The study of the interaction between the thermally treated Ti (TT-Ti) at 277 °C for 5 hours and the body fluids, ranging from the simplest to the most complex solution is analysed. Electrochemical techniques such as the measurement of the corrosion potential, electrochemical impedance spectroscopy and the polarization curves have been used. The characterization of TT-Ti has been performed by scanning electron microscopy, atomic force microscopy and X-ray Photoelectron Spectroscopy (XPS). The XPS reveals that the peak intensity associated with phosphate and calcium increases as immersion time does. However, the albumin covers rapidly the surface since the C peak intensity remains constant from the first day to the end of immersion time. The calcium ions have a bridging effect on the electrostatic adsorption of phosphate ions as well as that of albumin and the acidic hydroxyl groups of the oxide layer. The impedance measurement shows that the resistance of the oxide layer immersed in albumin and foetal bovine serum decrease probably due to the formation of organometallic complex. The polarization curves reveal that the presence of proteins decreases the current of anodic branch indicating that the proteins work as a barrier on the surface.
One of the most important stages in the life of an implant is the initial interaction between biomaterials and the physiological environment of surrounding tissues. Physiological environment is a complex medium composed of inorganic and organic compounds that compete to be adsorbed on the surface of the biomaterial. At the first step, proteins spontaneously adsorb onto the surface together with other inorganic compounds such as phosphates (Healy and Ducheyne,
The geometrical and chemical properties of biomaterial surfaces direct not only the characteristics of the protein layer but cellular functions such as cell migration, proliferation, modulate phenotypic differentiation and alter the responsiveness to extra-cellular signals (Kasemo,
Further, the lowest oxidation temperatures can have a beneficial effect on the formation of a high coverage of hydroxylated groups on the surface that act as covalent bonds between organometallic compounds and the oxidized titanium surface, increasing the stability of functional organic overlayers (Jones,
In this context, the aim of this work consists of the study of the electrochemical interaction between the Ti surfaces oxidized at low temperature and different inorganic and organic compounds presents in the body fluids, ranging from the simplest to the most complex solution. The influence of each component will be evaluated through the electrochemical response of the oxide layer grown after the oxidation treatment and the chemical composition of the surface by X-ray Photoelectron Spectroscopy (XPS) after several immersion times at the different compounds presents in the body fluids.
Commercial Ti disks (Goodfellow, France) of 25 mm diameter and 2 mm thickness were used as the test specimens. Their surface was ground in water with SiC abrasive paper of increasing fineness, from 400 to 1200 mesh and finally polished with 9 µm diamond. The Ti samples were then washed in distilled water and rinsed ultrasonically in ethanol for 10 minutes. The Ti disks were thermally treated at 277 °C for 5 hours (hereafter TT-Ti samples).
A scanning electron microscope Jeol-6500F equipped with a Field Emission Gun (FEG) coupled with an Energy Dispersive X-Ray (EDX) spectrometer was used to characterize the surface morphology of the TT-Ti samples after the thermal treatment. The images were taken by using secondary electrons.
