Effects of topography and composition of titanium surface oxides on osteoblast responses
Introduction
The biocompatibility of titanium as an implant material is attributed to surface oxides spontaneously formed in air and/or physiological fluids [1]. Cellular behaviours, e.g. adhesion, morphologic change, functional alteration, proliferation and differentiation are greatly affected by surface properties, including composition, roughness, hydrophilicity, texture, and morphology of the oxide on titanium [2], [3]. The natural oxide is thin (about 3–8 nm in thickness) and amorphous, stoichiometrically defective. It is known that the protective and stable oxides on titanium surfaces are able to provide favourable osseointegration [4], [5]. The stability of the oxide depends strongly on the composition, structure and thickness of the film [6]. As a consequence, great efforts have been devoted to thickening and stabilizing surface oxides on titanium to achieve desired biological responses.
The nature of the surface oxides can be manipulated by thermal oxidation and/or anodic oxidation. In vitro and in vivo studies show that alterations in the surface oxide of titanium implants strongly influence the tissue response [7]. The oxide film formed by thermal oxidation is typically thin due to its slow formation process by O2 diffusion through the titanium oxide film. In contrast, anodic oxidation is efficient to control the thickness, composition and topography of the oxide film on titanium [8], [9], [10] and can be applied for implant surface modification.
Among surface properties, surface roughness and composition have been considered the most important parameters for altering cell activity [11]. The biological response to titanium depends on the surface chemical composition, and the ability of titanium oxides to absorb molecules and incorporate elements [12]. Surface topography plays a fundamental role in regulating cell behaviour, e.g. the shape, orientation and adhesion of cells [13], [14], [15]. As a surface begins to contact with biological tissues, water molecules first reach the surface. Hence, surface wettability, initially, may play a major role in adsorption of proteins onto the surface, as well as cell adhesion. Cell adhesion is generally better on hydrophilic surfaces. It is known that changes in the physicochemical properties, which influence the hydrophilicity of Ti dioxide will modulate the protein adsorption and further cell attachment [16]. By anodic oxidation, elements such as Ca and P can be imported into the surface oxide on titanium and the micro-topography can be varied through regulating electrolyte and electrochemical conditions. The presence of Ca-ions has been reported to be advantageous to cell growth [17], [18], and in vivo data show implant surfaces containing both Ca and P enhance bone apposition on the implant surface [19]. Although in vitro and in vivo studies have shown that the anodic oxides of titanium demonstrate positive biological responses [20], [21], no detailed in vitro biological responses have been investigated to anodic oxides with different compositions and topographies. Since micro-pores of anodic oxides are comparable with biological entities in size, the biological interactions induced by such surfaces would most likely be different from those occurring on the flat surface.
As titanium with high-energy stable natural or anodic oxides is well known for its strong affinity to ambient elements such as oxygen, hydrogen, nitrogen, and carbon, absorption of ubiquitous hydrocarbon always occurs under normal conditions in air or solution containing organic contaminants. Surface contamination can be produced during the processing of titanium such as machining, surface treatment and sterilization. Surface contamination could be responsible for the loss of an implant a few of years after surgery [22]. More attentions have been paid to probably negative effects from surface contamination, and different chemical and physical cleaning techniques are applied to decontaminate titanium implants. It is reported that initial implant surface free energy and surface cleaning play a significant role in the healing and generation of host-tissue cells adjacent to the implant surface [22], [23]. In vitro studies indicate that surface contamination lowers the percent of cell attachment and spreading [24].
In the present study, to evaluate effects of surface characteristics and composition of anodic oxides of titanium on osteoblast responses, various anodic oxides on titanium were prepared and biologically tested in vitro. Additionally, chemical compositions and states of the elements, morphologies, roughness and contact angles were analysed on the anodic oxides. Biological responses to the oxides which were investigated include the metabolic activity, cell adhesion, proliferation and alkaline phosphatase (ALP) activity.
Section snippets
Preparation of specimens
Rectangular specimens with 20×10×1 mm in size were cut from cp titanium plate (ASTM B265 GR. 2) (TITANIA Products, Essen, Germany). The pre-treatment procedure was that specimens were mechanically polished to sandpaper No. 1200, etched by mixed HF/HNO3 solution, and cleaned by ethanol and deionized water, then air-dried. In electrolyte No. 1: 0.2 m H3PO4 solution [8] and electrolyte No. 2: the mixture of 0.03 m calcium glycerophosphate (Ca-GP) and 0.15 m calcium acetate (CA) [10], respectively, the
Surface characterization
The morphology of the prepared groups was analysed by SEM in Fig. 2. The control surface had parallel grooves oriented along the polishing direction. The surfaces G-2 and G-5 displayed island-shaped films. The increase of the anodizing voltage resulted in the gradual formation of the anodic oxide film, i.e. from islands to the whole surface, with more and larger irregular micro-pores generated by sparking during anodic oxidation. The grooves produced by polishing were removed gradually as the
Discussion
The surface properties of an implant play a critical role in the biological responses it induces and its ultimate success. In the present study, anodic oxidation produces different topographies and chemical compositions of surface oxides on titanium. SEM analyses show micro-pores in the surface oxides of titanium except for the control and G-5. With an increase of anodizing voltages, the geometry of micro-pores in the anodic oxides increases in 0.2 m H3PO4 (up to ca. 0.5 μm) as well as in 0.03 m
Conclusions
In the present study, surface oxides of titanium were varied in topography and chemical composition by anodic oxidation in 0.2 m H3PO4, and in 0.03 m Ca-GP and 0.15 m CA. P (ca.10 at%) or Ca (1–6 at%) and P (3–6 at%) were incorporated into the surface oxides and by anodic oxidation, surface roughness became lower in 0.2 m H3PO4 but enhanced in 0.03 m Ca-GP and 0.15 m CA. Phosphorus and calcium existed in the form of phosphate and calcium phosphate, respectively, in the anodic oxides. Anodized
Acknowledgements
The work was financially supported by the Fortuene Foundation (No. 989-0-0, Medical Clinic, University of Tuebingen). The authors gratefully acknowledge the assistance of Ms. Christine Schille in measurement of roughness, Mrs. Evi Kimmerle in cell culture as well as Prof. Wolfgang Lindemann in SEM.
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