Background <p>Osteoarthritis (OA) is characterized by a loss of the joint viscoelastic properties. Hyaluronic acid (HA) intraarticular injections restore viscoelasticity, alleviating pain and improving patient function. Larger amounts of HA in a single injection could lead to greater efficacy, although the mechanism responsible for the long-term analgesic effect remains uncertain. We evaluated the effect of high concentrations of intra-articular HA on the innate immune response in synovial tissue in experimental OA and in cultured ATDC5 chondrocytes. </p> Methods <p>Experimental OA was induced by destabilization surgery in the right knee of the rabbits. Four weeks after surgery, some rabbits received weekly intra-articular injections of 0.5 mL of 2% hyaluronic acid (HA, as commercially available formulation Adant Plus) for three weeks, while others received saline (vehicle). Eight weeks after surgery, right tibias, femurs, and synovial membranes were isolated for histopathology and molecular biology studies. In addition, the effect of 0.2% and 0.1% concentrations of HA on chondrocyte viability and oxidative stress was analyzed using ATDC5 chondrocytes in culture.</p> Results <p>2% HA did not alter cartilage or synovial damage, although it demonstrated a good safety profile. 2% HA administration reduced IL-1β protein synthesis in the synovial membrane of OA rabbits. In cultured ATDC5 chondrocytes, 0.1% and 0.2% HA partially counteracted the deleterious effects of H₂O₂ on the gene expression of superoxide dismutase (SOD)-1 and heme oxygenase (HMOX)-1.</p> Conclusion <p>Intra-articular administration of high concentrations of HA reduced IL-1β concentration in the OA synovium despite no observable improvement in cartilage or synovial lesions was observed. We suggest that the long-term beneficial effect of HA in human OA could be attributed to a “memory fingerprint” in the innate immune response, potentially leading to sustained reduced activity over an extended period of treatment.</p>