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Contrary Effects of UV-Irradiation on In Vitro Apatite-Forming Ability of TiO2 Layer in Simulated Body Fluid

K. Uetsuki1,2*, Y. Shirosaki1, S. Hayakawa1, and A. Osaka1

1Graduate School of Natural Science and Technology, Okayama University, 3-1-1, Tsushima-naka, kita-ku, Okayama 700-8530, Japan

2Nakashima Medical Co., 688-1, Joto-Kitagata, Higashi-ku, Okayama 709-0625, Japan

*Corresponding Author:
K. Uetsuki
[email protected]

Received date: 14 January 2011; Accepted date: 03 February 2011

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titanium dioxide; UV-irradiation; apatite; surface modification


Titanium and its alloys are commonly employed for such orthopedic and dental implants as artificial joints, tooth roots, screws, and pins, because they have a high corrosion resistance, good mechanical properties, and biocompatibility. It is essential that the materials should achieve stronger and earlier bonding to living bone without loosening during a long-term implantation. Those metals are bio-inert, that is, they are stable in physiological conditions, and an intervening thin fibrous tissue layer develops at the interface between the bone and materials when they are implanted in the body [1]. This means that they cannot directly bond to living bone tissue, or they are not osteoconductive [9]. Deposition of bone-like apatite on the surface of materials is considered to be prerequisite for implant materials to be able to bond to bone tissue. Soaking materials in Kokubo’s simulated body fluid (SBF; ISO 23317) is a method commonly used to evaluate their in vitro apatite-forming ability [10,12]. Many methods have been proposed to provide the material surface with apatiteforming ability, such as chemical treatment with NaOH [6,7,8,11] or H2O2 [4,14,15,18], and sol-gel coating of the titanium layer [13,16], to name a few. Recently, Shozui et al. [17] reported that UV-irradiation in air enhanced the in vitro apatite-forming ability of the TiO2 (rutile) layer prepared by heat treatment of cp-Ti in air, and that apatite particles were easily deposited on the TiO2. They suggested that the UV-irradiation increased the surface free energy of the rutile layer. Kasuga et al. [5] observed in vitro apatite formation on pressed TiO2 discs of 20% rutile and 80% anatase mixed phases when the samples were irradiated with UV-light in 1.5 SBF for 5 h and held in 1.5 SBF at 37 °C for various periods from 5 to 30 days. They suggested that UV-irradiation induced the apatite nucleation. Thus, UV-irradiation seems to be an effective strategy for the enhancement of apatite-forming ability of TiO2 on various substrates. However, the ability might be affected by the surrounding conditions for the UV-irradiation of the samples. In this study, we examined how the UV-irradiation and its surrounding conditions control the in vitro apatiteforming ability of the anatase layer prepared by the H2O2 chemical treatment of Ti that is reported byWang et al. [18].

Materials and methods

Mirror polished cp-Ti substrates (φ15 × 10 mm) were washed with acetone and ultra-pure water in an ultrasonic cleaner three times each for 10min. Then, anatase-type TiO2 layer was prepared by the method reported by Wang et al. [9]: cp-Ti substrates were treated with 3% H2O2 solution at 80 °C for 3 h and then heated at 400 °C for 1 h. The obtained samples were coded as CHT nt. Then, they were subsequently exposed to UV-light from a mercury lamp (HLR100T-2, SEN LIGHT Corp., Osaka, Japan; primary wavelength 365 nm, 170 mW/cm2) in air or in ultra-pure water for 1 h. The samples were coded as CHT UVa and CHT UVw, respectively. After the UV-irradiation, the samples were soaked in Kokubo’s simulated body fluid (SBF; pH 7.4) at 36.5 °C for 1 day in order to evaluate the apatite-forming ability. SBF contains the same inorganic components as human blood plasma in similar concentration. Table 1 shows the ion concentration of SBF and human plasma [10,12]. After soaking in SBF at 36.5 °C for 1 day, the samples were gently rinsed with ultra-pure water, dried in air, and stored in a vacuum desiccator until further characterization. Crystalline phases of the surface were identified with a thin film X-ray diffractometer (TFXRD, X’Pert PRO, PANalytical B.V., Netherland; CuKα, 45 kV, 40 mA) with a thin film apparatus, with the incident angle of 0.5° in θ. Surface morphology of those samples was observed under a scanning electron microscope (SEM, JSM- 6300, JEOL, Tokyo, Japan; 20 kV, 300 mA) with the samples sputter-coated with a 30 nm (approximate) gold layer.

