Hydrogel viscoelasticity regulates human monocyte-derived macrophage morphology and phenotype in 2D culture

Macrophages are a key cell type in the foreign body reaction (FBR), playing a central role in the inflammatory response to implanted biomaterials and in fibrous capsule formation. However, the influence of biomaterial viscoelasticity on macrophages remains poorly understood. Only a very limited number of studies have investigated the viscoelastic-macrophage interactions, using cell lines or murine cells with ill-defined material viscoelasticity. Here, we investigated how hydrogels with tuneable viscoelasticity, featuring low initial moduli mimicking soft tissues mechanics and a stress relaxation timescale comparable to molecular clutch binding dynamics, modulate human monocyte-derived macrophage responses in 2D culture. Polyacrylamide (PAAm) hydrogels with comparable initial moduli, mesh size, chemistry and surface adsorbed proteins but distinct viscoelasticities were prepared by tuning monomer (acrylamide) and crosslinker (bisacrylamide) concentrations. While the elastic PAAm composition displayed minimal stress relaxation, the viscoelastic PAAm hydrogel showed pronounced stress relaxation with ∼70% decrease of its initial modulus within 5 min and τ_1/2 ≈ 15.8 s (time to reach 50% of original modulus), a physiologically relevant timescale for cell-matrix binding dynamics and mechanotransduction. Human peripheral blood monocyte-derived macrophages cultured on the viscoelastic hydrogel spread more extensively and displayed higher eccentricity than those on elastic substrates. In contrast, macrophages on the elastic hydrogel showed reduced spreading, smaller cell area and increased clustering. Phenotype analysis revealed that elastic hydrogels promoted a pro-inflammatory response, characterised by elevated calprotectin expression and significantly higher secretion of TNF-α and IL-6, whereas anti-inflammatory surface markers and cytokines were not statistically different between the two hydrogel types. Collectively, these findings suggest that viscoelasticity is a key factor regulating macrophage phenotype, with effects dependent on initial modulus and stress relaxation rate, and highlight its relevance as a design parameter in immunomodulatory biomaterials with potential to mitigate the FBR. STATEMENT OF SIGNIFICANCE: This study reveals, for the first time, how viscoelastic hydrogels with physiologically relevant stress-relaxation timescales modulate the morphology and activation of human monocyte-derived macrophages. By engineering polyacrylamide hydrogels with comparable initial moduli, porosity and surface chemistry but distinct stress relaxation profiles, we isolated the specific contribution of viscoelasticity to macrophage mechanotransduction. Our findings demonstrate that substrate viscoelasticity critically governs macrophage morphology and inflammatory polarisation, with viscoelastic matrices attenuating pro-inflammation activation. This work bridges the gap between material mechanics and human macrophage polarisation, highlighting viscoelasticity as a tuneable design parameter for immunomodulatory biomaterials. These insights provide a foundation for developing next-generation implants and tissue scaffolds that leverage viscoelastic control to mitigate the foreign body response.