Composition of surface oxide film of titanium with culturing murine fibroblasts L929

Sachiko Hiromoto^{, , a}, Takao Hanawa^a and Katsuhiko Asami<sup>b

a</sup> Biomaterials Center, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan
^b Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku Sendai 980-8577, Japan

Received 2 March 2003;  accepted 22 July 2003. ; Available online 24 September 2003.
Biomaterials
Volume 25, Issue 6 , March 2004, Pages 979-986

1. Abstract

Changes in the composition of surface oxide film on titanium specimens in the presence of amino acids, serum proteins, and cells were characterized using X-ray photoelectron spectroscopy. The surface oxide film on titanium formed in the air is so protective that the further oxidation of titanium is prevented in various circumstances. During immersion of the specimen in Hanks' solution, Eagle's minimum essential medium (MEM), and MEM with the addition of fetal bovine serum (MEM+FBS), calcium phosphate precipitated, causing the increase in thickness of the surface oxide film. Calcium phosphate was also precipitated with culturing murine fibroblast L929, but the amount of the calcium phosphate was smaller than those in Hanks' solution, MEM, and MEM+FBS. The relative concentration ratio of calcium to phosphorous, [Ca]/[P], increased with proteins charging negatively, while the ratio decreased with the cells whose extracellular matrix charging positively. In addition, sulfur precipitated as S⁰ and/or S²⁻ only with culturing the cells. Sulfate ions in the MEM+FBS are reduced at the interface between titanium and the solution with the existence of cells.

Author Keywords: Titanium; Surface analysis; Amino acid; Protein; Cell culture; Biomaterial

2. Article Outline

1. Introduction

2. Experimental methods

2.1. Specimens

2.2. X-ray photoelectron spectroscopy

3. Results and discussion

3.1. X-ray photoelectron spectroscopy spectra

3.2. Composition and thickness of surface oxide film

3.3. Oxygen in surface oxide film

4. Conclusions

References

3. 1. Introduction

Tissue compatibility of materials is governed by the interactions between the surfaces of the materials and the tissues at the initial stage. Titanium and its alloys show good tissue compatibility in the human body, which is owned by their high corrosion resistance and probably by the precipitation of calcium phosphate on themselves [1, 2 and 3]. High corrosion resistance of titanium and its alloys is attributed to thin surface oxide film, so-called passive film. However, the passive film is continuously dissolved and reconstructed in aqueous solutions from the microscopical viewpoint [4]. These partial reactions cause the dissolution of alloying elements and incorporation of various elements from solutions into the film, leading to the change of surface composition.

Precipitation of calcium phosphate on titanium is observed mainly in Hanks' solution [2, 5, 6, 7 and 8] and it is considered that preferential adsorption of phosphate ions is the first stage of the precipitation followed by binding of calcium ions [2 and 6]. Here, Hanks' solution contains only inorganic ions such as sodium, chloride, calcium, and phosphate ions and its ion composition is similar to that of extracellular fluid. On the other hand, in the human body, the body fluids contain not only inorganic ions but also biomolecules such as amino acids and proteins. Also, tissues (cells) attach to the materials implanted in the body. Surface compositions of titanium and its alloys in the presence of biomolecules and cells are different from that in Hanks' solution. When titanium is immersed in Hanks' solution containing albumin, albumin is incorporated into a heterogeneous and porous apatite formed [9]. Relative concentration ratio of calcium to phosphorous of calcium phosphate on titanium implanted in human jaw is higher than that on titanium immersed in Hanks' solution [1, 2, 6 and 10]. Also in the human jaw, sulfur, probably originated from proteoglycans, is usually incorporated into the surface oxide film of titanium as well as calcium and phosphorous [10 and 11]. Incorporation of sulfur is also observed on the intramedullary nail of Ti-6Al-4V alloy implanted in human bone marrow [12]. However, surface and interfacial reactions of titanium and its alloys in the presence of biomolecules and cells are not precisely characterized. To perform the characterization of metallic biomaterials, the effects of biomolecules and cells on the surface composition must be investigated.

In the case of 316L stainless steel and Co-Cr-Mo alloy, surface characterization was performed on each specimen after culturing murine fibroblasts L929 on the surface [13 and 14]. The existence of L929 causes the precipitation of sulfite and/or sulfide on the surface oxide film of 316L steel although only sulfate was on the film without L929 [13]. The stainless steel incorporates sulfur also when it is implanted in soft tissue [10]. On the Co-Cr-Mo alloy, L929 retards the precipitation of calcium phosphate on the surface oxide film [14], indicating that the existence of cells prevent the adsorption of calcium and phosphate ions. The culturing cells on the metallic specimen gives the chemically different environment from that without cells, that may be the similar environment to that in vivo.

