morphology and chemical characterization of ti surfaces modified for biomedical applications

8/13/2019 Morphology and Chemical Characterization of Ti Surfaces Modified for Biomedical Applications

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2. Experimental

For the investigations a pure titanium (Grade 2) was used in the form of a

6 mm rod cut into cylinders 2 mm in thickness. The samples were polished,

degreased with detergent and rinsed in distilledwater. Next, theywere subjected

to four different chemical surface modifications: (1) etching in a piranha

solution (98% H2SO4+ 30% H2O2 mixture, the volume ratio 1:1) at room

temperature for 4 h (this pretreatment is assigned as PRT), (2) etching in

piranha boiling solution (PBS) for 10 min, (3) soaking in 5.0 M NaOH aq. at60 8C for 24 h, washing in deionized water at 40 8C for 48 h, heating with a rate

of 5 8C/h up to 600 8C, annealing at this temperature for 1 h, and then cooling

with a furnace (this pretreatment is assigned as SH, standing for sodium

hydroxide), and (4) immersing in a water dextran solution (0.05 wt.%) for

10 min (assigned as D, standing for dextran). An unmodified titanium surface

was used as a reference.

Morphology of the modified titanium surfaces was examined by a scanning

electron microscope (SEM, S-3500N, Hitachi). The chemical compositions of

the modified surface layers were analyzed by Auger electron spectroscopy

(AES) and X-ray photoelectron spectroscopy (XPS) (Briggs and Grant, 2003).

For this purpose an Auger microprobe analyzer, a Microlab 350 (Thermo

Electron) with XPS optional function was applied with a lateral resolution of

about 20 nm forAES andseveral mmfor XPS. The chemical state of thesurface

species was identified using the high-energy resolution of the Auger spectro-

meter (the energy resolution of the spherical sector analyzer is continuously

variable between 0.6% and 0.06%) and of the XPS spectrometer (the maximum

energy resolution is 0.83 eV). Appropriate standards for AES and XPS refer-

ence spectra were also used. XPS spectra were excited using Al Ka

(hn= 1486.6 eV) radiation as a source. The measured binding energies were

corrected referring to the energy of C 1s signal at 285 eV. An Avantage based

data system was usedfor data acquisition and processing. The crystalline phases

of thelayers were identifiedby Raman spectroscopy. Spectra were recorded on a

Nicolet Almega XR apparatus (laser: 532 nm; exposure time: 30 s; laser power:

25 mW; spectrograph aperture: 50 mm pinhole). In order to evaluate the

wettability of the modified titanium surfaces, contact angle measurements

were carried out at a constant room temperature. A given volume of DMEM

(Dulbeccos Modified Eagle Medium) was placed on the surface in order to

form a drop. Then, the contact angle was measured on both sides of the drop

using the goniometric method.

3. Results

Fig. 1 shows SEM images of titanium surfaces. In the as-

received state characteristic, oriented grooves resulting from

grinding can be seen (Fig. 1a). The morphology of the polymer-

coated surfaces (Fig. 1b) is similar to that in the as-received state.

The soaking in NaOH aq. solution (SH) results in a developed

morphology similar to a honeycomb (Fig. 1c). The surface

layer seems also quite porous. Pores of diameter 0.10.2 mm are

well visible in the honeycomb islands, surrounded by

elongated depressions and cracks, several mm long and about

0.5 mm wide. After etching in piranha solution at room

temperature (PRT) or in boiling piranha solution (PSB) the

morphology is quite different and less developed than thatproduced in the SH pretreatment. However, sub-microporosity

effects on the piranha treated surfaces can also be seen, e.g. in

boiling piranha solution a high population of shallow

depressions about 0.51 mm in diameter is formed (Fig. 1d).

In order to determine the chemical composition of the

titanium surfaces before and after modification, an AES study

was performed. The AES survey spectra taken from the surface

Fig. 1. SEM images of typical morphologiesof Ti surface subjectedto different chemical modifications: (a) as-received, (b) after immersingin water dextran solution

(D), (c) after NaOH and heat pretreatment (SH), and (d) after piranha pretreatment in boiling solution (PBS).

