Investigating biomaterial-based biophysical cues for modulating macrophage polarization towards bone regeneration applications

As one of the first cells to respond to biomaterial implantation, macrophages, through polarization into pro- (M1) and anti-inflammatory (M2) states, secrete cytokines and chemokines that determine the subsequent immune response and eventual success of an implanted biomaterial. Little is known about how biomaterial properties, especially biophysical cues, modulate the macrophage response and their interaction with mesenchymal stem cells (MSCs), another important cell type in the implant environment. The overall objective of the research presented in this thesis was to understand the role of biophysical cues presented by biomaterials in directing macrophage polarization, function, migration and interaction with MSCs, and to investigate the role of these biomaterial-based immune responses in promoting bone regeneration. The objective of Chapter 2 was to investigate the role of substrate stiffness (of collagen-coated 2D polyacrylamide hydrogels) in modulating macrophage morphology and polarization. Substrate stiffness ‘primed’ macrophages to a rounded cell shape and a predominantly anti-inflammatory M2-like phenotype on soft (11kPa) and medium (88kPa) stiffness gels and a spread shape and a pro-inflammatory M1-like phenotype on stiff (323kPa) gels. Soft gels also promoted a pro-inflammatory response in addition to an anti-inflammatory response from macrophages, resulting in a ‘hybrid’ phenotype. Building on these results, a comprehensive assessment of the role of substrate stiffness in modulating macrophage function and migration mode was undertaken in Chapter 3, with the aim of elucidating the mechanism of macrophage mechanosensing. It was shown that macrophage functions (phagocytosis and migration) were enhanced on softer gels, and impaired on stiff gels. Moreover, macrophages migrated using the RhoA Kinase (ROCK)-dependent amoeboid migration mode on softer gels and a proteolytic podosome-dependent mesenchymal migration mode on stiff gels. ROCK signalling was also shown to be involved in both macrophage polarization and migration, providing a mechanistic understanding of the role of the cytoskeleton in macrophage mechanosensing. Chapter 4 aimed to assess the role of substrate stiffness in MSC immunomodulation and their crosstalk with macrophages, in addition to evaluating the role of biomaterial-based macrophage responses in driving downstream events involved in bone regeneration. A stiffness-dependent cross-talk between MSCs and macrophages was established, with macrophages on softer gels producing decreased pro-inflammatory and increased anti-inflammatory factors in the presence of MSCs. It was also shown that factors secreted by the ‘hybrid’ macrophage phenotype on soft gels played an important role in mediating MSC osteogenesis. An alternative approach to investigate the immune response to biomaterial-based biophysical cues was undertaken in Chapter 5, which aimed to evaluate the role of material properties of collagen-based scaffolds such as stiffness, crosslinking and incorporated particle size in directing macrophage polarization and macrophage-mediated MSC osteogenesis. Macrophage response to 3D porous scaffold stiffness was shown to be dependent on the crosslinking method employed to modulate the stiffness, with no evident stiffness-related responses. Needle-shaped 5μm particles embedded in collagen-hydroxyapatite (Coll-HA) scaffolds promoted a ‘hybrid’ phenotype in macrophages, with upregulation of pro- and anti-inflammatory markers, which was not observed in scaffolds with spherical particles (5μm and 30μm). MSC osteogenesis was significantly increased when co-cultured with macrophages, with scaffolds with needle-shaped particles further enhancing this response through a pro-osteogenic macrophage phenotype. The differences between scaffold groups and between MSC mono-culture and co-culture with macrophages highlights the importance of analysing the macrophage response (in addition to MSCs alone) to biomaterial physical properties in directing subsequent osteogenic responses. In summary, this thesis has definitively demonstrated that biophysical properties of biomaterials like stiffness and particle size can be tailored to modulate macrophage polarization, function, migration and their interaction with MSCs, and has highlighted the role of biomaterial-based macrophage responses in mediating bone regeneration outcomes.