Tissue response to biphasic calcium phosphate covalently modified with either heparin or hyaluronic acid in a mouse subcutaneous implantation model.

Biphasic calcium phosphate (BCP) materials are widely employed as bone substitute materials due to their resorption/degradation properties. Inflammation after implantation of such materials represents a prerequisite for bone tissue repair and regeneration but can be also problematic if it is not only transient and if it is followed by fibrosis and scarring. Here, we modified BCP covalently with hyaluronan (HA) and heparin (Hep), glycosaminoglycans that possess anti-inflammatory properties. Beside the characterization of particle surface properties, the focus was on in vivo tissue response after subcutaneous implantation in mice. Histological analysis revealed a decrease in signs of inflammatory response to BCP when modified with either HA or Hep. Reduced vascularization after 30 days was noticed when BCP was modified with either HA or Hep with greater cellularity in all examined time points. Compared to plain BCP, expression of endothelial-related genes Flt1 and Vcam1 was higher in BCP-HA and BCP-Hep group at day 30. Expression of osteogenesis-related genes Sp7 and Bglap after 30 days was the highest in BCP group, followed by BCP-Hep, while the lowest expression was in BCP-HA group which correlates with collagen amount. Hence, coating of BCP particles with HA seems to suppress inflammatory response together with formation of new bone-like tissue, while the presence of Hep delays the onset of inflammatory response but permits osteogenesis in this subcutaneous bone-forming model. Transferring the results of this study to other coated materials intended for biomedical application may also pave the way to reduction of inflammation after their implantation.

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.