Biomaterials, Volume 24, Issue 1, January 2003, Pages 19-26

In vitro corrosion behaviour and osteoblast response of thermally oxidised Ti6Al4V alloy

M. C. García-Alonso^{, , a}, L. Saldaña^b, G. Vallés^b, J. L. González-Carrasco^a, J. González-Cabrero^c, M. E. Martínez^d, E. Gil-Garay^e and L. Munuera<sup>e

a</sup> Centro Nacional de Investigaciones Metalúrgicas, CENIM-CSIC, Avda. Gregorio del Amo 8, Madrid 28040, Spain
^b Unidad de Investigación, Hospital La Paz, Paseo de la Castellana 261, Madrid 28046, Spain
^c Unidad de Investigación, Fundación Jiménez Diaz, Avda. Reyes Católicos 2, Madrid 28040, Spain
^d Departamento de Bioquímica, Hospital La Paz, Paseo de la Castellana 261, Madrid 28046, Spain
^e Departamento de Traumatología y Cirugía Ortopédica, Hospital La Paz, Paseo de la Castellana 261, Madrid 28046, Spain

Received 14 January 2002;  accepted 10 June 2002.  Available online 2 October 2002.

1. Abstract

In this work, the influence of thermal oxidation treatments of Ti6Al4V at 500°C and 700°C for 1 h on the in vitro corrosion behaviour and osteoblast response is studied. The potential of these treatments, aimed to improve the wear surface performance as biomaterial, relies in the formation of an outer "ceramic" layer of rutile. The corrosion behaviour was evaluated in simulated human fluids by electrochemical impedance spectroscopy and anodic polarisation tests. The effect of these thermal oxidation treatments on osteoblastic behaviour was studied in primary cultures of human osteoblastic cells. Results show that thermal oxidation treatments do not decrease the high in vitro corrosion resistance of the Ti6Al4V alloy. Osteoblast adhesion studies indicate that thermal oxidation treatments do not impair the material biocompatibility. Moreover, the thermal oxidation at 700°C enhances the in vitro osteoblastic cell attachment compared to the thermal oxidation at 500°C.

Author Keywords: Corrosion behaviour; Human osteoblastic cells; Thermal oxidation treatment; Ti64V alloy; Cell attachment

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Microstructural characterisation

2.2. Corrosion tests

2.3. Cell culture

2.4. Attachment assays

3. Results

3.1. Microstructural characterisation

3.2. Corrosion characterisation

3.3. Cell attachment

4. Discussion

5. Conclusions

Acknowledgements

References

3. 1. Introduction

Ti6Al4V, hereafter Ti64, is among the most commonly used implant materials, particularly for orthopaedic and osteosynthesis applications. One of the main reasons is the fact that passive surfaces, typically 4-6 nm thick films of amorphous or poorly crystallised and nonstochiometric TiO₂, promote a high stability and a high in vitro corrosion resistance. However, there are evidences that the in vivo conditions can alter the stability of the passive layer. Analysis of retrieval implants points out to a possible accumulation of ions in tissue adjacent to the implant [1]. A probable explanation is that adherence of the passive film is rather poor, and it may be disrupted at very low shear stresses even rubbing against soft tissues [2], which precludes its use for articulating surfaces. The new passive film forms in milliseconds from reaction with the local environment but metal ions are released in the process. The dissolution of titanium into the human body can induce the release of potentially osteolytic cytokines involved in the implant loosening.

In the light of these observations the search for new treatments that improve the abrasive resistance and decrease the titanium release is increasingly important. A collection of surface treatment processes (mechanical treatments, anodising, ultrapassivation, nitriding, ion implantation, etc.) have been all studied to achieve desired surface properties [3]. Thermal oxidation treatments aimed to obtain "in situ" ceramic coatings, mainly based on rutile, can offer thick, highly crystalline oxide films with very good protective performances. Consequently, the use of thermal oxidation treatments has been studied for their possible application in wear- and corrosion-resistant technologies [4 and 5] or implants [6, 7, 8, 9 and 10]. From the biomedical point of view, the study of the corrosion and biological response of these oxide layers is essential to better understand and exploit those surface properties.

