Live endothelial cells on plasma-nitrided and oxidized titanium: An approach for evaluating biocompatibility.

Abstract

We evaluated the effects of titanium plasma nitriding and oxidation on live endothelial cell viscoelasticity. For this, mechanically polished titanium surfaces and two surfaces treated by planar cathode discharge in nitriding (36N2 and 24H2) and oxidant (36O2 and 24H2). Surfaces were characterized regarding wettability, roughness and chemical composition. Rabbit aortic endothelial cells (RAECs) were cultured on the titanium surfaces. Cell morphology, viability and viscoelasticity were evaluated by scanning electron microscopy (SEM), methyl thiazolyl tetrazolium (MTT) assay and atomic force microscopy (AFM), respectively. Grazing Incidence X-ray Diffraction confirmed the presence of TiN0,26 on the surface (grazing angle theta 1°) of the nitrided samples, decreasing with depth. On the oxidized surface had the formation of TiO3 on the material surface (Theta 1°) and in the deeper layers was noted, with a marked presence of Ti (Theta 3°). Both plasma treatments increased surface roughness and they are hydrophilic (angle <90°). However, oxidation led to a more hydrophilic titanium surface (66.59°±3.65 vs. 76.88°±2.68; p=0.001) due to titanium oxide films in their stoichiometric varieties (Ti3O, TiO2, Ti6O), especially Ti3O. Despite focal adhesion on the surfaces, viability was different after 24h, as cell viability on the oxidized surface was higher than on the nitrided surface (9.1×103 vs. 4.5×103cells; p<0.05). This can be explained by analyzing the viscoelastic property of the cellular cytoskeleton (nuclear and peripheral) by AFM. Surface oxidation significantly increased RAECs viscoelasticity at cell periphery, in comparison to the nucleus (2.36±0.3 vs. 1.5±0.4; p<0.05), and to the RAECs periphery in contact with nitrided surfaces (1.36±0.7; p<0.05) and polished surfaces (1.55±0.6; p<0.05). Taken together, our results have shown that titanium plasma treatment directly increased cell viscoelasticity via surface oxidation, and this mechanobiological property subsequently increased biocompatibility.