Dynamics of Fibronectin Adsorption on TiO₂ Surfaces

Abstract

In the present work we analyze the dynamics of fibronectin (FN) adsorption on two different stable titanium oxides, with varied surface roughness, and chemically similar to those used in clinical practice. The two types of titanium oxide surfaces used were TiO2 sputtered on Si (TiO2 sp) and TiO2 formed on commercially pure titanium after immersion in H2O2 (TiO2 cp). Surface characterization was previously carried out using different techniques (Sousa, S. R.; Moradas-Ferreira, P.; Melo, L. V.; Saramago, B.; Barbosa, M. A. Langmuir 2004, 20 (22), 9745-9754). Imaging and roughness analysis before and after FN adsorption used atomic force microscopy (AFM) in tapping mode, in air, and in magnetic alternating current mode, in liquid (water). FN adsorption as a function of time was followed by X-ray photoelectron spectroscopy (XPS), by radiolabeling of FN with 125I (125I-FN), and by ellipsometry. Exchangeability studies were performed using FN and HSA. AFM roughness analysis revealed that, before FN adsorption, both TiO2 surfaces exhibited a lower root-mean-square (Rq) and maximum peak with the depth of the maximum valley (Rmax) roughness in air than in water, due to TiO2 hydration. After protein adsorption, the same behavior was observed for the TiO2 sp substrate, while Rq and Rmax roughness values in air and in water were similar in the case of the TiO2 cp substrate, for the higher FN concentration used. Surface roughness was always significantly higher on the TiO2 cp surfaces. AFM led to direct visualization of adsorbed FN on both surfaces tested, indicating that after 10 min of FN incubation the TiO2 sp surface was partially covered by FN. The adsorbed protein seems to form globular aggregates or ellipsoids, and FN aggregates coalesce, forming clusters as the time of adsorption and the concentration increase. Radiolabeling of FN revealed that a rapid adsorption occurs on both surfaces and the amount adsorbed increased with time, reaching a maximum after 60 min of incubation. Time dependence is also observed for the evolution of the atomic (%) of N determined by XPS and by the increase of the thickness by ellipsometry. TiO2 cp adsorbs more FN than the TiO2 sp surfaces, after 60 min of adsorption, as shown by the radiolabeling data. FN molecules are also more strongly attached to the former surface as indicated by the exchangeability studies. The overall results provide novel evidence that FN spontaneously adsorbs as a self-assembly at TiO2 surfaces as a function of time. The aggregate structure is an intermediate feature shared by some protein fibrillar assemblies at interfaces, which is believed to promote cell adhesion and cytoskeleton organization (Pellenc, D.; Berry, H.; Gallet, O. J. Colloid Interface Sci. 2006, 298 (1), 132-144. Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell Sci. 2000, 113 (10), 1677-1686).

Osteogenic response of human mesenchymal stem cells to well-defined nanoscale topography in vitro

“…Cell adhesion to several material surfaces is dependent on the prior adsorption of specific serum proteins, including fibronectin and vitronectin, 53 which support cell attachment to the surface and influence cell morphology. [54][55][56] In this study, we demonstrated that hMSC morphology was affected by the underlying surface topography, specifically in terms of cell spread and the number of filopodia.…”

Electrospray deposition in vacuum as method to create functionally active protein immobilization on polymeric substrates

“…It is of interest to note that the measured roughness value for the lower protein surface coverage corresponding to Ra = 1.8 ± 0.2 nm is lower than for the bare surface. This could indicate a partial filling of "valleys" with FN molecules as was observed for the adsorption of FN molecules into valleys on titanium oxide surface [32] or the "cavities" on a poly(ethylene glycol) substrate [33]. The increased deposition of fibronectin on the substrate in the middle of the deposit (as previously confirmed from the fluorescence data analysis) led to the increased roughness observed in this area which may be related to disorder in the arrangement of adsorbed protein molecules [33,34].…”

“…The deposition of fibronectin was Previous studies [30,32,33] have demonstrated that proteins may be deposited with functional integrity using various methods to maintain protein stability. In this study the assessment of protein function has been extended by studying the cellular response to electrospray deposits.…”

Large-scale functionalization of biomedical porous titanium scaffolds surface with TiO2 nanostructures

“…5) as well as the aspect ratio of the nanostructures. And many researches demonstrated that the protein adsorbed on the nanostructures would mediate the cell nanotopographical interactions via the mechanotransductive cascade [27,28].…”