An Agilent Atomic Force Microscope (AFM) 5100 equipped with a scanner of maximum ranges of 10 µm in the “x” and “y” directions and 4 µm in the “z” direction was used to obtain roughness data and surface images. The images were acquired by using silicon nitride cantilevers with a nominal probe curvature radius of 10 nm and a force constant of 40 N m−1. Images were acquired at a resolution of 512 × 512 points. WSxM software of Nanotec was used (Horcas
The chemical composition at the surface was analyzed using X-ray Photoelectron Spectroscopy (XPS). Photoelectron spectra were obtained with a VG Escalab 200R spectrometer equipped with a hemispherical electron analyzer (pass energy of 50 eV) and a MgKα (hν = 1254.6 eV, 1 eV = 1.6302 × 10−19 J) X-ray source, powered at 120 W. The kinetic energies of photoelectrons were measured using a hemispherical electron analyser working in the constant pass energy mode. The background pressure in the analysis chamber was kept below 2 × 10−8 mbar during data acquisition. The XPS data signals were taken in increments of 0.1 eV with dwell times of 50 ms. Binding energies were calibrated relative to the C1s peak at 284.9 eV. High resolution spectra envelopes were obtained by curve fitting synthetic peak components using the software “XPS peak”. The raw data were used with no preliminary smoothing. Symmetric Gaussian-Lorentzian product functions were used to approximate the line shapes of the fitting components. Atomic ratios were computed from experimental intensity ratios and normalized by atomic sensitivity factors (Wagner
The electrochemical cell with a three-electrode setup was used (Alonso
All the solutions were prepared with ultrapure water by means of a Millipore Milli-Q system (18.2 MΩ-1). Electrochemical tests were performed in different solutions, composed of the same concentration of inorganic and organic compounds as present in Dulbecco's Modified Eagle's Medium (DMEM) culture medium from the most simple to the most complex composition (
Composition of the media (analytical grade reagents)
Solutions | NaCl (mM) | NaH2PO4 (mM) | CaCl2 (mM) | Glucose (mM) | BSA (g l−1) |
---|---|---|---|---|---|
NaH2PO4 | 110 | 0.91 | – | – | – |
NaH2PO4 + CaCl2 | 110 | 0.91 | 1.80 | – | – |
NaH2PO4 + CaCl2 + Glucose | 110 | 0.91 | 1.80 | 25.00 | – |
NaH2PO4 + CaCl2 + BSA | 110 | 0.91 | 1.80 | – | 2.52 |
The measurement of corrosion potential and Electrochemical Impedance Spectroscopy (EIS) was daily performed during 7 days of testing time. On the last day of testing, quasi-steady-state linear polarization measurements were registered. Before the EIS measurements, the corrosion potential was registered for at least 30 minutes until the potential was stabilized. The EIS experiments were performed at the corrosion potential by applying 10 mV amplitude sinusoidal wave at a frequency range from 105 Hz to 10−3 Hz spaced logarithmically (five per decade). Gamry equipment was used to perform the electrochemical tests.
The EIS results were analyzed by fitting the experimental impedance data with electrical equivalent circuit models. The equivalent circuit parameters were calculated by fitting the impedance function to the measured spectra by a Non-Linear Least-Squares program (NLLS program) using Z-plot/Z-view for all the frequencies measured. The criteria used in estimating the quality of the fitting were evaluated firstly, with the lower chi-square value and secondly, with the lower estimative errors (in%) for all the components.
Linear sweep voltammetry was used to record the polarization curves at ±0.5V with respect to Ecorr of TT-Ti electrode after seven days of immersion on each component of the corrosive media (DMEM).
SEM image of Ti sample thermally treated (TT-Ti) at 277 °C during 5 hours.
Dimensions of grooves verified by AFM were established between a range of 350 nm and 500 nm in deep and 2–2.5 µm in width. As an example,
Tapping mode AFM image of TT-Ti sample with height profile.