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of the materials analyzed indicate that Ti was oxidized on all thesurfaces investigated (the signals from Ti and O are well

distinguishable). Moreover, a beneficial incorporation of Ca is

also noted for the SH-modified surface, which apparently

results from the preparation procedure.

Fig. 2 presents a set of high-resolution Ti LMM spectra

showing Ti LMM signals for the samples under investigation,

as well as Ti metallic and TiO2oxide reference spectra. Careful

inspection of all of them confirms that there are Ti-oxides on all

the surfaces investigated before and/or after the NaOH or

piranha pretreatment. Comparison of these spectra does not

reveal any very distinct difference in their shape suggesting that

similar Ti-oxides are formed at the surface as a result of each ofthe pre-treatments.

In order to get a better insight into the chemical state of

titanium in the Ti-oxygen surface compounds, additional XPS

measurements were performed on NaOH aq. and piranha

treated pure Ti rods.Fig. 3demonstrates the corresponding Ti

2p spectra before Ar+ ion sputtering. These results confirm that

TiO2 is the main component of the chemically modified Ti

surface. Deconvolution of the Ti 2p signals suggests, however,

that some lower Ti-oxides are also present in the pretreated

samples. The stoichiometry of these oxides and their volume

fractions, as estimated from the XPS data, are listed in Table 1.

Contamination of the samples with carbon, in particular thosepretreated with piranha, made the analysis difficult. In order

to clean the surface, Ar+ ion bombardment was used. Analysis

of spectra after the ion bombardment may provide some

information about the stability of oxide layers. NaOH treated

samples produced an oxide layer which was rather quite stable

against Ar+ ion bombardment. The spectra did not change much

after sputtering. Piranha-treated Ti shows a more complex

XPS spectra. This may reflect the presence of a surface oxide

which is more prone to the modifications introduced by Ar+ ion

sputtering than that produced by the SH pretreatment.

The crystallographic structure of the titanium oxide layers

was analyzed by Raman spectroscopy. The results of theseinvestigations are presented in Fig. 4. Raman spectra of the

titanium surface before and after modification consist of a band

at 143 cm1 which correspond to anatase. The spectra of the

SH-modified surface consist additionally of two strong bands at

440 and 608 cm1 and a weaker band at 248 cm1, which

indicate rutile to be present at the surface. The bands

corresponding to anatase vary in intensity, which can be

attributed to the different content of crystalline anatase phase in

the surface layer.

Fig. 2. High-resolution Auger spectra (energy analyzer resolution 0.1 eV

within the energy range 350440 eV) taken at Ti surface chemically modified

in different solutions. For comparison, Ti LMM reference spectra for metallic Ti

and titanium oxide TiOx are given, as indicated in the figure.

Fig. 4. Raman spectra of Ti surface chemically modified in different solutions.

A, anatase; R, rutile.

Table 1

Ti 2p3/2binding energies as measured from the corrected XPS spectra and the

surface TiO compounds evaluated from deconvolution procedure

Samples

pretreatment

Binding energy

(eV), Ti 2p3/2

Compound

As-received 458.9 TiO2

PRT 458.3 TiO2 67%456.2 Ti2O3 21%

454.6 TiO 12%

PBS 458.5 TiO2

SH 458.8 TiO2 77%

457.8 Ti2O3 23%

Fig. 3. Ti 2p XPS spectra taken at Ti surface chemically modified in different

solutions: as-received, after piranha pretreatment at room temperature

(PRT), after piranha pretreatment in boiling solution (PBS), and after NaOH

and heat pretreatment (SH).

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Fig. 5 shows the Raman spectrum of a Ti surface with a

dextran coating. Bands from OH groups (3500 cm1) and

CH2 groups (2900 cm1) indicate that dextran is present at the

surface. However, there is also a clear distinguishable band

corresponding to anatase (143 cm1) in the spectrum. This

implies that the polymer film is not homogenously distributed

at the surface, and remains discontinuous leaving some areas

not covered at all.

