Electrochemical characterization of MC3T3-E1 cells cultured on γTiAl and Ti-6Al-4V alloys

Abstract

Electrochemical impedance spectroscopy (EIS) was used to study the behavior of MC3T3-E1 cells cultured in an αMEM+FBS solution on two Ti-based alloys (Ti-6Al-4V and γTiAl) for 4, 7 and 14 days. EIS measurements were carried out at an open-circuit potential in a 1 mHz to 100 kHz frequency range. Results indicate a general increase in impedance on the Ti alloy surfaces with cells as a function of time. Bode plots indicate changes corresponding to the passive oxide film, adsorption of proteins and cell tissue on surfaces with the passage of time. Normal cellular activity based on the polygonal morphology, with long and fine cytoplasmic prolongations of the cells on Ti-6Al-4V and γTiAl was observed from SEM images. Similarly, mineralization nodules corresponding to cell differentiation associated with the osseogenetic process were observed confirmed by Alizarin Red S staining. Immunofluorescence analysis to detect the presence of collagen Type I showed an increase in the segregation of collagen as a function of time. The impedance values obtained from EIS testing are indicative of the corrosion protection offered to the Ti alloy substrates by the cell layer. This study shows that γTiAl has better corrosion resistance than that of Ti-6Al-4V in the αMEM+FBS environment in the presence of MC3T3-E1 cells.

Keywords: EIS; Electrochemical characterization; MC3T3-E1 cells; Ti alloys.

Figure 1

Schematic diagram of the electrolytic cell.

Figure 2

SEM images of (a) Ti-6Al-4V without cells, (b) Ti-6Al-4V with cells at 4 days, (c) γTiAl without cells and (d) γTiAl with cells at 4 days. Red arrow heads indicate inorganic constituents and blue arrow heads indicate nodules of mineralization. A high magnification image of the mineralized nodule (e) and its corresponding energy dispersive spectrum (f) are also shown.

Figure 3

SEM images of (a) Ti-6Al-4V with cells for 4 day incubation, (b) Ti-6Al-4V with cells for 14 day incubation, (c) γTiAl with cells for 4 day incubation and (d) γTiAl with cells for 14 day incubation. F: Filopodia, L: Lamellipodia, B: Blebs; Semicircles represent intercellular connections. Samples at 7 days of incubation were not included since they exhibit similar morphology.

Figure 4

Alizarin Red S staining of cell culture incubated on glass coverslip (control) for (a) 4 days, (d) 7 days, (g) 14 days, on Ti-6Al-4V for (b) 4 days, (e) 7 days, (h) 14 days and on γTiAl for (c) 4 days, (f) 7 days and (i) 14 days. Pinkish stains are generally observed on all surfaces. Dark particles appear to correspond to mineralized nodules.

Figure 5

Confocal Laser Scanning Microscopy images corresponding to nucleus of the cell (DAPI labeled, blue) and collagen type I (Rhodamine labeled, red) in surfaces with MC3T3-E1 cells cultured at 5% CO₂ in air atmosphere and at 37°C on Ti-6Al-4V for (a) 4 days, (b) 7 days and (c) 14 days and γTiAl for (d) 4 days, (e) 7 days and (f) 14 days. Magnification 60X.

Figure 6

Intensity of rhodamine fluorescence corresponding to the segregation of collagen on glass coverslip, Ti-6Al-4V and γTiAl.

Figure 7

Bode impedance (a) Bode phase angle (b) and Nyquist (c) plots for Ti-6Al-4V without cells in αMEM+FBS solution.

Figure 8

Bode impedance (a) Bode phase angle (b) and Nyquist (c) plots for γTiAl without cells in αMEM+FBS solution.

Figure 8

Bode impedance (a) Bode phase angle (b) and Nyquist (c) plots for γTiAl without cells in αMEM+FBS solution.

Figure 9

Bode impedance (a) Bode phase angle (b) and Nyquist (c) plots for Ti-6Al-4V incubated with cells in αMEM+FBS solution.

Figure 10

Bode impedance (a) Bode phase angle (b) and Nyquist (c) plots for γTiAl incubated with cells in αMEM+FBS solution.

Figure 11

Cyclic voltammograms for collagen detection in MC3T3 mouse preosteoblasts cultured on Ti-6Al-4V and γTiAl samples for (a) 4 days, (b) 7 days and (c) 14 days. Data for control sample of collagen and αMEM+FBS solution are also shown for comparison. Glassy carbon was used as working electrode and Ag/AgCl as reference electrode.