High-resolution XPS of the Ti2p signal demonstrated that the chemical composition of the oxide film was TiO2 (Ti2p3/2 458.6 eV in
XPS binding energies for different peak components of TT-Ti, P/TT-Ti, PCa/Ti-TT, PCaG/Ti-TT, BSA/Ti-TT and FBS/Ti-TT samples
Surface | Element | Atomic (%) | Assignment | Energy /eV | Intensity (a.u.) |
---|---|---|---|---|---|
TT-Ti | C1s | 3.5 | C-C, C-H | 284.8 | 4366 |
O1s | 73.0 | TiO2 | 529.9 | 20762 | |
Ti2p | 23.5 | Ti-OH | 531.4 | 4598 | |
TiO2 | 458.6 | 16510 | |||
TT-Ti/P | C1s | 36.9 | C-C, C-H | 284.8 | 4438 |
C = O | 286.3 | 1172 | |||
O-C = O | 288.4 | 760 | |||
O1s | 42.7 | TiO2 | 529.9 | 21600 | |
Ti-OH | 531.4 | 7874 | |||
Ti2p | 18.9 | TiO2 | 458.5 | 17613 | |
P2p | 1.5 | PO4 3− | 133.3 | 342 | |
TT-Ti/PCa | C1s | 26.7 | C-C, C-H | 284.8 | 3490 |
C = O | 286.5 | 1095 | |||
O-C = O | 288.5 | 666 | |||
O1s | 53.8 | TiO2 | 529.9 | 21200 | |
Ti-OH | 531.4 | 6863 | |||
Ti2p | 16.8 | TiO2 | 458.6 | 17305 | |
P2p | 1.3 | PO4 3− | 133.5 | 316 | |
Ca2p | 1.4 | CaHPO4 | 347.9 | 1092 | |
TT-Ti/PCaG | C1s | 60.4 | C-C, C-H | 284.8 | 4902 |
C = O | 286.3 | 3771 | |||
O-C = O | 288.2 | 2155 | |||
O1s | 30.7 | TiO2 | 530.1 | 8894 | |
O = C-O | 531.8 | 5820 | |||
Ti2p | 7.5 | TiO2 | 458.6 | 7955 | |
P2p | 0.8 | PO4 3− | 133.5 | 189 | |
Ca2p | 0.6 | Ca2 + | 347.1 | 375 | |
TT-Ti/BSA | C1s | 63.3 | C-C, C-H | 284.8 | 5797 |
C-NH-, C-O | 286.3 | 3810 | |||
CO-NH-, COOH | 288.1 | 2856 | |||
O1s | 13.7 | TiO2 | 529.9 | 5247 | |
C = O, CO-NH-, COOH | 531.6 | 8806 | |||
Ti2p | 5.2 | TiO2 | 458.6 | 4750 | |
N1s | 17.8 | -O = C-NH-, -NH2 | 400.4 | 4711 | |
TT-Ti/FBS | C1s | 57.7 | C-C, C-H | 284.8 | 5085 |
C-NH-, C-O | 286.4 | 3347 | |||
CO-NH-, COOH | 288.1 | 2409 | |||
O1s | 23.8 | TiO2 | 530.1 | 4281 | |
C = O | 531.9 | 7984 | |||
Ti2p | 3.4 | TiO2 | 458.6 | 3478 | |
P2p | 0.3 | PO4 3− | 133.6 | 70 | |
Ca2p | 0.2 | Ca2 + | 347.7 | 144 | |
N1s | 14.6 | -O = C-NH-, -NH2 | 400.8 | 4441 |
The high resolution O1s spectrum shows the separation of the O1s band into two components assigned to TiO2 (529.9 eV) and Ti-OH (531.4 eV in
The high resolution C1s spectrum shows only one peak at 284.8 eV representing carbons in a hydrocarbon environment (C-C, C-H) (Wagner
The XPS spectrum of TT-Ti immersed in the P solution is similar to that obtained for TT-Ti surface in air. Ti2p signal appears with a similar intensity, but lower atomic percentage (
High-resolution XPS of O1s and C1s for PCa, PCaG, and BSA after 7 days of immersion.