The degree of integration of an implant with the human body depends strongly on the surface characteristics. The initial interaction of bone cell with the biomaterial will influence the cell capacity to proliferate and to differentiate itself on contact with the implant. This is essential for successful osseointegration, in which mature living bone lies in direct apposition to the component without intervening fibrous tissue interface. The diversity of cell responses to the different materials tested highlighted the capacity of cells to discriminate between different chemistries [11].

Previous reports have described the relative influence of the topography and chemistry of Ti64 surfaces on osteoblastic cell proliferation and adhesion [12]. This paper assesses the surface modification after thermal oxidation treatments at two different temperatures on the in vitro corrosion resistance and its effect on osteoblastic cell attachment in a primary culture of human osteoblastic cells.

4. 2. Materials and methods

The material used in this research was Ti6Al4V (wt%) alloy in the shape of extruded bars of 10 mm diameter. For the oxidation experiments, discs about 1 mm thick were used. Before oxidation, all major surfaces were abraded on successive finer silicon carbide papers, and then mechanically polished with 1 
m diamond paste to achieve a mirror-like finish. Specimens were washed in running water, and ultrasonically cleaned with alcohol. The microstructure was revealed with an etchant consisting of HNO₃+HCl+HF+water.

The oxidation treatment was performed at 500°C and 700°C for 1 h, in air. After oxidation, samples were removed from the furnace and cooled at room temperature.

2.1. Microstructural characterisation

Microstructural characterisation has been performed using scanning electron microscopy (SEM) and glancing angle X-ray diffraction (GAXRD). Element distribution through the scale was studied by means of a radiofrequency glow discharge optical emission source (GDOES), using a LECO GDS-750A.

2.2. Corrosion tests

The corrosion behaviour was studied by electrochemical impedance spectroscopy (EIS) and anodic polarisation tests of both the as-received state (polished condition) and thermally oxidised samples. The Ringer's solution, which simulates the human body fluids, was used as electrolytic medium. The chemical composition of Ringer's solution is: 8 g/l NaCl, 0.2 g/l CaCl₂, 0.2 g/l KCl, 1 g/l NaHCO₃. Saturated calomel and platinum wire electrodes were used as reference and auxiliary electrodes, respectively. Prior to the beginning of the corrosion measurements, the specimens were maintained for 1 h in the solution. Impedance measurements were performed by applying a sinusoidal wave of 10 mV in amplitude to the working electrode at a frequency range from 64 kHz to 1 mHz. Anodic polarisation curves were drawn from the corrosion potential to the breakdown potential. At this potential, a cathodic sense was imposed. The curves were registered at a scanning rate of 0.6V/h. All corrosion experiments were performed at ambient temperature.

2.3. Cell culture

For the osteoblast attachment tests discs of 6 mm diameter and 1 mm height were used. Both the as-received (polished condition) and thermally oxidised Ti64 samples were studied. The samples were sterilised under ultraviolet light overnight before testing.

Human osteoblastic cells were derived from fresh trabecular bone explants from knee obtained during arthroplasty procedures. Bone samples were obtained from 6 osteoarthritic patients aged 75±5 years. Each sample was processed in a separated primary culture. So as to avoid variations between cultures due to possible heterogeneity, all were taken from the knees of similar aged patients' [13]. None of the patients had evidence of any metabolic bone disease that might have affected their calcium metabolism. The bone explants were cultured as previously described [13]. Briefly, trabecular bone was minced into 0.3-0.5 cm pieces and washed thoroughly in phosphate-buffered saline (PBS) to remove adherent bone marrow cells. The fragments were seeded into 75 cm² tissue culture flasks and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum (FBS), 100 IU/ml of penicillin, and 100 
g/ml of streptomycin, and incubated at 37°C in a humidified atmosphere with 5% CO₂. The medium was changed twice weekly and a confluent monolayer was obtained after 4-6 weeks.

