Engineering macrophage responses through 3D scaffold microarchitecture

Biomaterial implantation in living tissues triggers a physiological response known as foreign body reaction, leading to the recruitment of macrophages, that can polarize either into a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. Currently, there is growing interest in tailoring the physical properties of biomaterials to promote efficient tissue regeneration. Tridimensionality can profoundly influence macrophage behaviour; however, there is no clear consensus on the underlying mechanisms. 3D microstructures may play a crucial role in modulating immune cells, promoting anti-inflammatory responses, and supporting effective tissue repair and regeneration. In this study, we designed and fabricated 3D scaffolds with large pores (50x50x20 μm 3 ) and small pores (15x15x15 μm 3 ) by two-photon polymerization. Both microstructures effectively influenced macrophage cytoskeletal organization and cellular metabolic activity. Notably, they were not sufficient to induce spontaneous macrophage polarization, indicating that they are intrinsically immunologically inert. When combined with chemical stimulation, as typically occurs physiologically, they elicited distinct responses. The investigation of two pore sizes allowed us to find a balance between the anti-inflammatory and pro-inflammatory phenotypes, with a slight upregulation of Arg1 by large pores, and a marked increase of iNOS expression by small pores. Our results demonstrate that 3D microstructures are versatile tools for multiple applications. Their precisely tunable architecture enables fine control over macrophage behaviour, opening new avenues both for in vivo tissue engineering, by preventing fibrosis and promoting anti-inflammatory and pro-regenerative responses, and for the development of in vitro platforms to model inflamed tissues for screening anti-inflammatory drugs.