After immersion in the PCa solution, the Ti2p spectrum shows the characteristic peak of Ti-O-Ti with similar intensity (
The general spectrum when samples are immersed in the PCaG solution changes drastically. The high resolution C1s spectrum shows a broadening with a contribution of three different components corresponding to carbon in different environments: the first peak, at the lowest binding energy, is assigned to carbon bonded to C or H (C-C, C-H groups); the second peak is attributed to carbon in C = O bond and the third peak, at the highest binding energy, includes the signal for carbon in O-C = O (Mantel and Wightman,
The spectrum measured after seven days in BSA solution is similar to that obtained for Ti in glucose solution. After the deconvolution of the C1s spectrum three peaks are obtained: (1) 284.7 eV for C-C, C = C and C-H bonds; (2) 286.3 eV being C = O bond; (3) 288.1 eV includes signals from peptide bond (CO-NH-) and acidic groups (COOH). These well-identified bonds correspond to the different chemical groups present in the albumin molecule. Both C1s signal and N1s band come from adsorbed protein. The N1s peak is symmetric, centred at 400.4 eV, corresponding to −NH3
+. In fact, the strong adsorption of albumin was most likely due to protonated and positively charged amino groups (e.g., histidine, lysine, and arginine). TT-Ti has a negative charge, and positively charged amino groups in albumin act as anchoring sites in the region of contact between the protein and titanium surface. In accordance with these results the O1s band shows two components: 529.9 eV (Ti-O-Ti) and 531.6 eV (O = C-OH, -O = C-NH) (
No significant differences with respect to the albumin spectra were obtained for TT-Ti surfaces immersed in FBS after seven days (
The heterogeneity of the roughness in TT-Ti surfaces can promote that other species, such as Ca and P, can be also adsorbed on specific sites where adsorption of organic molecules is not so facilitated. On the other hand, the oxidation treatment provides a higher amount of OH-group on the surfaces that can be used to promote the adsorption not only of the organic molecules but also inorganic species.
OCP vs Time of P/TT-Ti (
The evolution of the impedance modulus and shift-phase angle versus frequency Bode diagrams of the TT-Ti samples after seven days of immersion in the PCa, BSA and FBS media appear in
Impedance modulus and phase angle diagrams versus frequency of the Bode plots for: a) PCa/TT-Ti, b) BSA/Ti-TT and c) FBS/TT-Ti, during 7 days.
The impedance measurements in the P and PCa media show the same behaviour so only the results from the PCa medium are shown (
In general, the EIS results for TT-Ti immersed in inorganic media reveal capacitive behaviour due to the control exerted by the formation of TiO2 generated with the oxidation treatment.
The impedance plots of TT-Ti surfaces immersed in BSA and FBS solution for 1 and 7 days (
The impedance diagrams have been fitted considering the electrical equivalent circuits of
Equivalent electrical circuits used for fitting.
Evolution of electric components of the equivalent circuit versus immersion time.
Experimental data from the impedance diagrams and fitting results, obtained from the simulation by using equivalent circuits of