2.4. Attachment assays

Cells were subcultured from those initially isolated and seeded at a density of 8000 cells/well onto polished as-received Ti64 (Ti64), and thermally treated Ti64 at 500°C (Ti64 500) or 700°C (Ti64 700) in 96 well plates. Tissue culture polystyrene (PS) was used as a control substrate. The cells were incubated at 30 min, 1, 2, and 24 h at 37°C in DMEM with 15% FBS. After the incubation period, cells were gently rinsed away with serum-free medium to eliminate any unattached cell. The attached osteoblasts were incubated for 45 min with 8 

2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester (BCECF-AM, Sigma. St. Louis, MO) in serum-free medium. This nonfluorescent esterified dye, when internalised by living cells, is hydrolysed by cellular esterases to a membrane-impermeable fluorescent species and serves as a sensitive quantitative indicator of cell number. Following this, the cells were washed twice in serum-free medium and the attached osteoblasts were quantified in a spectrofluorometer (Luminescence Spectrometer LS50B, Perkin-Elmer, Woburn, MA). All assays were run in triplicate and results were expressed as a percentage of the amount of osteoblasts in the control substrate.

5. 3. Results

3.1. Microstructural characterisation

In the as-received condition the Ti64 alloy clearly shows the expected duplex microstructure containing
- and
-phase, Fig. 1. Confidence about the stability of the microstructure during the thermal oxidation treatments was provided by no significant differences in hardness from 329 to 320 HV0.5 after thermal oxidation at the highest temperature for 1 h.

(21K)

Fig. 1. Microstructure of Ti64 alloy.

SEM examination of the oxidised specimens revealed the presence of a smooth oxide scale without signs of spallation, irrespective of the oxidation temperature. A close examination, Fig. 2, reveals that the scale is decorated with small oxide islands, which morphology, size and distribution are consistent with that of the
-phase in the underlying substrate. Although a denser scale seems to be formed on these zones, GAXRD analysis failed to reveal the presence of other oxidation products than rutile, as can be seen in Fig. 3. However, the presence of other oxidation products cannot be fully discarded regarding the small volume fraction of the islands. As a matter of fact, quantitative depth profiles obtained by GDOES, Fig. 4, reveals the formation of a rutile scale which composition gradually evolves. On the outer surface an aluminium-rich oxide zone is observed, followed by an inner zone in which concentrations of both aluminium and vanadium are low. For the sake of clarity it should be remarked that existence of variations of Al and V in the vicinity of the outer surface is highlighted by the amplification of the scale (×5 and ×10, respectively). It is interesting to note that beneath the scale, about 1 
m thick, an oxygen-enriched zone of less than 2 
m thick is observed. The excess in oxygen in this zone is counterbalanced by depletion in the alloying forming elements. During oxidation, oxygen advance via diffusion ahead of the TiO₂ surface layer hardens and strengths the immediate substrate and provides a built-in interlayer.

(28K)

Fig. 2. SEM examination of the surface of thermally treated Ti64 alloy at 700°C for 1 h.

(8K)

Fig. 3. GADRX difractogram of the thermally treated Ti64 surface at 700°C for 1 h.

(11K)

Fig. 4. Depth profile obtained by GDOES of thermally treated Ti64 alloy at 700°C for 1 h.

3.2. Corrosion characterisation

Fig. 5 shows representative electrochemical impedance diagrams obtained for Ti64 in Ringer's solution, for as-received condition and after thermal treatment at 500°C and 700°C for 1 h. Experimental data are shown together with the fitted data generated using the equivalent circuits shown in Fig. 6. A very good agreement between the fitted and experimental data is obtained, as it can be confirmed by the
² values given in Table 1.

(18K)

Fig. 5. Experimental and fitted impedance Bode diagrams obtained for Ti64 alloy in as-received condition, (a); and after thermal treatment at 500°C and 700°C for 1 h, (b).

