Effect of silver ion incorporation into a bioactive glass surface on the adsorption of albumin

Introduction Nowadays, bacterial infection is one of the major causes for orthopedic implants failure. While it is well known how to manufacture materials that are able to stimulate osseointegration and to firmly bond with the bone, the fight against pathogenic microorganisms is carried on mainly by antibiotics, with consequent problems of poorly localized actions and antibiotic-resistance. Therefore, novel antibacterial strategies have been deeply researched. Among those, incorporation of silver in biomaterials, such as bioactive glasses, is acknowledged as an effective way to reduce bacteria proliferation. Osseointegration of biomaterials is dependent on the surface properties of the implants and on the interactions with the biological environment. In particular, a protein layer is formed on the surface within minutes after the contact between the surface and the biological fluids and it will dictate how the cells will respond to the implanted foreign body. As consequence, it is important to understand how antibacterial modifications of bioactive materials affect their interactions with proteins. In this work, the adsorption of albumin was investigated onto a silica-based bioactive glass where silver ions were incorporated through ionic exchange (Ag-SBA2) in order to understand eventual differences with the untreated surface (SBA2)[1]. Experimental Methods SBA2 bioactive glass (mol %: 48% SiO2, 18% Na2O, 30% CaO, 3% P2O5, 0.43% B2O3, 0.57% Al2O3) was prepared via precursors melting and casting, cut into disks and grinded (up to 1000 grit). Ag-SBA2 was prepared by soaking glass slices for 1h in 0.03M AgNO3 solution. Protein adsorption was obtained by soaking the samples for 2h at 37°C in albumin solution in PBS, in near physiological conditions (20 mg/ml, pH 7.4). The glass substrates were characterized in terms of topography and roughness (SEM, AFM and confocal microscopy), chemical composition (EDS and XPS), surface charge and potential (solid surface zeta potential, Kelvin Probe Force Microscopy (KPFM)) and surface energy (contact angle, Owens-Wendt method). The adsorbed proteins were quantified by using different methods (BCA assay, fluorescent proteins and XPS) and the BSA layer was also imaged (fluorescent microscopy and KPFM). Substrate-protein interactions and albumin conformation were investigated, too (solid surface zeta potential and ATR-FTIR). Results and Discussion After silver incorporation, confirmed by chemical analysis, the surface properties of Ag-SBA2 were mostly similar to the undoped glass. In particular, topography and roughness were unchanged during soaking in the silver solution, as expected. Wettability and surface free energy, both the dispersive and polar components, were also similar between the two substrates. Instead, zeta potential titration curve showed that the incorporation of Ag3+ ions increased the surface potential, in particular around physiological pH (7.4). Quantification of adsorbed BSA showed that both surfaces adsorb a similar amount of albumin, with a little higher amount on Ag-SBA2. This can be possibly related to a couple of different factors: the high affinity of silver for proteins and the presence of a more positive charge on the surface, which is able to attract the negatively charged albumin. On both surfaces, albumin forms a complete and homogeneous layer, as detected by imaging techniques. Adsorption of proteins was confirmed also by zeta potential measurement on the surfaces, with a shift of the IEP of both glassestowards the IEP of albumin. Thanks to ATR-FTIR measurement, it was found that albumin retains more its native conformation on the undoped glass with respect to the silver containing glass, where a more disordered structure was found. This fact can be ascribed to a greater interaction between the proteins and the doped surface, due to the presence of metal ions and more positive charges. Conclusion In conclusion, even though the incorporation of silver ions in a bioactive glass surface does not affect surface properties that are usually addressed as pivotal in protein adsorption, such as roughness and surface energy, the presence of a more positive charge on the surface of the glass and affinity of proteins towards metallic ions seems to be enough to increase adsorption of albumin and strength of the protein-biomaterial interaction. The increased interaction with proteins may be beneficial for the cells response to antibacterial silver containing materials.