Different diameters of titanium dioxide nanotubes modulate Saos-2 osteoblast-like cell adhesion and osteogenic differentiation and nanomechanical properties of the surface

Abstract

The formation of nanostructures on titanium implant surfaces is a promising strategy to modulate cell adhesion and differentiation, which are crucial for future application in bone regeneration. The aim of this study was to investigate how the nanotube diameter and/or nanomechanical properties alter human osteoblast like cell (Saos-2) adhesion, growth and osteogenic differentiation in vitro. Nanotubes, with diameters ranging from 24 to 66 nm, were fabricated on a commercially pure titanium (cpTi) surface using anodic oxidation with selected end potentials of 10 V, 15 V and 20 V. The cell response was studied in vitro on untreated and nanostructured samples using a measurement of metabolic activity, cell proliferation, alkaline phosphatase activity and qRT-PCR, which was used for the evaluation of osteogenic marker expression (collagen type I, osteocalcin, RunX2). Early cell adhesion was investigated using SEM and ELISA. Adhesive molecules (vinculin, talin), collagen and osteocalcin were also visualized using confocal microscopy. Moreover, the reduced elastic modulus and indentation hardness of nanotubes were assessed using a TriboIndenter™. Smooth and nanostructured cpTi both supported cell adhesion, proliferation and bone-specific mRNA expression. The nanotubes enhanced collagen type I and osteocalcin synthesis, compared to untreated cpTi, and the highest synthesis was observed on samples modified with 20 V nanotubes. Significant differences were found in the cell adhesion, where the vinculin and talin showed a dot-like distribution. Both the lowest reduced elastic modulus and indentation hardness were assessed from 20 V samples. The nanotubes of mainly 20 V samples showed a high potential for their use in bone implantation.

Fig. 1. Histograms of nanotube inner diameters and scanning electron microscopy visualization of nanostructures, created by anodic oxidation at 10 V, 15 V, 20 V on cpTi. Magnification 50k×.

Fig. 2. Concentration of focal adhesion proteins, vinculin (A), talin (B), measured by the enzyme-linked immunosorbent assay (ELISA) in human Saos-2 osteoblast-like cells cultured 24 hours on untreated cpTi, differently nanostructured cpTi by anodic oxidation at 10 V, 15 V, 20 V and control glass. P values < 0.05 (marked by group name) and P < 0.01 (marked by group name and an asterisk) considered significant versus samples labelled above.

Fig. 3. Immunofluorescence staining of vinculin (A–F) and talin (G–L) in human Saos-2 osteoblast-like cells cultured on cpTi samples 48 h after seeding. Untreated cpTi (A and G), nanostructured cpTi by anodic oxidation at 10 V (B and H), 15 V (C and I), 20 V (D and J) and control glass (E and K). Separated channels in photomicrographs of 15 V sample (F and L). Vinculin and talin (green), F-actin (red) and nucleus (blue). Objective 63× , magnification 2× , scale 20 μm, immerse oil.

Fig. 4. Visualization of human Saos-2 osteoblast-like cells adhered on untreated and nanostructured cpTi scaffolds 48 hours after seeding, scanning electron microscopy. Cells cultured on untreated cpTi (A), nanostructured cpTi by anodic oxidation at 10 V (B), 15 V (C), 20 V (D) and control glass (E). Magnification 2k×. The marked squares of SEM photographs of the upper row are magnified in a lower row.

Fig. 5. Proliferation (A), metabolic activity (B) and osteogenic differentiation (C–E) of human Saos-2 osteoblast-like cells cultured on untreated and differently nanostructured cpTi samples. Untreated cpTi (Ti), differently nanostructured cpTi samples by anodic oxidation at 10 V, 15 V, 20 V and control glass. Day (D), no significant differences were observed in the cell proliferation. (B) P values < 0.05 (marked by group name) considered significant versus samples labelled above columns. (C–E) Relative expression of RunX2 mRNA (C), relative expression of osteocalcin mRNA (D), relative expression of osteocalcin mRNA (E) P values < 0.01 (marked by group name) and <0.005 (marked as name of the group with a star) considered significant versus samples labelled above columns.

Fig. 6. Alkaline phosphatase activity of human Saos-2 osteoblast-like cells cultured on untreated and differently nanostructured cpTi samples. Untreated cpTi (Ti), differently nanostructured cpTi samples by anodic oxidation at 10 V, 15 V, 20 V and control glass. P values < 0.05 (marked by group name) and <0.01 (marked as name of the group with a star) considered significant versus samples labelled above.

Fig. 7. Detection of collagen type I (A and B) and osteocalcin (C) in human Saos-2 osteoblast-like cells cultured on untreated cpTi (Ti), differently nanostructured cpTi scaffolds by anodic oxidation at 10 V, 15 V, 20 V and control glass (G). Data shown as collagen type I and osteocalcin fluorescence intensity calculated from photomicrographs. Day (D), P values < 0.05 (marked by group name) considered significant versus samples labelled above columns.

Fig. 8. Immunohistochemical staining of collagen type I in Saos-2 cells cultured on untreated cpTi and differently nanostructured cpTi scaffolds on day 7 (A–E) and day 11 (F–J) of culture. Collagen type I (green) and propidium iodide staining (cell nuclei, red), untreated cpTi (A and F), cpTi modified by anodic oxidation at 10 V (B and G), 15 V (C and H), 20 V (D and I) and control glass (E and J). Objective 20×, magnification 2×, scale bar 20 μm.

Fig. 9. Immunohistochemical staining of osteocalcin (green) and propidium iodide staining (cell nuclei, red) in human osteosarcoma cells Saos-2 cultured on untreated cpTi (A), cpTi modified by anodic oxidation at 10 V (B), 15 V (C), 20 V (D) and control glass (E) on day 11. Objective 40×, magnification 1×, scale bar 20 μm, immerse oil.

Fig. 10. Depth profiles of mechanical properties differently nanostructured cpTi. There is clearly visible influence of porosity on graphs. Mechanical properties decrease with increasing porosity. Based on rule the 1/10 and length of nanotubes in the Table 2 we can accept measured data up to the contact depth h_c = 46.7 ± 2.4 nm for 10 V, h_c = 73.1 ± 3.2 nm for 15 V and h_c = 109.7 ± 7.5 nm for 20 V.

Fig. 11. Depth profile of hardness. Zone I: initial compression of nanotubes in axial direction. Zone II: combination of compression and lateral bending of nanotubes. Zone III: combination of densification and influence of substrate.