Nanoscale TiO2 nanotubes govern the biological behavior of human glioma and osteosarcoma cells

Abstract

Cells respond to their surroundings through an interactive adhesion process that has direct effects on cell proliferation and migration. This research was designed to investigate the effects of TiO2 nanotubes with different topographies and structures on the biological behavior of cultured cells. The results demonstrated that the nanotube diameter, rather than the crystalline structure of the coatings, was a major factor for the biological behavior of the cultured cells. The optimal diameter of the nanotubes was 20 nm for cell adhesion, migration, and proliferation in both glioma and osteosarcoma cells. The expression levels of vitronectin and phosphor-focal adhesion kinase were affected by the nanotube diameter; therefore, it is proposed that the responses of vitronectin and phosphor-focal adhesion kinase to the nanotube could modulate cell fate. In addition, the geometry and size of the nanotube coating could regulate the degree of expression of acetylated α-tubulin, thus indirectly modulating cell migration behavior. Moreover, the expression levels of apoptosis-associated proteins were influenced by the topography. In conclusion, a nanotube diameter of 20 nm was the critical threshold that upregulated the expression level of Bcl-2 and obviously decreased the expression levels of Bax and caspase-3. This information will be useful for future biomedical and clinical applications.

Keywords: adhesion; apoptosis; migration; nanotopography; proliferation.

Figure 1

Sectional layer of vertically oriented TiO₂ nanotubes with different diameters.

Figure 2

Scanning electron microscopic images of U87 cells cultured on amorphous nanotube coatings. Abbreviation: Ti, titanium.

Figure 3

High magnification scanning electron microscopic images of U87 cells. Notes: (A) U87 cells cultured on 20 nm nanotubes. The red circles indicate the tiny filiform pseudopodia. (B) The ECM component is deposited on the 20 nm nanotubes. (C) U87 cells cultured on 120 nm nanotubes. The red circles indicate the fracture of the cells. (D) Cell extensions protrude to the tube walls. (E) U87 cells cultured on smooth titanium substrate. (F) Microfilaments and pseudopodia stretched on the surface of smooth titanium. Abbreviation: ECM, extracellular matrix.

Figure 4

Expression of vitronectin and p-FAK in U87 glioma cells cultured on nanotubes with different diameters. Notes: (A) Western blot analysis of the expression of vitronectin and p-FAK. (B) Statistical analysis of vitronectin and p-FAK expression. The densities of the vitronectin and p-FAK bands were measured, and the ratio was calculated. *P<0.05, compared with titanium and 20 nm nanotubes. Abbreviations: p-FAK, phosphor-focal adhesion kinase; VN, vitronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ti, titanium.

Figure 5

Cell morphology, crystallinity, and contact angle of the TiO₂ nanotubes. Notes: (A) Scanning electron micrographs of U87 glioma cells on the 20 nm nanotubes with annealing at 450°C. (B) Scanning electron micrographs of U87 cells on the 120 nm nanotubes with annealing at 450°C. (C) X-ray diffraction patterns for amorphous nanotubes and annealed nanotubes. (D) Contact angles of amorphous nanotubes and annealed nanotubes. (E) Expression levels of vitronectin and p-FAK in U87 glioma cells cultured on 20 nm and 120 nm nanotubes with or without annealing. (F) Statistical analysis of vitronectin and p-FAK expression. The densities of vitronectin and p-FAK bands were measured, and the ratio was calculated. *P<0.05, compared with the 20 nm nanotubes. Abbreviations: au, absorbance units; p-FAK, phosphor-focal adhesion kinase; VN, vitronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ti, titanium.

Figure 6

Viability of U87 (A) and MG-63 (B) cells on nanotubes with different diameters after cell culture for 24 hours. The data are presented as the mean ± standard deviation (n=3). *P<0.05 versus titanium, 50 nm, 70 nm, 100 nm, and 120 nm samples. Abbreviations: Ti, titanium; OD, optical density.

Figure 7

(A) Adhesion of U87 cells to titanium and 20 nm and 120 nm nanotubes. *P<0.05 compared with titanium and 120 nm nanotubes. (B, C) Migratory capacity of U87 cells on nanotubes with different diameters without (B) and with (C) annealing. (D, E) Immunofluorescence of acetylated α-tubulin expression in U87 cells cultured on nanotubes with different diameters without (D) and with (E) annealing. Abbreviation: Ti, titanium.

Figure 8

Effect of nanotubes with different diameters on the apoptosis of U87 cells. Notes: (A) Expression of Bcl-2, Bax, and caspase-3 in U87 cells cultured on nanotubes with different diameters without annealing. (B) Statistical analysis of Bcl-2, Bax, and caspase-3 expression. *P<0.05, compared with the 20 nm nanotubes. (C, D) Immunostaining of caspase-3 in U87 cells cultured on nanotubes with different diameters without (C) and with (D) annealing. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ti, titanium.

Figure 9

Double staining of caspase-3 and acetylated α-tubulin expression in MG-63 cells cultured on nanotubes with different diameters without (A) and with (B) annealing. Abbreviations: Ac, acetylated; Ti, titanium.