Soft tissue-mimicking hydrogel stiffness modulates polarisation of human monocyte-derived macrophages

Minimising the foreign body response (FBR) to implants remains an unmet clinical challenge. The mechanical properties of biomaterials, particularly stiffness, have been shown to influence macrophage activation and FBR. However, current literature on the effects of material stiffness on macrophage activation presents conflicting results. These inconsistencies may stem from the use of cell lines or murine primary cells, which do not fully represent the behaviour of primary human monocyte-derived macrophages. Additionally, many previous studies have focused on stiffness values that fall outside the physiological range of soft tissues. In this study, we investigated how variations in stiffness affect the activation status of human monocyte-derived macrophages using alginate methacrylate (ALMA) hydrogels. The hydrogel stiffness was tuned within a physiologically relevant range (0.25 – 4.5 kPa) to mimic the mechanical properties of soft tissues. We found that increasing hydrogel stiffness consistently upregulated pro-inflammatory markers. Specifically, the stiffest hydrogel (ALMA 6% w/v) induced higher secretion of TNF-α and increased the calprotectin-to-mannose surface maker ratio, both hallmarks of inflammatory macrophages. Moreover, macrophages cultured on stiffer hydrogels exhibited a more elongated morphology and greater spreading. These findings provide new insight into how small changes in stiffness, within a soft tissue-relevant range, can modulate the inflammatory behaviour of human macrophages. Our results, together with findings from the literature, suggest that the contradictory data on the effects of stiffness on macrophages may be attributed to other factors, such as viscoelasticity, surface chemistry, and protein absorption, which warrant further investigation.