Effect of various surface modifications on c.p. Ti and Ti6Al4V on the adsorption of proteins relevant for osseointegration and inflammatory response

In the race towards the perfect implantable material, titanium and its alloys have been subjected to a great number of different surface treatments. A fundamental step in this way is to understand how biological fluids and proteins interact with biomaterials, since the layer of adsorbed proteins strongly influences the cell-surface interfacial region. In this work, c.p. Ti and Ti6Al4V alloy with different surface modifications are investigated to understand how the various surface characteristics, such as roughness, chemistry, zeta potential, hydroxylation degree, wettability and surface energy, affect protein adsorption. A set of conventional and innovative techniques is here proposed to characterize different aspects of protein adsorption on surfaces designed for implantation. Titanium samples were subjected to three different surface modifications for bioactivity enhancement: acid etching followed by controlled oxidation in hydrogen peroxide on Ti6Al4V: combined alkali-acid-heat treatment on Ti; alkali-heat treatment followed by Sr and Ag ionic exchange, for adding antibacterial properties, on Ti6Al4V. Adsorption properties of these surfaces were evaluated using two proteins, albumin and fibronectin, which are relevant in the osseointegration and inflammatory pathway of implantable biomaterials. Quantification and assessment of adsorbed proteins was obtained by more conventional techniques such as bicinchoninic acid assay, XPS or fluorescent-labeled proteins and it was found that surface energy and micro- and/or nano-structuration play a major role in determining the amount of adsorbed proteins. An innovative approach to surface potential imaging, by means of Kelvin Probe Force Microscopy, allowed to visualize the layer of adsorbed proteins and to study its homogeneity, while proteins conformation and orientation upon adsorption was investigated thanks to novel Surface Enhanced Raman Spectroscopy method and to solid surface zeta potential titration measurements. As to get closer to the implantation environment, competitive and subsequent adsorption of albumin and fibronectin were also investigated.