Media | Time |
Cexp (398Hz) |
Re |
Zthreshold, |
Rs |
R2 |
CPE2 |
n2 | R1 |
CPE1 |
n1 | χ2 |
---|---|---|---|---|---|---|---|---|---|---|---|---|
|
1 | 5.96 | 137.3 | 1.33·107 | 141.9 | – | – | – | 1.82·108 | 9.48 | 0.954 | 9.26·10−3 |
2 | 5.99 | 136.5 | 1.24·107 | 140.7 | – | – | – | 5.48·107 | 9.53 | 0.954 | 8.69·10−3 | |
4 | 6.06 | 135.8 | 1.16·107 | 140.3 | – | – | – | 2.97·107 | 9.49 | 0.957 | 8.88·10−3 | |
5 | 5.98 | 135.0 | 9.23·106 | 139.8 | – | – | – | 9.64·107 | 9.11 | 0.963 | 1.28·10−2 | |
6 | 5.92 | 136.0 | 1.48·107 | 140.6 | – | – | – | 1.89·108 | 8.92 | 0.965 | 1.41·10−2 | |
7 | 6.01 | 136.2 | 5.93·106 | 141.4 | – | – | – | 1.38·108 | 9.20 | 0.963 | 1.47·10−2 | |
|
1 | 4.59 | 100.7 | 1.77·107 | 104.4 | – | – | – | 4.49·107 | 6.72 | 0.963 | 1.39·10−2 |
2 | 4.81 | 117.9 | 1.51·107 | 122.7 | – | – | – | 3.78·107 | 7.24 | 0.962 | 1.48·10−2 | |
3 | 4.79 | 111.3 | 1.17·107 | 115.9 | – | – | – | 1.05·108 | 7.04 | 0.963 | 1.50·10−2 | |
4 | 4.76 | 122.2 | 7.95·106 | 127.9 | – | – | – | 1.89·108 | 7.10 | 0.965 | 2.03·10−2 | |
5 | 5.16 | 123.6 | 1.15·107 | 128.2 | – | – | – | 9.21·107 | 7.28 | 0.967 | 1.14·10−2 | |
6 | 4.95 | 119.4 | 1.83·107 | 124.6 | – | – | – | 2.64·108 | 7.29 | 0.966 | 1.82·10−2 | |
7 | 4.91 | 120.4 | 1.19·107 | 125.8 | – | – | – | 4.87·108 | 7.27 | 0.966 | 2.07·10−2 | |
|
1 | 6.17 | 98.5 | 1.03·107 | 99.2 | 148.60 | 4.50 | 1.000 | 7.32·107 | 3.73 | 0.930 | 1.12·10−3 |
2 | 6.08 | 104.8 | 1.03·107 | 105.4 | 155.79 | 4.61 | 1.000 | 6.05·107 | 3.62 | 0.935 | 1.51·10−3 | |
3 | 5.99 | 106.3 | 1.07·107 | 106.9 | 161.08 | 4.54 | 1.000 | 1.33·108 | 3.52 | 0.938 | 1.88·10−3 | |
4 | 6.15 | 104.2 | 1.01·107 | 105.0 | 160.45 | 4.68 | 1.000 | 4.63·107 | 3.64 | 0.936 | 1.90·10−3 | |
5 | 6.23 | 105.8 | 1.00·107 | 106.7 | 165.90 | 5.05 | 0.997 | 4.99·107 | 3.37 | 0.943 | 1.80·10−3 | |
6 | 6.14 | 112.3 | 1.08·107 | 112.8 | 188.18 | 4.95 | 1.000 | 3.52·108 | 3.24 | 0.943 | 1.15·10−3 | |
7 | 6.16 | 118.7 | 1.06·107 | 119.3 | 198.21 | 5.02 | 1.000 | 3.07·108 | 3.27 | 0.943 | 1.13·10−3 | |
|
1 | 7.40 | 93.3 | 4.76·106 | 93.5 | 3.83·106 | 25.42 | 0.933 | 2.44·106 | 14.16 | 1.000 | 1.19·10−3 |
2 | 6.90 | 88.6 | 4.76·106 | 88.9 | 3.46·106 | 28.07 | 0.923 | 2.65·106 | 12.30 | 1.000 | 4.45·10−4 | |
3 | 6.84 | 87.4 | 4.65·106 | 87.6 | 3.26·106 | 31.05 | 0.916 | 2.77·106 | 11.67 | 1.000 | 4.26·10−4 | |
4 | 6.78 | 88.0 | 4.59·106 | 88.2 | 3.10·106 | 32.96 | 0.912 | 2.88·106 | 11.30 | 1.000 | 3.94·10−4 | |
5 | 6.78 | 87.6 | 4.56·106 | 87.8 | 3.07·106 | 32.92 | 0.912 | 2.85·106 | 11.31 | 1.000 | 3.92·10−4 | |
6 | 6.78 | 85.7 | 4.09·106 | 85.9 | 3.01·106 | 26.85 | 0.925 | 2.52·106 | 12.27 | 1.000 | 2.03·10−4 | |
7 | 6.76 | 84.5 | 4.10·106 | 84.7 | 3.00·106 | 26.68 | 0.927 | 2.46·106 | 12.47 | 1.000 | 2.00·10−4 | |
|
1 | 7.97 | 95.9 | 2.45·106 | 97.1 | 1.27·106 | 12.25 | 0.983 | 2.44·106 | 55.36 | 0.928 | 1.28·10−3 |
2 | 7.71 | 95.5 | 2.77·106 | 96.6 | 9.24·105 | 13.61 | 0.973 | 2.44·106 | 34.59 | 0.961 | 2.33·10−3 | |
3 | 7.74 | 94.7 | 2.68·106 | 95.8 | 9.72·105 | 13.26 | 0.971 | 2.32·106 | 38.73 | 0.961 | 2.32·10−3 | |
4 | 7.64 | 95.2 | 2.85·106 | 96.3 | 8.04·105 | 14.46 | 0.974 | 2.67·106 | 29.91 | 0.957 | 2.73·10−3 | |
5 | 7.51 | 94.1 | 3.30·106 | 95.1 | 5.97·105 | 16.91 | 0.980 | 3.42·106 | 22.00 | 0.955 | 2.91·10−3 | |