L929 is cultured on titanium plate, followed by anodic and cathodic polarization test and AC impedance measurement [15]. Cathodic reactions, probably a reduction of dissolved oxygen, decreases and diffusion resistance parameter in impedance measurement increases with the existence of L929. Those indicate that the diffusion of dissolved oxygen to titanium is prevented with L929, leading to the lack of dissolved oxygen at the interface between titanium and L929. As the result, the reducing environment is generated at the interface. Therefore, the existence of cells must influence the surface composition of titanium.

In the present study, effects of amino acids, proteins, and cells on the surface composition of titanium specimen were examined. Titanium plate was immersed in Hanks' solution, Eagle's minimum essential medium (MEM), and MEM with the addition of fetal bovine serum (MEM+FBS) for 605 ks (7 days). In addition, murine fibroblast L929 was cultured on the plate. Subsequently, the surface composition of titanium was characterized using X-ray photoelectron spectroscopy (XPS). The obtained data should be useful to understand the change in surface composition of titanium and its alloys in the human body.

4. 2. Experimental methods

2.1. Specimens

Commercially pure titanium plate (99.5%) (5 mm×5 mm×1 mm) was polished with 600 grid SiC paper in deionized water. The specimens were ultrasonically rinsed in deionized water and acetone and dried for a few days in a dessicator to generate air-formed passive film. The specimen polished was named "Polished". The "Polished" specimen was sterilized in an autoclave under a saturated water vapor pressure at 120°C for 1.8 ks. The specimen after autoclaving was named "Autoclaved".

Subsequently, the "Autoclaved" specimen was immersed in 5 ml of Hanks' solution (Hanks), Eagle's MEM, or MEM with the addition of 10 vol% fetal bovine serum (MEM+FBS). In addition, the "Autoclaved" specimen was placed in the cell culture dish with 27-mm diameter and 3×10⁴ cells of murine fibroblast L929 were seeded uniformly in the dish with 5 ml of MEM+FBS. The number of seeded cells was determined to show confluence on polystyrene dish with 27-mm diameter after 605 ks (7 days). The immersion in the solutions and incubation of L929 were performed in the air containing 5% CO₂ and a saturated water vapor at 37°C for 605 ks in an incubator. At least two specimens were prepared for each condition to examine the reproducibility. The specimens after immersion in Hanks, MEM, and MEM+FBS are named "Hanks", "MEM", and "MEM+FBS", respectively. The specimen after incubation of L929 was named "L929". The compositions of Hanks, MEM, and MEM+FBS are summarized in Table 1.

Table 1. Composition of Hanks and MEM+FBS

2.2. X-ray photoelectron spectroscopy

XPS was performed with an electron spectrometer (SSI-SSX100). X-ray source was a monochromatized Al K
line (1486.61 eV) and take-off angle of photoelectron was 35° to specimen surface. All binding energies in this paper are relative to the Fermi level. The spectrometer was calibrated against Au 4f_7/2 (binding energy, 84.07 eV) and Au 4f_5/2 (87.74 eV) of pure gold and Cu 2p_3/2 (932.53 eV), Cu 2p_1/2 (952.35 eV), and Cu Auger L₃M_4,5M_4,5 line (kinetic energy, 918.65 eV) of pure copper. The energy values were based on published data [16]. Composition and thickness of surface oxide film were calculated according to the method described elsewhere [17 and 18]. Empirical data [19, 20, 21, 22 and 23] and theoretically calculated data [24] of relative photoionization cross-sections were used for quantification. The relative photoionization cross-sections,
_ij/
_O1s, are summarized in Table 2, where relative photoionization cross-section of a level j electron of an element i to that of O 1s electrons.

Table 2. Values of
_ij/
_O1s used in the quantification of the surface composition

