Macrophages, the key players in immunoregulation, are actively involved in tissue remodelling and vascularization. Recent advances in tissue engineering and regenerative medicine illustrate the importance of "immuno-informed" biomaterials to regulate the microenvironment of biomedical implants. In the current study, silk-based 3D hydrogels were utilized to regulate cytokine delivery for macrophage, a type of immune cell, differentiation and polarization. Three different hydrogel variants, silk-poly(ethylene glycol) (PEG) (SP), silk-horseradish peroxidase (HRP) (SH) and silk-sonicated (SS) hydrogels were studied. Hydrogels were loaded with the M1 and M2 polarizing cytokines interferon-γ (IFN-γ) and interleukin-4 (IL-4), respectively. Functional cytokine release and macrophage polarization studies were conducted using three cytokine exposure approaches: only cytokine encapsulation (macrophage in culture well), only macrophage encapsulation (cytokine in culture media) and cytokine with macrophage encapsulation. The extent of macrophage polarization by cytokine-eluting and macrophage-encapsulating hydrogels was investigated using gene expression analysis for C-C chemokine receptor 7 (CCR7), Interleukin-1 beta (IL-1β), cluster of differentiation 206 (CD206) and cluster of differentiation 209 (CD209). The released cytokines polarized macrophages from an M0 phenotype to an M1/M2 phenotype. Also, lineage committed M1/M2 macrophages could be "switched" to their M2/M1 counterparts (M1-to-M2 or M2-to-M1 transition) exhibiting their well-established plasticity. When macrophages were encapsulated in hydrogels, polarization could be induced to the lineage committed M1 or M2 phenotypes either in polarizing media or when coencapsulated with cytokines. Through this study, silk hydrogels demonstrated utility as a novel system for focal delivery of cytokines and macrophages as "immuno-informed" 3D silk-biomaterials.
Keywords: cytokine; hydrogel; macrophage polarization; silk; tissue engineering.