Macrophage Polarization in Response to Collagen Scaffold Stiffness Is Dependent on Cross-Linking Agent Used To Modulate the Stiffness.

Macrophages are the first responders to biomaterial implantation and determine the success or failure of an implant through their polarization into proinflammatory (M1) and anti-inflammatory (M2) states. It is known that material properties such as stiffness can influence this response, with these properties typically modulated using a cross-linking agent. However, the cellular response comparing different cross-linking agents is often not analyzed. In this study, collagen scaffolds were cross-linked with one physical (DHT) and two chemical cross-linking methods (EDAC and genipin) in order to independently modulate the stiffness of scaffolds. The physical and structural properties of the scaffolds were thoroughly characterized to ensure that macrophage behaviors toward scaffold stiffness and cross-linking agent employed could be evaluated independent of each other. Through gene expression and protein secretion analysis of THP1 cultures, we demonstrate that the macrophage response to collagen scaffold stiffness is dependent on the cross-linking agent used. Macrophages respond similarly to scaffolds of increasing stiffness generated using the same cross-linking agent. However, when exposed to scaffolds of similar bulk modulus and degradation characteristics cross-linked using different cross-linkers, the cells responded to the cross-linking agent used rather than to the bulk modulus of the scaffolds. Moreover, while genipin cross-linking suppressed both proinflammatory and anti-inflammatory responses from macrophages, EDAC cross-linking promoted a robust proinflammatory and anti-inflammatory response to M1 and M2 factors, respectively. The results demonstrate the potential of using tailored individual cross-linking treatments depending on the clinical indication. Taken together, the results from this study highlight the importance of understanding the macrophage response to both chemical and physical properties of scaffolds in order to promote positive remodeling outcomes after biomaterial implantation.