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Macrophages are essential in the initiation, maintenance, and transition of inflammatory processes like foreign body response and wound healing. These sentinel cells are very motile, susceptible, and adaptive to environmental cues. Mounting evidence suggests that physical factors also modulate macrophage activation in vitro and in vivo. 2D in vitro systems have demonstrated that constraining macrophages to small areas or channels modulates their phenotypes and changes their responses to known inflammatory agents such as lipopolysaccharide. However, macrophage responses in these 2D systems cannot be directly correlated to an in vivo response. Thus, the study of macrophage confinement in a 3D environment that can be directly used in vivo is needed and would allow the design of more translatable biomaterials for clinical applications. In this work, we explored the change in M1/M2 polarization when macrophages were confined in microporous annealed particle scaffolds (MAPS), which are granular hydrogels generated from annealed spherical microgels. We cultured primary murine macrophages within MAPS constructed of microgels with diameters of 40 μm, 70 μm, and 130 μm. Spatially confining macrophages in scaffolds with pore size on the scale of cells led to a reduced level of the inflammatory response, which was correlated with a change in cell morphology and motility.

Macrophages are essential in the initiation, maintenance, and transition of inflammatory processes such as foreign body response and wound healing. Mounting evidence suggests that physical factors also modulate macrophage activation. 2D in vitro systems demonstrate that constraining macrophages to small areas or channels modulates their phenotypes and changes their responses to known inflammatory agents such as lipopolysaccharide. However, how dimensionality and pore size affect macrophage phenotype is less explored. In this work, the change in macrophage M1/M2 polarization when confined in microporous annealed particle (MAP) scaffolds is studied. Particles sizes (40, 70, and 130 µm) are selected using outputs from software LOVAMAP that analyzes the characteristics of 3D pores in MAP gels. As the size of building block particle correlates with pore size inside the scaffolds, the three  types of scaffold allow us to study how the degree of spatial confinement modulates the behavior of embedded macrophages. Spatially confining macrophages in scaffolds with pore size on the scale of cells leads to a reduced level of the inflammatory response, which is correlated with a change in cell morphology and motility.

Void volume fraction (VVF) of granular biomaterials is a global measurement frequently used to characterize void space. There is currently no gold standard for measuring the VVF of granular scaffolds made in lab. To help the biomaterials field, we provide a library of different simulated scaffolds with known VVF for easy look-up. We use our simulated data to explore the relationship between microscope magnification and VVF, and we study the accuracy of approximating VVF using 2-D z-slice images, which is the most common approach for computing VVF of real scaffolds. Lastly, we test four different approaches for computing VVF using microscope images of real granular scaffolds and reveal that VVF may be an unreliable descriptor.