The effects of substrate stiffness on the in vitro activation of macrophages and in vivo host response to poly(ethylene glycol)-based hydrogels.

Poly(ethylene glycol) (PEG) hydrogels, modified with RGD, are promising platforms for cell encapsulation and tissue engineering. While these hydrogels offer tunable mechanical properties, the extent of the host response may limit their in vivo applicability. The overall objective was to characterize the effects of hydrogel stiffness on the in vitro macrophage response and in vivo host response. We hypothesized that stiffer substrates induce better attachment, adhesion, and increased cell spreading, which elevates the macrophage classically activated phenotype and leads to a more severe foreign body reaction (FBR). PEG-RGD hydrogels were fabricated with compressive moduli of 130, 240, and 840 kPa, and the same RGD concentration. Hydrogel stiffness did not impact macrophage attachment, but elicited differences in cell morphology. Cells retained a round morphology on 130 kPa substrates, with localized and dense F-actin and localized α(V) integrin stainings. Contrarily, cells on stiffer substrates were more spread, with filopodia protruding from the cell, a more defined F-actin, and greater α(V) integrin staining. When stimulated with lipopolysaccharide, macrophages had a classical activation phenotype, with increased expression of TNF-α, IL-1β, and IL-6, however the degree of activation was significantly reduced with the softest hydrogels. A FBR ensued in response to all hydrogels when implanted subcutaneously in mice, but 28 days postimplantation the layer of macrophages at the implant surface was significantly lower in the softest hydrogels. In conclusion, hydrogels with lower stiffness led to reduced macrophage activation and a less severe and more typical FBR, and therefore are more suited for in vivo tissue engineering applications.