LAUSR

Biomaterial implantation is followed by an inflammatory cascade dominated by macrophages, which determine implant acceptance or rejection through pro- and anti-inflammatory polarization states (Anderson et al., 2008; Brown and Badylak, 2013). It is known that chemical signals such as bacterial endotoxins and cytokines (IL4) can direct macrophage polarization (Mantovani et al., 2004); however, recent evidence implicates biophysical cues in this process (McWhorter et al., 2015; Patel et al., 2012). Here we report that THP-1 derived macrophages cultured on collagen-coated polyacrylamide gels of varying stiffness adapt their polarization state, functional roles and migration mode according to the stiffness of the underlying substrate. Through gene expression and protein secretion analysis, we show that stiff polyacrylamide gels (323 kPa) prime macrophages towards a pro-inflammatory phenotype with impaired phagocytosis in macrophages, while soft (11 kPa) and medium (88 kPa) stiffness gels prime cells towards an anti-inflammatory, highly phagocytic phenotype. Furthermore, we show that stiffness dictates the migration mode of macrophages; on soft and medium stiffness gels, cells display Rho-A kinase (ROCK)-dependent, podosome-independent fast amoeboid migration and on stiff gels they adopt a ROCK-independent, podosome-dependent slow mesenchymal migration mode. We also provide a mechanistic insight into this process by showing that the anti-inflammatory property of macrophages on soft and medium gels is ROCK-dependent and independent of the ligand presented to them. Together, our results demonstrate that macrophages adapt their polarization, function and migration mode in response to the stiffness of the underlying substrate and suggest that biomaterial stiffness is capable of directing macrophage behaviour independent of the biochemical cues being presented to them. The results from this study establish an important role for substrate stiffness in directing macrophage behaviour, and will lead to the design of immuno-informed biomaterials that are capable of modulating the macrophage response after implantation. STATEMENT OF SIGNIFICANCE: Biomaterial implantation is followed by an inflammatory cascade dominated by macrophages, which determine implant acceptance or rejection through pro- and anti-inflammatory polarization states. It is known that chemical signals can direct macrophage polarization; however, recent evidence implicates biophysical cues in this process. Here we report that macrophages cultured on gels of varying stiffness adapt their polarization state, functional roles and migration mode according to the stiffness of the underlying substrate. The results from this study establish an important role for substrate stiffness in directing macrophage behaviour, and will lead to the design of immuno-informed biomaterials that are capable of modulating the macrophage response after implantation.