p21 Knockdown as a Therapeutic Strategy for Focal Cartilage Injury Repair

Focal articular cartilage defects lack intrinsic regenerative capacity and can progress to osteoarthritis as no effective treatment exists to slow, stop or reverse cartilage degeneration. Cellular senescence, characterized by elevated p21 (CDKN1A) expression, impairs chondrocyte proliferation and cartilage repair. We hypothesized that targeted p21 suppression via lentiviral delivery would enhance cartilage regeneration. Lentiviral shRNA vectors targeting p21 or non-sense controls with a tdTomato reporter were constructed. In vitro, murine synovial progenitor cells and MC3T3E1 preosteoblasts were transduced and p21 mRNA was quantified by qRT-PCR. Cell cycle analysis was performed using flow cytometry. In vivo, immunocompromised (B6.Cg-Prkdc^scid/SzJ) and immunocompetent (C57BL6) mice received full-thickness cartilage defects followed by intraarticular p21 shRNA or nonsense control injection. Cartilage repair was assessed by Safranin O staining and tissue cytometry quantified lentiviral transduction and p21 knockdown in chondrocytes. In vitro, p21 shRNA achieved ~80% reduction in p21 mRNA and increased G2M phase cells. In vivo, p21 shRNA treatment significantly improved cartilage repair in both strains. Tissue cytometry revealed 90% transduction efficiency with p21⁺ cells reduced from ~90% to ~30%. Spearman correlation showed significant negative correlation between p21 expression and repair outcomes. Hepatic off-target transduction was minimal in uninjured animals but increased in injured animals, yet morphological hepatotoxicity was not observed. p21 knockdown via lentiviral shRNA effectively promotes cartilage regeneration, supporting the concept of targeting p21 and/or the p21 pathway as a therapeutic strategy for cartilage repair.