TiO2 Nanotopography-Driven Osteoblast Adhesion through Coulomb's Force Evolution

Abstract

Nanotopography is an effective method to regulate cells' behaviors to improve Ti orthopaedic implants' in vivo performance. However, the mechanism underlying cellular matrix-nanotopography interactions that allows the modulation of cell adhesion has remained elusive. In this study, we have developed novel nanotopographic features on Ti substrates and studied human osteoblast (HOb) adhesion on nanotopographies to reveal the interactive mechanism regulating cell adhesion and spreading. Through nanoflat, nanoconvex, and nanoconcave TiO₂ nanotopographies, the evolution of Coulomb's force between the extracellular matrix and nanotopographies has been estimated and comparatively analyzed, along with the assessment of cellular responses of HOb. We show that HObs exhibited greater adhesion and spreading on nanoconvex surfaces where they formed super matured focal adhesions and an ordered actin cytoskeleton. It also demonstrated that Coulomb's force on nanoconvex features exhibits a more intense and concentrated evolution than that of nanoconcave features, which may result in a high dense distribution of fibronectin. Thus, this work is meaningful for novel Ti-based orthopaedic implants' surface designs for enhancing their in vivo performance.

Keywords: Ti implant; cell adhesion; cell−material interaction; nanotopography; protein adsorption.

Figure 1

Topographical characterization of nanoflat, nanoconvex, and nanoconcave. (a) Topography of nanoflat, nanoconvex, and nanoconcave by SEM, illustrates the nano scale flat of nanoflat, and highly ordered arrangement of subunits on nanoconvex and nanoconcave. (b) Three-dimensional illustration of nanoflat, nanoconvex, and nanoconcave by AFM tapping mode in the 500 × 500 nm² square area. (c) Sectional dimensions of three topographies. (d) Based on the AFM section measurement, the statistic dimensions of nanoflat (height), each subunit (height and width) of nanoconcave and nanoconcave (height and width). Ten subunits on different nanotopographies were analyzed (n = 10). ***, p < 0.001; n.s., not significant. Error bars represent the standard error with the mean (s.e.m). (e) Surface area of nanoconvex and nanoconcave. Nanoconvex and nanoconcave are seen as a spherical dome. (f) Schematic illustration of dimensions of nanoconvex and nanoconcave in average, respectively.

Figure 2

Chemical composition analysis by XPS. (a) High-resolution scan of O1s of nanoflat. (b) High-resolution scan of O 1s of nanoconvex. (c) High-resolution scan of O 1s of nanoconcave. (d) High-resolution scan of Ti 2p of nanoflat. (e) High-resolution scan of Ti 2p of nanoconvex. (f) High-resolution scan of Ti 2p of nanoconcave. (g) Full spectra scan of nanotopographies.

Figure 3

Relative surface potential distribution on nanoflat, nanoconvex, and nanoconcave. (a) AFM topographical characterization of nanoflat, nanoconvex, and nanoconcave by the tapping mode in 1 × 1 μm² square area. (b) Surface potential distribution measured by KPFM with the sample bias model in identical topography 1 × 1 μm² square area. The surface potential of each topography displays highly correlated with the topographical features. With the sample bias model, topographical bumps on nanoconvex are shown bowls in potential distribution, and nanoconcave displays a constant shape in both topography and surface potential distribution. (c) Sectional potential distribution in relative value on nanoflat, nanoconvex, and nanoconcave. (d) Absolute potential difference on each subunit of nanoflat, nanoconvex, and nanoconcave. Ten subunits on different nanotopographies were analyzed (n = 10). ***, p < 0.001; n.s., not significant. Error bars represent standard error compared with the mean (s.e.m). (e) Measurement of surface potential of nanoconvex and nanoconcave calibrated by HOPG.

Figure 4

F-actin and FA quantification. (a) HObs after 3 h on FN adsorbed nanoflat, nanoconvex, and nanoconcave. First column shows F-actin cytoskeleton arrangement, second one is FA plaques (vinculin), and third one is merged image. (b) Cell spreading area measurement. (c) Aspect ratio measurement per cell. (d) The length of F-actin per cell. (e) F-actin cluster width per cell. (f) FA size per cell. (g) Focal adhesion quantification per cell. 15 cells on different nanotopographies were analyzed (n = 15). ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant.

Figure 5

Cell morphological features by SEM. (a) HObs after 3 h on FN adsorbed nanoflat. (b) Cell morphological features on nanoconvex. (c) Cell morphological features on nanoconcave. (d) Filopodia extensions on nanoflat. (e) Filopodia extensions on nanoconvex. (f) Filopodia extensions on nanoconcave. (g) Filopodia features on nanoflat. (h) Filopodia features on nanoconvex. (i) Filopodia features on nanoconcave.

Figure 6

Charge density evolution on nanoconvex and nanoconcave of FN adsorption. (a) Distribution of charge density and maximum charge areas while FN from 100 to 5 nm with excursion of 10 nm. (b) Schematic illustration of Coulomb’s force (F_C), horizontal (F_H), and vertical (F_V) vector. (c) Variation of Coulomb’s force (F_C) and the distance between FN and nanoconvex/nanoconcave. The F_C of nanoconvex increased dramatically with the FN attracted by the surface from 100 to 5 nm, and the maximum of F_C reaches to 2.85 nN at d = 5 nm.

Figure 7

Simulation and experimental investigation of FN–subunit interactions. (a) Dynamic map of the charge density of regions on a subunit of nanoconvex and nanoconcave when FN is adsorbing from 100 to 0 nm. When FN is attracted to the subunit, the charge density on nanoconvex top (A) is greatly increased, the charge density on ridge (B) is slightly decreased. Charge density on nanoconcave bottom region (A) is increased in the range from 100 to 20 nm, and decreased from 20 to 0 nm, charge density on ridge (B) is decreased from 100 to 5 nm, and slightly increased from 5 to 0 nm. (b) In situ topographical characterization of nanoconvex and nanoconcave in PBS solution and in FN solution. Sectional dimension has shown that the FN is mainly adsorbed at the top region on the nanoconvex, and the FN was distributed homogeneously on the nanoconcave. (c) Illustration of the FN adsorptive distribution of nanoconvex and nanoconcave, the high dense distribution (<80 nm) could increase the ligand density and form matured FA, the low density of distribution (>80 nm) forms dot FA.

Figure 8

Morphological comparison analysis of nanoflat in PBS/FN and the conformation of FN adsorbed on nanoflat. (a) Topological characterization of nanoflat in PBS. (b) Topological characterization of nanoflat with adsorbed FN, the FN on nanoflat exhibited compact conformation. 1, 2, and 3 are the sectional tracks of morphological comparison between nanoflat in PBS/FN.

Figure 9

Morphological analysis of FN adsorption on nanoconvex and nanoconcave surfaces. (a) AFM imaging of FN on nanoconvex in PBS and FN, nanoconvex tip is smooth in PBS and rough in FN. (b) AFM imaging of FN on nanoconcave in PBS and FN, the morphology of different regions on concave have shown identical features.