Surface Modification of Bioactive Glasses and Titanium Alloys for Controlled Proteins Adsorption : Towards predictable cell and bacteria fate

Abstract

Musculoskeletal diseases have been impacting stronger on the worldwide society and healthcare system, due to the continuous population ageing occurring in the last decades. In order to guarantee a proper wellbeing to patients while optimizing the healthcare expenses, a wide range of innovative biomaterials for tissue engineering have been recently developed aiming to a further improvement of the current state of art. Moreover, recent studies have been focusing also on surface modification, with the goal of further tailor the physicochemical properties of the material, while maintaining the optimized bulk features. This leads to a higher control over the biological response. However, it has been proven that, regardless of the promising results after <i>in vitro</i> studies, the 50% of the approved biomaterials are failing <i>in vivo</i>. This critical issue was mainly attributed to the lack of knowledge concerning the interaction of the biomaterials with the proteins adsorbing on them from the surrounding environment, strongly influencing the cell behavior and, consequently, the fate of the implant. Indeed, cells are not interacting directly with the material surface, but with the proteins adsorbing on it at the very early stage after the implantation. Protein distribution, orientation and conformation, as well as the eventual denaturation due to contact with the material, are then crucial in influencing cell adhesion and spreading, and the consequent integration of the implant in the surrounding tissue. In the context of the current work, protein-biomaterial biosystems have been studied to investigate the influence of the material composition and its surface physicochemical properties on protein adsorption. The thesis mainly focuses on bioactive glasses (BGs) and titanium alloys. Four BG compositions, two silicates, one borosilicate and one phosphate glass, have been surface modified to obtain five different surface conditions (bare, treated in two buffer solutions simulating body fluids, and grafted with two different aminosilanes). All the surfaces were physicochemically characterized (SEM/EDS, contact angle, zeta potential, FTIR-ATR spectroscopy, XPS, ICP-OES) and coated with three model proteins (fibronectin, chimeric avidin, streptavidin). The protein quantity, distribution and clustering were evaluated by confocal microscopy as a function of the material composition and surface properties. Successively, proteins were adsorbed on the considered substrates in dynamic conditions to better mimic the adsorption kinetics occurring in vivo, and the surface properties and protein conformation were studied. Cell viability, proliferation, morphology and osteogenesis were evaluated using human adipose stem cells (hASCs), showing a significant impact of protein adsorption on cell orientation and differentiation. Titanium alloys were surface treated to provide bioactivity to the material using a patented protocol and characterized by SEM/EDS, confocal microscopy, profilometry, contact angle and zeta potential. The substrate functionalization with antimicrobial peptides (nisin) was then optimized to add antibacterial properties to the engineered surface for future application in bone tissue regeneration. The successful functionalization with nisin was confirmed and characterized by zeta potential, XPS, surface free energy analysis, and the maintenance of the antimicrobial activity of the peptide even after the coating was confirmed by antibacterial tests performed using <i>Staphylococcus aureus</i>.