Free-standing multilayer films as growth factor reservoirs for future wound dressing applications.

Chronic skin wounds place a high burden on patients and health care systems. The use of angiogenic and mitogenic growth factors can facilitate the healing but growth factors are quickly inactivated by the wound environment if added exogenously. Here, free-standing multilayer films (FSF) are fabricated from chitosan and alginate as opposing polyelectrolytes in an alternating manner using layer-by-layer technique. One hundred bilayers form an about 450 µm thick, detachable free-standing film that is subsequently crosslinked by either ethyl (dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide (E-FSF) or genipin (G-FSF). The characterization of swelling, oxygen permeability and crosslinking density shows reduced swelling and oxygen permeability for both crosslinked films compared to non-crosslinked films (N-FSF). Loading of fibroblast growth factor 2 (FGF2) into the films results in a sustained release from crosslinked FSF in comparison to non-crosslinked FSF. Biocompatibility studies in vitro with human dermal fibroblasts cultured underneath the films demonstrate increased cell growth and cell migration for all films with and without FGF2. Especially G-FSF loaded with FGF2 greatly increases cell proliferation and migration. In vivo biocompatibility studies by subcutaneous implantation in mice show that E-FSF causes an inflammatory tissue response that is absent in the case of G-FSF. N-FSF also represents a biocompatible film but shows early degradation. All FSF possess antibacterial properties against gram+ and gram- bacteria demonstrated by an agar diffusion disc assay. In summary, FSF made of alginate and chitosan crosslinked with genipin can act as a reservoir for the sustained release of FGF2, possessing high biocompatibility in vitro and in vivo. Moreover, G-FSF promotes growth and migration of human dermal fibroblasts and has antibacterial properties, which makes it an interesting candidate for bioactive wound.

Studies on the Mechanisms of Anti-Inflammatory Activity of Heparin- and Hyaluronan-Containing Multilayer Coatings-Targeting NF-κB Signalling Pathway.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1ß release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

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-1ß 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-κB (p65 subunit) together with immunoblotting. We studied also association and translocation of FITC-labeled GAG. The results show a significant decrease in NF-κB 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.