(6K)

Fig. 6. Equivalent electrochemical circuits used to simulate the experimental data for Ti64 in as-received conditions, (a); and after thermal treatment at 500°C and 700°C for 1 h, (b).

Table 1. Main parameters obtained from experimental impedance data using the equivalent circuits of Fig. 6

The impedance data obtained for nontreated Ti64 are simulated by a simple RCPE couple (Fig. 6a), in which R_e is the electrolytic resistance, CPE_film corresponds to the double-layer capacity and R_film is the charge transference resistance of the passive layer. In the Bode diagram (Fig. 5a), impedance modulus at the lowest frequencies of about 10⁷ 
and phase angles near -90° at the lowest frequencies are observed. The high R_film values, about 10⁷ 
(see Table 1 and impedance Bode diagram), indicate the formation of a protective passive layer that confers a high corrosion resistance to the Ti64 alloy.

With respect to the data obtained for thermally treated Ti64 samples, the electrochemical equivalent circuit that simulates the corrosion behaviour of the oxidised samples is somewhat different, as it can be seen in Fig. 6b. In this last case, a new box composed of CPE_oxide and R_oxide elements is needed to add to the electrochemical equivalent circuit to fit the experimental data. The presence of this new box reveals the existence of two different time constants that control the corrosion mechanism, as it will be discussed later.

Fig. 7 shows the anodic polarisation curves for the Ti64 alloy in the as-received condition and after heat treatment at 500°C and 700°C for 1 h. In all cases, the passive current density is very low, which indicates the high corrosion resistance of the Ti64 alloy. The polarisation that the Ti64 alloy, without and with heat treatment, is able to withstand before the increase of the current density is very high for every case, irrespective of the oxidation temperature. From this point, the decrease of the polarisation applied in the three conditions produces a decrease of the current density until achieving the previous passive current density values.

(8K)

Fig. 7. Anodic polarisation curves for the Ti64 alloy in as-received condition and after thermal treatment at 500°C and 700°C for 1 h.

3.3. Cell attachment

Fig. 8 shows the temporal profile of the osteoblastic cell attachment on various substrates: PS (control), Ti64, Ti64 500, Ti64 700. The results are expressed as percentage of the osteoblast attachment on PS control at each time. Time course experiments on all tested substrates show that the percentage of attached cells increased with time until 2 h. After this time, there was no significant difference in the percentage of osteoblasts attached on the different substrates.

(8K)

Fig. 8. Osteoblasts attachment on different substrates: PS (Control) (
), polished untreated Ti64 (
), Ti64 500 (
), Ti64 700 (
). Cell attachment under standard cell culture conditions was determined at 30 min, 1, 2 and 24 h. The attached osteoblasts labelled with BCECF-AM were quantified by spectrofluorimetry. The results indicate the percentage of osteoblast attached to Ti64, Ti64 500 and Ti64 700 in relation to PS control. Results are mean ± SE. n=6. (*) p<0.001: Control vs. Ti64, Ti64 500 and Ti64 700 in all time matched values. (#) p<0.05: Ti64 500 vs. Ti64 700 at 1 and 2 h.

Osteoblast attachment on all the implant materials after 30 min, 1 and 2 h was significantly greater than attachment on control (p<0.001). However, after a 24 h attachment period there was no significant difference in the attachment of osteoblasts on any of the materials as compared to control. There was no significant difference between the percentage of attached cells on Ti64 and both thermal oxidation treatments at each time point. Furthermore, the percentage of attachment after 1 and 2 h was significantly (p<0.05) greater on Ti64 700 than time matched Ti64 500.

6. 4. Discussion

Ti64 alloy is widely used as an implant due to the excellent corrosion, biocompatibility and mechanical properties. However, the mechanical properties of the surface, i.e. wear resistance, are very poor. This bad performance can induce to a continuous release of metal debris or corrosion products that both modify the topography of the surface of the implant and are accumulated in the adjacent tissues or migrate to other body organs. For this reason, alternative treatments pointed at improving the wear resistance without compromising the good corrosion and biocompatibility properties are welcomed.

