We have previously shown that implantation of a fragmented chitosan hydrogel suspension (chitosan-FPHS) into a traumatic spinal cord lesion in adult rats led to significant axon regrowth and functional recovery, which was associated to a modulation of inflammation. Using an in vitro culture system, we show here that polarization of bone marrow-derived macrophages is indeed modified by direct contact with chitosan-FPHS. Reducing the degree of acetylation (DA), and raising the concentration of chitosan (Cp, from 1.5% to 3%), favors macrophage polarization towards anti-inflammatory subtypes. These latter also migrate and adhere efficiently on low, but not high DA chitosan-FPHS, both in vitro and in vivo, while inflammatory macrophages rarely invade a chitosan-FPHS implant in vivo, no matter the DA. Our in vitro model setup should prove a valuable tool for screening diverse biomaterial formulations and combinations thereof for their inflammatory potential prior to implantation in vivo.