6 | 7.58 | 93.0 | 3.12·106 | 94.0 | 5.32·105 | 17.64 | 0.983 | 3.19·106 | 21.40 | 0.953 | 2.60·10−3 | |
7 | 7.60 | 91.6 | 3.01·106 | 92.7 | 4.76·105 | 18.33 | 0.986 | 3.06·106 | 20.51 | 0.952 | 2.25·10−3 |
The results obtained from the impedance spectra measured for TT-Ti immersed in NaH2PO4 and (NaH2PO4 + CaCl2) can be assigned to the circuit shown in
When the calcium is added to the phosphate solution, R1 is practically constant over exposure time and CPE1 slightly increase with the exposure time, especially from the first to the fourth day of immersion (
In the presence of glucose the physical arrangement of the electrical equivalent circuit has slightly changed to get the best fitting. The physical arrangement of the electrical equivalent circuit in the presence of glucose to get the best fitting has slightly changed. Taking into account the high resolution XPS spectra, some P and Ca elements could be seen on the TT-Ti surface (
The change in R1 over immersion time of TT-Ti in P, PCa and PCaG solutions agrees with the results obtained by Contu
When titanium is immersed in a solution containing BSA the circuit of
Finally, when TT-Ti samples are immersed in the FBS the impedance plots are very similar to those obtained for the BSA and the impedance spectra are adequately fitted by using the same equivalent circuit. The contribution of P and Ca incorporated into the passive film is so low (comparing P and Ca intensities for solutions containing glucose and FBS) that the best equivalent circuit to fit the experimental data is described in
Linear polarization curves of a) P/TT-Ti (
With respect to the anodic polarization from Ecorr, the passive region does not show significant differences between the polarization curves (
On the other hand, two regions can be identified in the cathodic part of the polarization curve when TT-Ti is immersed in the P, PCa and PCaG solutions (
Tafel slopes and open circuit potentials from polarization curves of P/TT-Ti, PCa/Ti-TT, PCaG/Ti-TT, BSA/Ti-TT and FBS/Ti-TT samples
Solutions | Ecorr (V) | ba (V) | bc (V) |
---|---|---|---|
P | 0.038 | 0.466 | 0.121 |
PCa | 0.079 | 0.421 | 0.187 |
PCaG | 0.104 | 0.360 | 0.160 |
BSA | −0.082 | 0.454 | 0.228 |
FBS | −0.081 | 0.416 | 0.213 |
The
In summary, the electrochemical properties of the physiological medium/TT-Ti surface are completely controlled by the formation of a homogeneously distributed enriched TiO2 surface that provides a high corrosion resistance, even in the presence of proteins that seems to be able to react with the Ti lead to the organometallic complex and producing some decrease in the oxide resistance.
Each component of the culture medium interacts with the TT-Ti surface in different ways:
The Ca and P of the physiological medium are incorporated to the titanium oxide layer slightly increasing the corrosion resistance.
The BSA covers the Ti surface independently of the presence of calcium ions in the solution. The adsorption of BSA in the presence of calcium and phosphate ions inhibits the oxygen diffusion to the electrode surface.
The TT-Ti surface interacts with albumin and fetal bovine serum probably giving rise to the formation of organometallic complex.
The authors thank the financial support under project MAT2011-29152-C02-01.