In this work the oxidation treatment has been used in order to generate a more wear-resistant surface making use of the crystalline nature of the thicker oxide layers without minimising the excellent corrosion resistance and biocompatibility of the Ti64 alloy. For this reason, we have tried to confirm both the excellent corrosion and biocompatibility characteristics of the oxidised Ti64 alloy through the electrochemical techniques and osteoblast response analysis, respectively.

As was shown in Fig. 1, Ti64 alloy has a duplex microstructure. It is well known the excellent corrosion behaviour of the Ti64 alloy, which relies on the formation of an amorphous oxide layer of 4-6 nm at the surface. The composition of the oxide passive layer is widely known in the literature [14 and 15]. Studies performed by XPS show that the oxide layer on top of Ti64 alloy is predominantly TiO₂, with lower amounts of suboxides TiO and Ti₂O₃ close to the metal/oxide interface. However, also a little proportion of Al, in the oxidation state of Al³⁺ to form Al₂O₃ has been observed in the oxide/air interface.

The corrosion behaviour of as-received Ti64 alloy studied by EIS indicates the contribution of one time constant associated with the formation of the layer of Ti-oxides that controls the corrosion process and provides a very high corrosion resistance to the alloy. Some authors [16 and 17] have detected a second time constant due to a distinct porous layer that dominates the impedance in the high frequency region in contrast with our experiments. The different results can be explained by considering the nature of the anions in the solution, in our experiments without phosphate ions.

The oxidation treatment at 500°C and 700°C for 1 h promotes the formation of a relative thick oxide layer with different structure and composition to the passive layer. The oxidation in the earlier stages of the Ti64 at high temperatures promotes the oxidation of both Ti and Al in preferential regions of the substrate. At higher temperature and exposure time, the probability to observe also an alumina contribution at the oxide/air interface increases considerably [18]. In Fig. 2, we could see that apparently nodular nuclei are developed on the sample surface together with a thin film of TiO₂. Considering the heterogeneous composition of the duplex microstructure, consisting of platelets of pure
-phase, Al-enriched, surrounded by a mixture of
- and
-phase, a possible explanation of the different morphology of the oxides could be the formation of Al-enriched oxides on the
-phase of the substrate surrounded by Ti-enriched oxides. Nevertheless, the proportion of Al-enriched oxide zones is very low, as could be deduced by GAXRD results. However, the GDOES detected an Al-enrichment on the outer surface, which confirm the presence of Al-oxides on the surface. These Al₂O₃ nuclei grow with increasing exposure time and/or temperature and even could undergrow the TiO₂ scale. This growth mechanism gives rise to the formation of alternative layers of TiO₂ and Al₂O₃ [18]. In our tests, the oxidation treatments were not longer enough to observe these alternative layers. Instead of that, only small Al₂O₃ islands were generated.

Impedance spectroscopy reveals interesting information about the oxidation mechanism. After oxidation treatment, the electrochemical equivalent circuit used to simulate the experimental data was somewhat different to those obtained for Ti64 without oxidation treatment, Fig. 6. The equivalent circuit was composed of two different boxes: R_oxide and CPE_oxide, and R_film and CPE_film. At 500°C, a rutile scale covers the surface in which the Al₂O₃ nuclei begin to appear. At this temperature and treatment time, the proportion of the Al₂O₃ nuclei is very low, thus only a slight deviation of the master line at the intermediate frequencies in the impedance modulus Bode diagram is observed. This feature indicates the existence of two time constants that are very similar and difficult to distinguish in the impedance diagrams. At 700°C for 1 h, the proportion of Al₂O₃ nuclei is higher and its contribution is clearly reflected at the intermediate frequencies in the impedance diagrams. In this case, the deviation from the master line at the intermediate frequencies in the impedance modulus Bode diagram is more evident, revealing two time constants separated enough. The two time constants are associated with the two boxes, R_oxide and CPE_oxide, and R_film and CPE_film, i.e., associated with the oxide layer and the passive film of the Ti64 substrate, respectively. The physical meaning of this equivalent circuit can be the following. In the earlier stages of oxidation treatment a rutile layer covers the preoxidised samples that offers a high corrosion resistance. In this rutile layer, alumina nuclei of lower resistance begin to grow. These nuclei on the rutile surface with a lower resistance (see the R_oxide values), act as diffusion easy-paths through which the electric signal preferentially pass until achieving the Ti64 substrate. The electrochemical impedance spectroscopy is able to detect possible defects or diffusion easy-paths in the thick oxide layer of the thermally treated Ti64 alloy.