5. 3. Results and discussion

3.1. X-ray photoelectron spectroscopy spectra

XPS spectra of binding energy region of Ti 2p, O 1s, N 1s, and C 1s were obtained on all the specimens. Those of Ca 2p and P 2p were obtained from the specimens immersed in the solutions and "L929" and that of S 2p was only from "L929" specimen. The XPS spectra of Ti 2p, O 1s, Ca 2p, and P 2p electrons on "Hanks" and that of S 2p electrons on "L929" are shown in Fig. 1(a)-(e). The Ti 2p spectra were decomposed into four doublet spectra originating from Ti⁰, Ti²⁺, Ti³⁺, and Ti⁴⁺ according to the binding energy data [25]. The O 1s spectrum was decomposed into spectra originating from O²⁻, hydroxide or hydroxyl group (OH⁻), and hydrate and/or adsorbed water (H₂O) according to the binding energy data concerning metal oxides [26]. The N 1s spectrum appeared in the binding energy region of 400.4-401.1 eV originating from contaminant and/or proteins. Binding energies of Ca 2p_3/2 and P 2p electrons were 348.0-348.3 and 133.6-134.8 eV, respectively, indicating that calcium phosphate was formed [27]. The binding energy of S 2p spectrum was 163.8 eV, originating from sulfur in S⁰ state and/or sulfide (S²⁻) according to the binding energy data [28]. However, S/N ratio of the spectrum is very small in this energy region, so the exact chemical state of sulfur was not determined.

(18K)

Fig. 1. XPS spectra of Ti 2p, O 1s, Ca 2p, and P 2p electrons on "Hanks" and S 2p electrons on "L929": (a) Ti 2p; (b) O 1s; (c) Ca 2p; (d) P 2p; (e) S 2p.

3.2. Composition and thickness of surface oxide film

Compositions and thickness (t) of the surface oxide film were calculated, assuming that adsorbed biomolecules are not included in the film and all oxygen obtained are in the film although a certain amount of oxygen are originated from adsorbed biomolecules. The schematic illustration of the surface according to this assumption is shown in Fig. 2(a). In this assumption, carbon and nitrogen originating from biomolecules are included in a so-called contaminant. The compositions, relative concentration ratio of calcium to phosphorous, [Ca]/[P], and that of Ti⁴⁺ to total titanium cations, [Ti⁴⁺]/[Ti²⁺+Ti³⁺+Ti⁴⁺], and t are summarized in Table 3.

(25K)

Fig. 2. Schematic illustration of the surface of titanium: (a) surface assuming that adsorbed biomolecules are included in contaminant layer; (b) surface assuming that adsorbed biomolecules are uniformly included in the surface oxide film.

Table 3. Compositions, [Ca]/[P], and t of the surface oxide film assuming that all oxygen are from the film and adsorbed amino acids and proteins are not in the film (mean±standard deviation, SD)

n: number of experiments.

On "MEM", "MEM+FBS", and "L929" specimens, the following assumption was also used to calculate surface composition. Amino acids and serum proteins contained in the MEM and FBS and extracellular matrix (ECM) produced by L929 are uniformly included in the surface oxide film and the contaminant layer does not apparently exist, as shown in Fig. 2(b). Compositions of the surface oxide film calculated using this assumption are summarized in Table 4. Composition of "Autoclaved" was also calculated as the reference. Concentrations of nitrogen, [N], and carbon, [C], of "MEM+FBS" and "L929" were the same as each other and were higher than those of "MEM". A large amount of proteins and ECM was adsorbed by titanium. The [N] and [C] of "MEM" were the same as those of "Autoclaved", indicating that amount of adsorbed amino acids on "MEM" is as small as contaminant on "Autoclaved". In any case, concentrations of elements except nitrogen and carbon were too small to be used for discussion (Table 4); therefore, the former assumption ( Table 3) that nitrogen and carbon are not in the film was employed for the following discussion.

Table 4. Compositions of the surface oxide film assuming that adsorbed amino acids and proteins are in the film (mean±SD, n=2)

As shown in Table 3, the differences in composition, [Ti⁴⁺]/[Ti²⁺+Ti³⁺+Ti⁴⁺] ratio, and t between "Polished" and "Autoclaved" specimens were very small. Thus, oxidation of titanium in the film itself does not proceed. In the case of 316L stainless steel and Co-Cr-Mo alloy, the surface oxide films were grown and dehydrated, and the fraction of alloying elements in the film varied after autoclaving [13 and 14]. Titanium forms thin surface oxide film in the air that shows higher protectiveness than 316L stainless steel and Co-Cr-Mo alloy. The film on titanium formed in the air is so protective that it can prevent the further oxidation of titanium for the film growth even in a saturated water vapor.

