Macrophage Phenotypic Changes on FN-Coated Physical Gradient Hydrogels

The chemical and physical properties are two crucial cues when designing tissue engineering scaffold to mimic living tissue. Macrophages, the major players in the immune response, react rapidly to microenvironmental signals, including gradients of physical or chemical cues. Spatiotemporal gradients can modulate cell behavior, such as polarization, proliferation, and adhesion. Here, we studied macrophage phenotypic changes on untreated and fibronectin (FN)-coated methacrylated gellan gum with varying stiffnesses. The compressive moduli of hydrogel with different stiffnesses ranged from ∼5 to 30 kPa. Fibronectin was chemically attached to the substrate to facilitate macrophage proliferation, adhesion, and polarization. Classically (M1) and alternatively (M2) activated macrophages were cultured on both untreated and FN-coated gels. FN-coated substrates elevated cell numbers and enhanced macrophage spreading. The urea/nitrite ratio indicated that untreated rigid substrates shifted both polarizations toward a more proinflammatory phenotype. FN-coated substrates had no impact on M1 polarization. In contrast, FN-coated stiffer gels polarized M2 cells toward an anti-proinflammatory state based on arginine activity and CD206 expression. In addition, macrophage polarization on the softer gel was not influenced by the neighboring cells cultured on the stiffer side of the gel. Using mechanical gradients to control macrophage polarization can be a useful tool in ensuring a proper healing response and for tissue engineering.