The contact angle was measured using a DMEM solution,

whose chemical composition is similar to human body fluids.

The values of contact angle obtained are presented in Fig. 6.

The results clearly indicate that SH and PBS pretreatment led toa higher contact angle than that for the other pretreated samples.

The samples subjected to these modifications are characterized

by sub-micro- and microporosity, which apparently influence

the wettability of the titanium surface. However, it should be

noted that wettability is an average surface property measured

on the macroscopic scale. The lowest value of contact angle

(458) is observed for the samples with a dextran coating.

4. Discussion

It is generally accepted that rough, textured and porous

surfaces are able to stimulate cell attachment, differentiation

and formation of the extracellular matrix (Boyan et al., 1996).

Moreover, an appropriate surface roughness can produce

beneficial mechanical interlocking at the initial adhesion stage

and aid in further cell adhesion (der Brugge et al., 2002). The

morphology of NaOH or piranha treated surfaces can be

generally described as microscopically rough due to the

grinding, and as porous on the sub-micrometric scale due to

chemical treatment (etching and oxidation). However, after

etching in PRTor in PBS, the morphology is quite different and

in fact less developed than that produced in the SH

pretreatment. The differences in surface morphology after

these chemical surface modifications may be caused by

different chemical reactions leading to oxide formation. In

the case of piranha solution, the oxidizing factor is H2O2,

whereas for alkaline solution is H2O (corrosion under water

decomposition, see Kaesche, 1979). The kinetics of these

reactions vary depending on temperature and environment.

Thus, the different topography of the surface may result from

various oxide thickness.

Recently, more attention has been paid to the role of surfacechemistry in the behavior of cells. Surface components

influence the response of cells to biomaterials (Tambasco de

Oliveira and Nanci, 2004; Feng et al., 2003). In the present

investigations Auger electron spectroscopy has shown that all

the chemical modifications of Ti surfaces resulted in formation

of a titanium oxide layer. XPS confirmed that TiO2is the main

component of the chemically modified Ti surface. However,

some lower Ti-oxides are also present for the samples

pretreated in the piranha solution at room temperature or

in the NaOH solution. More detailed discussion is given

elsewhere (Lewandowska, 2007).

The other factor which can influence cell behavior andgrowth is the crystal structure of TiO2. TiO2may occur in three

polymorphic forms: anatase, brookite and rutile. Rutile is

thermodynamically the most stable modification, whereas

anatase and brookite can undergo irreversible exothermic

transition to rutile over a wide range of temperatures (Diebold,

2003). It is generally accepted that the natural surface oxide

film of titanium is anatase. Raman spectroscopy investigations

showed that after alkali and subsequent heat treatment the

surface oxide is a mixture of rutile and anatase. This may be a

result of the heat treatment applied. Etching in piranha

solution (PRT and PBS) leaves anatase unchanged. Some

authors suggest that oxide structure plays an important role in

ion release into the physiological fluid (Lee et al., 2000). Thedifferent levels of ion release change the environmental fluid

condition and further influence the reaction between cell and

biomaterial. Rutile is denser with a closer packed structure.

Thus, ion diffusion will be more difficult than for anatase and

the corrosion resistance of rutile will be greater.

Analysis of the Raman spectroscopy data showed that the

titanium surface covered with dextran coating is rich in

hydroxyl groups. However, the polymer coating is not uniform,

but occurs locally at the surface (see the prominent Raman

signal from anatase,Fig. 5).

It is known from the literature that cell adhesion is generally

better on hydrophilic surfaces. Good wettability of biomater-

Fig. 5. Raman spectrum of Ti surface covered with a discontinuous dextran

coating. OH and CH2 signals from dextran-covered part of the surface are

assigned. A, anatase signal from the uncovered part is well distinguishable.

Fig. 6. Diagram presenting the values of contact angle for unmodified and

modified titanium surfaces.

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morphology and chemical characterization of ti surfaces modified for biomedical applications

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