Immersion in the solutions changed the composition of the surface oxide film and the t increased. On the other hand, the [Ti⁴⁺]/[Ti²⁺+Ti³⁺+Ti⁴⁺] ratio did not change, indicating that oxidation of titanium does not always proceed in the solutions. Then, the growth of the film is not caused by the oxidation of titanium. After the immersion, calcium phosphate was precipitated. Therefore, the growth of the film after immersion in Hanks' solution is caused by the precipitation of calcium phosphate. However, the large values of t of "MEM+FBS" and "L929" could not be explained only by the precipitation of calcium phosphate, because the amount of oxygen is one parameter for the quantification of t [17 and 18]. Also, a certain amount of oxygen on "MEM+FBS" and "L929" was originated from adsorbed biomolecules. Concentrations of calcium, [Ca], and phosphorous, [P], of "MEM", "MEM+FBS", and "L929" were smaller than those of "Hanks". It may be due to a large amount of adsorbed molecules such as serum proteins and ECM of L929 because the adsorbed molecules and ECM inhibits the precise detection of trace elements by XPS. Nevertheless, the adsorbed molecules such as amino acids, proteins, L929 itself, and ECM of L929 prevent the diffusion of molecules and ions to the surface [15], causing the decrease in [Ca] and [P].

The [Ca]/[P] ratios of "Hanks" and "MEM" were the same as those previously reported in Hanks' solution and
-MEM, ca. 1.0 [1, 6, 7 and 8], but were smaller than those in simulated body fluids with longer immersion period, ca. 1.5 [1, 5 and 6]. On the other hand, the [Ca]/[P] ratio of "MEM+FBS" was larger than those of "Hanks" and "MEM". The ratio of "L929" was smaller than those of "Hanks" and "MEM". Because phosphate ions initially adsorbed on titanium promote the adsorption of calcium ions, the [Ca]/[P] ratio reflects the effect of coexisting molecules on the adsorption behavior of calcium and phosphate ions [6]. The adsorbed serum proteins promote the preferential adsorption of calcium ions because [Ca] was the same as that of "MEM" although [P] was smaller than that of "MEM". On the other hand, L929 and its ECM reduce the adsorption of calcium ions because [Ca] decreased although [P] increased with L929. The serum proteins mainly consist of albumin whose isoelectric point is 4.9 [29]. This negative charge of adsorbed albumin causes the preferential adsorption of calcium ions in the same way as phosphate ions that are initially adsorbed on titanium and promote the adsorption of calcium ions due to their negative charge [6]. From the viewpoint of surface charge governing the adsorption of calcium ions, the [Ca] less than expected from the [P] on "L929" should be due to the rather positive charge of the adsorbed molecules. The ECM of L929 is mainly collagen [30] which shows isoelectric point of more than 8.0 because it has more amino side chains positively charged than side chains negatively charged [31]. Therefore, the precipitation mechanism of calcium phosphate depends on the charge of adsorbed molecules.

Sulfur precipitated on the surface oxide film only on "L929". We could not determine the exact chemical state of the sulfur due to the small S/N ratio of the spectrum, while the state of sulfur is attributed to be S⁰ and/or S²⁻ according to binding energy data. The employed cell culture medium, MEM+FBS, contains sulfate ions and very small amount of amino acids and proteins having thiol group (SH). On the other hand, L929 does not generate ECM containing sulfur. Thus, it cannot be denied that the existence of L929 promotes the preferential adsorption of the thiol group in biomolecules. However, the concentration of biomolecules containing thiol group such as cystine in MEM+FBS is much smaller than that of the other biomolecules [32]. In addition, the concentration of sulfate ions in MEM+FBS, 7.3×10⁻⁴ mol 
⁻¹, is much larger than that of the biomolecules containing thiol group, <30 mg 
⁻¹ [32]. Therefore, the source of sulfur precipitated on the film is mainly sulfate ions in MEM+FBS that is reduced and precipitated. The reducing environment is generated at the interface between titanium and cells because of the lack of dissolved oxygen due to the prevention of diffusion of ions and molecules with the existence of L929 cells [15]. The precipitation of sulfide and/or sulfite with the existence of L929 is revealed on the 316L stainless steel [13], indicating that the sulfate ions are also reduced at the interface between 316L and cells. Therefore, the sulfate ions should be reduced at the interface between materials and cells.

3.3. Oxygen in surface oxide film

The O 1s spectrum was decomposed into three spectra originating from O²⁻, OH⁻, and H₂O, as shown in Fig. 1(b). Proportions of these oxygen species are listed in Table 5. Here, a certain amount of oxygen species is originated from hydroxyl and carboxyl groups in calcium phosphate, adsorbed amino acids, proteins, and ECM. The oxygen species outside the surface oxide film causes the out of electric balance between positive and negative charges calculated using the values in Table 3 and Table 5.

