Effects of high-molecular-weight hyaluronic acid on inflammatory and matrix-associated responses in a chondrocyte-osteoclast co-culture model.

Osteoarthritis (OA) is a degenerative joint disorder marked by chronic inflammation, extracellular matrix (ECM) breakdown, and subchondral bone remodeling, ultimately leading to loss of cartilage integrity and joint function. Hyaluronic acid (HA), a key component of synovial fluid, contributes to joint lubrication and has been implicated in the regulation of inflammatory and anabolic processes. High-molecular-weight (HMW) HA (> 1 MDa) is reported to exert anti-inflammatory and matrix-preserving effects; however, its role in multicellular systems remains incompletely understood. In this study, we investigated the effects of HMW HA using an in vitro chondrocyte-osteoclast co-culture model designed to reflect key features of the osteoarthritic microenvironment. Bone marrow-derived mesenchymal stem cells were differentiated into chondrocytes, while RAW 264.7 macrophages were induced into osteoclast-like cells 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 HA treatment (50-500 µg/mL) for 24-48 h. Cell viability, morphology, and TRAP 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 using flow cytometry and agarose gel electrophoresis. LPS exposure reduced metabolic activity, altered morphology, and was associated with increased expression of NF-κB-related inflammatory markers and elevated expression of IL-1β, TNF-α, MMP-13, and ADAMTS-5, alongside reduced COL2A1 and ACAN levels. HA treatment improved metabolic activity and preserved cellular morphology in a concentration-dependent manner, with the most pronounced effects observed at 200-500 µg/mL. HA-treated cultures showed lower levels of inflammatory and catabolic markers together with increased expression of ECM-associated genes. In addition, HA treatment was associated with a more balanced cell-cycle profile and reduced DNA fragmentation. HMW HA was associated with coordinated modulation of inflammatory and matrix-related markers in an LPS-induced co-culture system. These findings suggest that HA may contribute to maintaining cellular homeostasis under inflammatory conditions in vitro. Further studies are needed to clarify the underlying mechanisms and translational relevance.