998 Research Article Received: 11 November 2009 Revised: 8 June 2010 Accepted: 19 July 2010 Published online in Wiley Online Library: 5 August 2010 (wileyonlinelibrary.com) DOI 10.1002/sia.3683 The effect of annealing temperatures on surface properties, hydroxyapatite growth and cell behaviors of TiO 2 nanotubes Yu Bai, a Il Song Park, a Hyeoung Ho Park, a Min Ho Lee, a* Tae Sung Bae, a Warwick Duncan b and Michael Swain b Well-ordered TiO 2 nanotubes were prepared by the electrochemical anodization of titanium in an ethylene glycol electrolyte containing 1 wt% NH 4 F and 10 wt% H 2 O at 20 V for 20 min, followed by annealing. The surface morphology and crystal structure of the samples were examined as a function of the annealing temperature by field emission scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD), respectively. Crystallization of the nanotubes to the anatase phase occurred at 450 ◦ C, while rutile formation was observed at 600 ◦ C. Disintegration of the nanotubes was observed at 600 ◦ C and the structure vanished completely at 750 ◦ C. Electrochemical corrosion studies showed that the annealed nanotubes exhibited higher corrosion resistance than the as-formed nanotubes. The growth of hydroxyapatite on the different TiO 2 nanotubes was also investigated by soaking them in simulated body fluid (SBF). The results indicated that the tubes annealed to a mixture of anatase and rutile was clearly more efficient than that in their amorphous or plain anatase state. The in vitro cell response in terms of cell morphology and proliferation was evaluated using osteoblast cells. The highest cell activity was observed on the TiO 2 nanotubes annealed at 600 ◦ C. Copyright c  2010 John Wiley & Sons, Ltd. Keywords: TiO 2 nanotubes; surface properties; hydroxyapatite growth; cell behaviors Introduction Titanium (Ti) and its alloys have been studied extensively for applications to orthopedic and dental implants owing to their excellent mechanical properties, corrosion resistance and outstanding biocompatibility. [1–4] However, Ti and its alloys are nonbioactive and lack rapid tissue integration, which results in the subsequent development of interfacial fibrous tissue, finally leading to isolation of the implants. [5,6] Therefore, it is essential to develop surface modification technologies to improve the bioactivity of titanium. One possible way of modifying the surface of the titanium substrate is the formation of self-organized TiO 2 nanotubes. There are a few articles that reported the formation of self-organized TiO 2 nanotubes by the anodization of Ti in a fluorine-containing electrolyte. [7 – 14] They demonstrated that modifying the titanium surfaces with TiO 2 nanotubes accelerated the rate of apatite formation, and enhanced bone cell adhesion and proliferation compared to a smooth control titanium surface. [15,16] It is well known that the successful implantation of an orthopedic material is dependent on close apposition between the bone and biomaterial. This is associated closely with two major events at the interface biomaterial/bone tissue: (i) the formation of an apatite layer; and (ii) bone cells anchorage, attachment, spreading and growth. [17] There is evidence suggesting that the surface structure has a significant effect over these two events. It was reported that apatite growth is closely related to the crystallographic phase of the substrate. As-formed TiO 2 nanotubes have been reported to be amorphous, and can transform to anatase or rutile depending on the annealing temperature. [18] According to the literature, this transformation is beneficial to apatite growth. [19] However, the changes in crystallinity caused by altering the annealing temperature will probably affect the surface structure and properties of the TiO 2 nanotubes. Changes in the surface properties have a strong effect on the osteoblast behavior. Therefore, understanding the relationship between the material surface properties and cellular responses is essential for designing optimal material surfaces for implantation. [20,21] This study examined the surface morphology, corrosion resistance and crystal structure of the nanotubes after annealing at different temperatures. The effect of annealed TiO 2 nanotubes on the in vitro apatite formation kinetics and cell behavior was also investigated systematically. Materials and Methods Fabrication of TiO 2 nanotubes The specimens were prepared from 0.4-mm-thick 99.5% titanium foil. Prior to anodizing, the specimens were degreased ultrasoni- cally in acetone and then in ethanol, followed by a final wash with deionized water and drying at 40 ◦ C. * Correspondence to: Min Ho Lee, Department of Dental Biomaterials, School of Dentistry and Institute of Oral Bioscience, Brain Korea 21 Project, Chonbuk National University, Jeonju 561-756, Korea. E-mail: mh@jbnu.ac.kr a Department of Dental Biomaterials, School of Dentistry and Institute of Oral Bioscience,BrainKorea21Project,ChonbukNationalUniversity,Jeonju561-756, Korea b DepartmentofOralSciences,UniversityofOtago,Dunedin,Otago,NewZealand Surf. Interface Anal. 2011, 43, 998–1005 Copyright c  2010 John Wiley & Sons, Ltd.