The thermal treatment on the Ti64 alloy does not impair the extremely low susceptibility to pitting corrosion of the untreated Ti64 alloy, as could be seen in Fig. 7. In this figure the passive current densities are practically the same as for the untreated alloy. Relevant for this research is that the tested thermal oxidation treatments do not impair the good corrosion behaviour of the Ti64 alloy in the tested time studied. Nevertheless, variations on the corrosion behaviour should not be seen over longer time periods of testing since thermal oxidation treatments on Ti64 alloys have proved [8 and 9] to decrease the metal ion release.

Our experimental findings regarding the chemical properties of thermally oxidised Ti64 alloy are believed to be of significance with respect to some biocompatibility issues in connection with the use of these materials in implants. Properties of the biomaterial surface have critical implications in the nature of cell substrate attachment and may influence in the cellular and molecular events at the bone-implant interface. One of the aims of our study was to assess the effect of the surface modification induced by the thermal oxidation treatments at two different temperatures on osteoblastic cell attachment. Our results show that osteoblast attachment on all the substrates studied was greater than attachment to PS control. In addition, there were no significant differences between the percentage of attachment on Ti64 alloy and both thermal oxidation treatments. However, our results suggest that thermal oxidation at 700°C enhances the osteoblastic cell attachment with respect to both, as-received and at 500°C. The significative differences in the cell attachment are reached when we compare both thermal treatments. Thus, the present findings demonstrate that the thermal oxidation treatments tested would not affect the biocompatibility of the material.

Consistent with our studies, other authors [6] have reported that thermal oxidation of Ti64 alloy at a lower temperature (400°C/0.75 h) does not seem to have any effect on cell activity when testing in femoral head trabecular bone. In addition, the good osteoblastic cell attachment of the specimens preoxidised at 700°C is consistent with the beneficial role of the thermally grown oxides observed on the specific protein adsorption pattern in contact with blood [19].

It is worth remarking that thermal oxidation causes a roughening of the surface due to the selective
-phase oxidation, the effect being more evident for the highest temperature. In previous studies, other authors [12, 20 and 21] have reported that topography, chemistry and surface roughness of orthopedic implants may regulate the proliferation, differentiation and cell attachment of osteoblastic cells. As to how far this feature accounts for the observed improvement of cell attachment should be controlled by testing different exposure times, which should yield to a different roughness.

7. 5. Conclusions

• Thermal oxidation of the Ti64 alloy gives rise to the formation of a rutile scale that has incorporated Al in the outer oxide/air interface.

• A different electrochemical response detected by electrochemical impedance spectroscopy is observed due to the alumina and rutile oxides formed on the Ti64 alloy after oxidation treatment.

• The thermal oxidation treatments do not impair the good in vitro corrosion behaviour and are not detrimental for the biocompatibility of the material. Moreover, the thermal oxidation at 700°C promotes the osteoblastic cell attachment compared to the thermal oxidation at 500°C.

8. Acknowledgements

The authors would like to thank CICYT (Spain) for financial support under Projects MAT99/1154/CO2/O2 and 2FD97-1357. Y. Díaz and M. Berlanga (CENIM) are acknowledged for the technical assistance with the oxidation experiments and Dr. P. Pérez (CENIM) for the useful discussion on the oxidation behaviour of Ti6Al4V alloy.

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