Morphological alterations of T24 cells on flat and nanotubular TiO2 surfaces

Abstract

Aim: To investigate morphological alterations of malignant cancer cells (T24) of urothelial origin seeded on flat titanium (Ti) and nanotubular TiO(2) (titanium dioxide) nanostructures.

Methods: Using anodization method, TiO(2) surfaces composed of vertically aligned nanotubes of 50-100 nm diameters were produced. The flat Ti surface was used as a reference. The alteration in the morphology of cancer cells was evaluated using scanning electron microscopy (SEM). A computational model, based on the theory of membrane elasticity, was constructed to shed light on the biophysical mechanisms responsible for the observed changes in the contact area of adhesion.

Results: Large diameter TiO(2) nanotubes exhibited a significantly smaller contact area of adhesion (P<0.0001) and had more membrane protrusions (eg, microvilli and intercellular membrane nanotubes) than on flat Ti surface. Numerical membrane dynamics simulations revealed that the low adhesion energy per unit area would hinder the cell spreading on the large diameter TiO(2) nanotubular surface, thus explaining the small contact area.

Conclusion: The reduction in the cell contact area in the case of large diameter TiO(2) nanotube surface, which does not enable formation of the large enough number of the focal adhesion points, prevents spreading of urothelial cells.

Figure 1

Scanning electron microscopy images of surface layers of self-assembled vertically aligned TiO₂ nanotubes synthesized by anodization method. Ethylene glycol solution with 0.3 wt % NH₄F and 1% volume water was used in preparation. High-resolution scanning electron microscopy FEG-SEM 7600F from JEOL was used. Images were taken at 100000× magnitude using low accelerating voltage of 2kV. The internal TiO₂ tube diameter was around 50-60 nm (A-D) and 100 nm (E-F) while the length could be up to 10 micrometers (B).

Figure 2

Urothelial T24 cells on (A) large diameter (of 50-100 nm diameter) TiO₂ nanotubes and on (B) flat titanium surfaces. The surface topography of urothelial cells reveals smaller cell diameter and numerous membrane protrusions (eg, nanotubular structures – thin arrows and pleomorphic microvilli – arrowheads) on the large diameter TiO₂ nanotubes (A), whereas, on the flat titanium surface, the cells are considerably larger in size and the cell membrane is smoother (asterisks) (B). Scale bars: 10 μm.

Figure 3

Urothelial T24 cells on TiO₂ and Ti scaffold. One half of the scaffold was flat titanium and another half was constructed into TiO₂ nanotubes. Urothelial cells growing on the TiO₂ nanotube surface are smaller, more spherical (ie, less flattened) and have numerous long membrane exvaginations. Their apical surface is covered by pleomorphic microvilli (arrowheads). The gondola-like structures are indicated by arrows. The urothelial cells on the flat titanium surface are flatter than cells on the large diameter TiO₂ nanotube surface. Their apical surface is smooth (asterisk), only few microvilli are seen. Scale bar: 10 μm.

Figure 4

Summary statistics of changes in surface area of T24 cells grown on large diameter (50-100 nm) nanotubular TiO₂ surfaces and on flat titanium surfaces. Note the clear difference in size between the traced cells (examples in yellow) on the nanotubular surface (A) in comparison to the cells (examples in red) on the flat titanium surface (B). Statistical analysis showed a significant (P < 0.0001***) increase in the surface area of T24 cells on flat surface when compared to T24 on 50-100 nm nanotubes (C). Data are reported as mean ± standard deviation.

Figure 5

The effects of small diameter (15 nm) and large diameter (100 nm) nanotubular TiO₂ surfaces, as well as flat (amorphous) titanium surfaces, on cell membrane adhesion. The modeled membrane segment distributed with integrins of positive spontaneous curvature is initially positioned 100 nm above the flat surface (A), the 15 nm diameter nanotube surface (B); see right inset), and the 100 nm diameter nanotube surface (C); see right inset). The membrane adhesion is driven by the negative adhesion energy and the positive spontaneous curvature of integrins. The membrane shapes and integrin density distributions are obtained by the minimization of the membrane free energy. Steady state membrane shapes h(x) (D-F) and integrin density distributions n(x) (G-I) of the cell membrane adhered to the corresponding surfaces are plotted. Note the cell membrane adhesion to the flat titanium surface and to the 15 nm TiO₂ nanotube surface, in comparison to the hindered adhesion to the 100 nm TiO₂ nanotube surface.

Figure 6

A possible mechanism underlying the weak adhesion of a T24 cell membrane to large diameter (100 nm) TiO₂ nanotubular surfaces. The low density of TiO₂ nanotube edges in the case of large diameter TiO₂ surface presents less adhesion areas for integrins and fibronectins, and thus, the adhesion to 100 nm nanotubes is weak (A). Furthermore, the long distance (100 nm) between neighboring nanotubes may not facilitate the cross-interaction between integrins, which may be crucial for the formation of a focal adhesion (in close-up). On the flat titanium surface, the even distribution of integrins that make contact with the substrate surface may facilitate the formation of focal adhesions, and thus, explaining the observed increase in cell diameter and cell flattening (in close-up) (B).