Table 5. Fraction of O²⁻, OH⁻, and H₂O in the surface oxide film and [OH⁻]/[O²⁻] (mean±SD)

Total positive charge in the surface oxide film was calculated with atomic concentration of cations and the valences of titanium atoms in the film as the following equation (Eq. (1)):

Totalpositivecharge=[Ti^4+]×4+[Ti^3+]×3+[Ti^2+]×2+[Ca^2+]×2.

(1)

In the case of total negative charge of the film, two cases were assumed. One of them is that all amount of OH⁻ in Table 5 is truly hydroxyl group in the surface oxide film ( Eq. (2)); the other is that all amount of OH⁻ in Table 5 is originated from oxygen species in calcium phosphate, adsorbed amino acids, proteins, and ECM ( Eq. (3)). In other words, total negative charge was calculated with and without [OH⁻] in Table 5 as the following equations:

Totalnegativecharge (1)=[O^2−]×2+[OH^−]×1+[PO4^3−]×3,

(2)

Totalnegativecharge (2)=[O^2−]×2+[PO4^3−]×3.

(3)

The calculated charges were summarized in Table 6. When total negative charge was calculated with [OH⁻] according to Eq. (2), positive and negative charges were balanced only on "Polished". Total negative charge was much larger than total positive charge on "MEM+FBS" and "L929". Also, total negative charge was larger than total positive charge on "Autoclaved", "Hanks", and "MEM". On the other hand, when the negative charge was calculated without [OH⁻] according to Eq. (3), the difference between positive and negative charges on the specimens except "Polished" decreased, meaning that a part of [OH⁻] on "Hanks" and "MEM" is originated from calcium phosphate formed and adsorbed amino acids and a great part of [OH⁻] on "MEM+FBS" and "L929" was originated from oxygen species in adsorbed proteins and ECM. On the other hand, total negative charge calculated with [OH⁻] on "Autoclaved" was large probably because water strongly adsorbed to the oxide film and a large amount of hydroxyl group was formed on the oxide.

Table 6. Total positive and negative charges of the surface oxide film

Although a certain amount of [OH⁻] on the specimens was not originated from hydroxyl group in the surface oxide film, relative concentration ratio of OH⁻ to O²⁻, [OH⁻]/[O²⁻], was calculated as listed in Table 5 to examine the degree of oxidation and dehydration in various conditions. The [OH⁻]/[O²⁻] ratio decreased after autoclaving, indicating that hydroxyl group is dehydrated. On the other hand, oxidation of titanium did not proceed after autoclaving, as shown in Table 3. Therefore, dehydration of hydroxyl group proceeds only on the film not in the film.

The [OH⁻]/[O²⁻] ratio increased after immersion in Hanks' solution because calcium phosphate is formed (Table 3). On the other hand, the [OH⁻]/[O²⁻] ratios on 316L stainless steel and Co-Cr-Mo alloy decrease after immersion in Hanks' solution [13 and 14], which is explained by the progress of dehydration and oxidation of the surface oxide film. In those cases, the increase in [OH⁻] with the formation of calcium phosphate was negligible because less calcium phosphate is formed on 316L steel and Co-Cr-Mo alloy than on titanium.

The [OH⁻]/[O²⁻] ratio of "MEM" was the same as that of "Autoclaved" although the adsorbed amino acids contain carboxyl group. The amount of amino acids was very small as shown in Table 4 and the [Ca] and [P] of "MEM" were smaller than those of "Hanks", indicating that the formation of calcium phosphate does not contribute to the increase in [OH⁻]. The [OH⁻]/[O²⁻] ratios of "MEM+FBS" and "L929" were higher than that of "Autoclaved" because the adsorbed biomolecules contains a certain amount of oxygen species such as carboxyl group.

6. 4. Conclusions

The surface oxide film on titanium formed in the air is so protective to prevent the further oxidation of titanium after autoclaving, immersion in various solutions, and incubation with L929 cells. Then, the growth of the film and the increase in the relative concentration ratio of OH⁻ in the film in the solutions are caused by the precipitation of calcium phosphate. The calcium phosphate precipitated on the film in the presence of amino acids and serum proteins and with the existence of cells, but the amount precipitated decreases with those molecules and cells. The [Ca]/[P] ratio increased with serum proteins charging negatively, while the ratio decreased with cells whose extracellular matrix charging positively. The precipitation mechanism of calcium phosphate depends on the charge of adsorbed molecules. Sulfur precipitated as S⁰ and/or S²⁻ on the film only when L929 cells existed. This is the first confirmation of the precipitation of sulfur on titanium specimen in vitro experiment. Sulfate ions in the medium should be reduced at the reducing environment generated at the interface between titanium and solution with the existence of cells.

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