Study on the potential mechanism of anti-inflammatory activity of covalently immobilized hyaluronan and heparin

Inflammation and subsequent fibrotic encapsulation that occur after implantation of biomaterials are issues that fostered efforts in designing novel biocompatible materials to modulate the immune response. In this study, glycosaminoglycans (GAG) like hyaluronic acid (HA) and heparin (Hep) that possess anti-inflammatory activity were covalently bound to NH2-modified surfaces using EDC/NHS cross-linking chemistry. Immobilization and physical surface properties were characterized by atomic forces microscopy, water contact angle studies and streaming potential measurements demonstrating the presence of GAG on the surfaces that became more hydrophilic and negatively charged compared to NH2-modified. THP-1 derived macrophages were used here to study the mechanism of action of GAG to affect the inflammatory responses illuminated by studying macrophage adhesion, the formation of multinucleated giant cells (MNGCs) and IL-1 beta release that were reduced on GAG-modified surfaces. Detailed investigation of the signal transduction processes related to macrophage activation was performed by immunofluorescence staining of NF-kappa B (p65 subunit) together with immunoblotting. We studied also association and translocation of FITC-labeled GAG. The results show a significant decrease in NF-kappa B level as well as the ability of macrophages to associate with and take up HA and Hep. These results illustrate that the anti-inflammatory activity of GAG is not only related to making surfaces more hydrophilic, but also their active involvement in signal transduction processes related to inflammatory reactions, which may pave the way to design new anti-inflammatory surface coatings for implantable biomedical devices.