Titanium surface functionalization via fibrinogen-coated nano-topography: Physicochemical surface characterization and pre-osteoblastic cell response

• Fibrinogen was adsorbed on the Nano-Ti surface at 0.7 mg/cm 2 . • The protein layer preserved the Nano-Ti topography characteristics. • Zeta potential confirmed the presence of a continuous fibrinogen layer. • FG/Nano-Ti enhanced osteogenic differentiation of pre-osteoblastic cells. • Fibrinogen coating may improve bone integration of Ti implants. The aim of this study was to evaluate fibrinogen (FG) coating on a nanostructured titanium surface (Ti) regarding its physicochemical properties and the response of pre-osteoblastic cells in vitro . Commercially pure Ti discs were ground and chemically treated with a 1:1 mixture of 30% hydrogen peroxide and concentrated sulfuric acid (Nano-Ti). FG coating was performed by simple adsorption at its plasma concentration (FG/Nano-Ti). Pre-osteoblastic MC3T3-E1 cells were plated on Nano-Ti and FG/Nano-Ti and cultured for up to 18 days. FG/Nano-Ti resulted in the availability of 0.7 mg/cm 2 of protein on FG/Nano-Ti (BCA). Nano-Ti and FG/Nano-Ti exhibited microscale grooves and a typical nano-topography, with a network of nanopores, which was partially masked for FG/Nano-Ti by the presence of granulated material of homogeneous distribution. The adsorbed protein layer was continuous and probably a few nanometers thick, not affecting the micro-grooves of Nano-Ti. The roughness parameters exhibited higher values for FG/Nano-Ti. Zeta potential titration curves showed an isoelectric point at pH 3.5 for Nano-Ti evidencing hydroxyl functional groups with a weak acidic reactivity and 5.5 for FG/Nano-Ti, which is the same as FG, in agreement with a continuous adsorbed layer. The presence of FG reduced the wettability of the nano-topography in contact with a drop of water or fetal bovine serum, but enhanced it when a blood drop was used. The biological results showed a higher expression of classical osteoblast markers – especially RUNX2 – on FG/Nano-Ti, which corresponded to higher values of alkaline phosphatase activity and mineralization of the cultures. When FG/Nano-Ti was exposed to exogenous thrombin, a homogeneous fibrin fibril was assembled. In conclusion, the strategy of coating FG/Nano-Ti with FG potentiates the capacity of Nano-Ti to promote osteogenic differentiation.