Ion concentration/Mm
  Na+ K+ Mg2+ Ca2+ Cl HCO3 HPO42– SO42–
SBF 142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5
Human plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5

Table 1: Ion concentration of SBF and human plasma [10,12].

Results and discussion

Figure 1 shows the TF-XRD patterns of samples CHT nt, CHT UVa and CHT UVw in the range from 20° to 40° after soaking in SBF for 1 day. All samples showed a diffraction of anatase (JCPDS #21-1272) at ca. 25.3° as well as the titanium diffractions (#44-1294) at ca. 35.1° and ca. 38.4° in 2θ. When soaked in SBF, broad peaks assignable to hydroxyapatite (JCPDS #72-1243) appeared at ca. 31.7° in 2θ for all samples. Additionally, CHT UVw showed diffraction at also ca. 31.7° in 2θ that is assigned to hydroxyapatite (JCPDS #72-1243). The X-ray diffraction intensity of the hydroxyapatite peaks increases in the order: CHT UVa < CHT nt < CHT UVw.


Figure 1: TF-XRD patterns of several CHT samples after soaking in SBF at 36.5 °C for 1 day. See the text for sample code.

Figure 2 shows the SEM photographs of the surface of CHT (a), CHT UVa (b) and CHT UVw (c) before soaking in SBF. The surfaces of all samples before soaking have sub-micrometer scale retiform pore structures. In addition, UV-irradiation did not change the morphology of those CHT samples. Figure 3 shows the SEM photographs of the surface of CHT (a), CHT UVa (b) and CHT UVw (c) after soaking in SBF for 1 day, where spherical particles were observed on the surface of sub-micrometer scale retiform pore structure. The increase of particles in number agreed with that of their X-ray intensity of the hydroxyapatite diffraction. That is, CHT UVw has a greater apatite-forming ability than CHT, whereas CHT UVa has a lesser apatite-forming ability than CHT.


Figure 2: SEM photographs of several CHT samples before soaking in SBF. See text for sample code.


Figure 3: SEM photographs of several CHT samples after soaking in SBF at 36.5 °C for 1 day. See text for sample code.

The TF-XRD and SEM analysis indicated that UV-irradiation demonstrated two-types effects for apatiteforming ability for anatase-type TiO2. UV-irradiation in air decreases the apatite-forming ability of the TiO2 (anatase). On the other hand, UV-irradiation in water enhances the ability. These phenomena can be explained by considering the photo-catalytic ability of TiO2. When TiO2 absorbs a photon with energy equal to or greater than its band gap, electron-hole pairs are generated [2,3]. These pairs react with water molecules or hydroxyl groups on or nearby the surface of the anatase layer. Some researchers suggested that hydroxyl groups were active sites for apatite formation [4,6,7,8,11,14,15,18]. Moreover, the CHT samples used in this study originally had a high apatite-forming ability [4,14,15,18], for which the presence of OH groups and sub-micrometer topography were responsible. As the CHT samples lost apatite-forming ability without clear change in topography, it is reasonable to consider that the UV-irradiation in air reduced the amount of the hydroxyl groups. Consequently, if it is proven that the hydroxyl groups are active site for apatite formation, UV-irradiation in air must induce the dehydration on material surface and lead to reducing hydroxyl groups. On the other hand, when UV-light is irradiated on the samples in water, photo-dissociation of water takes place very close to the material surface due to photo-catalytic ability of TiO2 [2,3], and then the generated hydroxyl radicals attack Ti4+ or Ti-O-Ti on the material surface, resulting in the increase of Ti-OH sites active for apatite nucleation.


UV-irradiation demonstrated contrary effects for apatite-forming ability of TiO2 (anatase) of CHT, depending on the conditions under which CHT samples were placed. UV-irradiation in air suppressed the apatite-forming ability of the TiO2 layer of CHT. In contrast, UV-irradiation in water enhanced the ability. It is speculated that the UV-irradiation in air decreased the amount of hydroxyl groups but increased when irradiated in water.

Acknowledgments A part of this study was supported by the Kirameki-Okayama-Creation-Fund, and Grant-in-Encouragement of Students, Graduate School of Natural Science and Technology, Okayama University.


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