. Gene and protein expressions of catabolic enzyme (<i>Mmp9</i>), hypertrophic chondrocyte marker (<i>Col10a1</i>), senescence marker, and cyclin-dependent kinase inhibitor 1A (<i>Cdkn1a</i>) were evaluated. Single-cell RNA sequencing (scRNA-seq) data from WT and <i>Sox9</i>-deficient cartilage were reanalyzed to identify <i>Ccn3</i>⁺ progenitor populations. Immunofluorescence staining assessed CD44 and Ki67 expression in articular cartilage. The effects of <i>Ccn3</i> knockdown on IL-1β-induced <i>Mmp13</i> and <i>Adamts5</i> expression in chondrocytes were examined <i>in vitro</i>. <i>Ccn3</i> KO mice exhibited reduced cartilage degradation and catabolic gene expression compared with WT mice post-DMM. scRNA-seq revealed enriched <i>Ccn3</i>-<i>Cd44</i> double-positive cells in osteoblast progenitor, synovial mesenchymal stem cell, and mesenchymal stem cell clusters. Immunofluorescence showed increased CCN3⁺/CD44⁺ cells in femoral and tibial cartilage and meniscus. Ki67⁺ cells were significantly increased in DMM-treated <i>Ccn3</i> KO cartilage, mostly CD44⁺. In vitro <i>Ccn3</i> knockdown attenuated IL-1β-induced <i>Mmp13</i> and <i>Adamts5</i> expressions in chondrocytes. <i>Ccn3</i> contributes to OA pathogenesis by promoting matrix degradation, inducing hypertrophic changes, and restricting progenitor cell proliferation, highlighting <i>Ccn3</i> as a potential therapeutic target for OA.