Therapeutic Potential of Hyaluronic Acid in Osteoarthritis: Insights from a Chondrocyte- Osteoclast Co-Culture System

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by chronic inflammation, extracellular matrix (ECM) degradation, and subchondral bone remodeling, leading to progressive loss of cartilage homeostasis and joint function. Hyaluronic acid (HA), a major component of synovial fluid, functions as a viscoelastic lubricant and has been associated with modulation of inflammatory and anabolic signaling. High-molecular-weight (HMW) HA (> 1 MDa) has been reported to exert anti-inflammatory and matrix-preserving effects; however, its mechanistic actions in multicellular systems remain incompletely understood. This study investigated the effects of HMW HA on inflammatory responses, osteoclastogenesis, and matrix-related markers using an in-vitro chondrocyte–osteoclast co-culture model that recapitulates key aspects of the osteoarthritic microenvironment. Methods: Bone-marrow-derived mesenchymal stem cells were differentiated into chondrocytes, and RAW 264.7 macrophages were induced into osteoclasts using M-CSF and RANKL. Cells were co-cultured at a 1:1 ratio and stimulated with lipopolysaccharide (LPS; 1 µg/mL) to induce inflammatory stress, followed by treatment with HA (50–500 µg/mL) for 24–48 h. Cell viability, morphology, and tartrate-resistant acid phosphatase staining were assessed. Expression of inflammatory and matrix-associated markers was analysed by Western blot, qPCR, and ELISA. Cell-cycle distribution and DNA integrity were evaluated by flow cytometry and agarose gel electrophoresis. Results: LPS exposure reduced cell viability, altered chondrocyte morphology, and increased NF-κB activation, accompanied by elevated IL-1β, TNF-α, MMP-13, and ADAMTS-5 expression and reduced COL2A1 and ACAN levels. HA treatment was associated with improved metabolic activity and preservation of cellular morphology in a concentration-dependent manner, with the greatest effects observed at 200–500 µg/mL. HA-treated cultures showed lower expression of inflammatory cytokines and catabolic enzymes together with comparatively higher levels of anabolic ECM markers. In addition, a more balanced cell-cycle distribution and maintenance of genomic integrity were observed. These findings were consistent with modulation of the TLR4/MyD88/NF-κB axis and a potential involvement of CD44-associated PI3K/Akt signaling under the present experimental conditions. Conclusion: HMW HA was associated with coordinated reductions in inflammatory mediators and matrix-degrading enzymes together with increased expression of cartilage matrix markers in an LPS-induced chondrocyte–osteoclast co-culture model. These findings suggest that HA may contribute to maintaining cartilage homeostasis through modulation of NF-κB–associated inflammatory signaling and CD44-mediated pathways within the osteoarthritic microenvironment. Further studies using advanced three-dimensional systems and in-vivo models are required to confirm the underlying mechanisms and translational relevance.