Voltage-controlled cellular viability of preosteoblasts on polarized cpTi with varying surface oxide thickness
Introduction
Titanium and its alloys due to their corrosion resistance and biocompatibility are among the most highly utilized metallic biomedical alloys [1]. The specific surface interactions between these alloys and the body similar to other biomaterials significantly affect their function inside body. To elicit a desired response or prevent detrimental interactions, surfaces of implants are often modified using coatings, grafts, or other surface treatment techniques [2], [3], [4], [5], [6]. In the case of Ti and its alloys, growing the thickness of the thin native surface oxide layer (2–10 nm) [7] through electrochemical anodization or thermal treatment is associated with favorable effects on gene expression [8], [9], [10], [11], bioactivity [12], cellular migration [13], adhesion [11], [14], [15], proliferation [16], differentiation [17], [18], and osseointegration [19].
On the other hand, the surface voltage of the metallic implants may digress from the open circuit potential (OCP) into more cathodic voltages due to fretting corrosion [20], [21], [22], and affect different aspects of the cellular behavior such as cell viability, cytoskeletal organization, and cell adhesion [23], [24], [25]. Negative voltages in the range of − 1000 mV vs. Ag/AgCl have been reported when the passive oxide films of Ti and CoCrMo alloys have been abraded [21], [22]. Likewise, a voltage shift in the anodic direction may occur due to the presence of hydrogen peroxide, as in the case of inflammation in the body [26]. It is shown that cells cultured on polarized metals can only remain viable if the electrode potential stays within a specific voltage range, known as voltage viability range. The upper and lower limits of the voltage viability range depend on the type of metal and the cell line used among other factors [23], [27], [28], [29].
The surface interactions at play and the parameters that regulate the cellular behavior on polarized metals have not been fully investigated. Two important parameters determining the cell-polarized metal interaction are the potential of the electrode and the electrochemical currents. Cells and the surrounding physiological solutions contain a myriad of active substances that can take part in redox reactions on polarized electrodes. While the voltage determines what type of redox reactions may occur on metallic surfaces, electrochemical currents measure the magnitude of such reactions. The thickness of the native surface oxide on metallic biomedical implants can modulate charge transfer properties of the surface and subsequently increase or decrease the scale of redox reactions.
For cpTi, the lower limit is believed to be at − 400 mV vs. Ag/AgCl and no upper limit has been reported up to 1000 mV vs. Ag/AgCl [23]. To date, there has been no study to investigate how the thickness of the passive oxide influences voltage viability range and cellular behavior on titanium under cathodic polarization. This is important as it is not clear how the improved cell–biomaterial interactions associated with anodizing titanium and its alloys can be affected by voltage shifts. Furthermore, there could be synergistic effects on directing the cell–metal interactions when cells are cultured on anodized cpTi surfaces at voltages within the viability range.
The objective of this study is to understand the effect of voltages at the cathodic edge of the voltage viability range for cpTi (~− 400 mV vs. Ag/AgCl) on cellular morphology, viability, organization of adhesion complexes, and actin cytoskeleton. Furthermore, the surface of the bare and anodized cpTi was characterized at cathodic voltages using electrochemical impedance spectroscopy (EIS), and the impedance parameters were related to the observed biological response. The surfaces of the bare and anodized titanium were additionally characterized using atomic force microscopy (AFM) and sessile drop method to measure the roughness and surface contact angle, respectively. We hypothesize that a thicker surface oxide layer on cpTi shifts the cathodic edge of voltage viability range in titanium to lower voltages hence providing better protection against the harmful effects of cathodic voltages on cellular viability.
Section snippets
Sample preparation
Disks of grade 4 commercially pure titanium (ASTM-F67) with a diameter of 2 cm were mechanically polished up to 600 grit. After rinsing the samples with de-ionized water (DI), they were sonicated in DI for 10 min. Bare cpTi samples were mounted in a two-electrode custom-made electrochemical setup and were anodized at a voltage of 9 V (0.7 ± 0.04 mA cm− 2) at room temperature in PBS. The anode was the titanium sample and the cathode a carbon rod. Samples were removed from the chambers post-anodization
Anodization and surface characterization
Anodization of cpTi surfaces in PBS at a voltage of 9 V grows a compact non-porous oxide layer whose surface pattern does not differ from the native surface oxide observed in bare samples (Fig. 1).
The thickness of the formed oxide based on the golden color of the anodized surface and the reported oxide growth rates for cpTi [1] (2 nm/V) is estimated to be about 20 nm. Characterization of the surfaces of bare and anodized cpTi shows that there is no significant difference between these two surfaces
Discussion
The effect of cathodic voltages on the biocompatibility of metallic biomaterials has recently gained importance in the context of corrosion of biomedical implants. The cathodic voltage shifts from the open circuit potential can readily happen due to fretting corrosion, and affect the properties of the surface oxide layer and the implant biocompatibility. That is why a great deal of effort has been exerted into modifying the surface oxide layer of metallic biomedical alloys in order to improve
Conclusion
Anodization of cpTi at a voltage of 9 V in PBS shifts the cathodic limit of the voltage viability range to − 500 mV vs. Ag/AgCl from − 400 mV vs. Ag/AgCl. Cells show signs of distress on the anodized surface at − 400 mV vs. Ag/AgCl despite showing high cell viability. Cells cultured on anodized cpTi at − 400 mV vs. Ag/AgCl had fewer focal adhesions than those cultured at OCP demonstrating this effect. At a higher cathodic voltage of − 500 mV vs. Ag/AgCl, cells were rendered non-viable with a rounded up
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