Molecular Weight-Driven Tunable Hyaluronic Acid–Based Hydrogels Modulate Immune Polarization in Three-Dimensional Microenvironments

Macrophages exhibit phenotypic plasticity that is strongly influenced by their surrounding microenvironment, including extracellular matrix (ECM) components. Hyaluronic acid (HA), a major glycosaminoglycan in ECM, has immunomodulatory effects that are highly dependent on its molecular weight (MW). However, most previous studies have been limited to two-dimensional (2D) culture systems, which were unable to accurately replicate the in vivo environment. In this study, we utilized a three-dimensional (3D) culture system based on HA-based hydrogels to better understand the MW-dependent immunomodulatory effects of HA on macrophages under more physiologically relevant conditions. Three different MWs of HA were chemically modified and cross-linked with PEG-SH₄ to form hydrogels with distinct biophysical properties. Immortalized macrophages were encapsulated within these hydrogels and assessed for the expression of both pro-inflammatory and anti-inflammatory markers. Notably, hydrogels with high-MW HA significantly upregulated the expression of anti-inflammatory markers, indicating that the immunomodulatory effects of HA in 3D culture are affected by its biophysical characteristics. Our findings demonstrate the potential of HA-based hydrogels as customizable ECM-mimetic scaffolds for modulating immune responses in regenerative medicine applications.