Electrochemical Surface Treatment for Controlling the Release of Silver Ion As Antibacterial Agent on Titanium
Authors: Shimabukuro Masaya , Tsutsumi Yusuke , Ashida Maki , Chen Peng , Doi Hisashi , Hanawa Takao 
Published: February 2020

＜Introduction＞
Titanium (Ti) and Ti alloys are widely used in both field of orthopedics and dentistry because of their good mechanical properties, high corrosion resistance, and biocompatibility. However, in recent years, biofilm formation due to bacterial adhesion and colonization on biomaterials has been recognized as a major cause of failure in implant surgeries. There is no way to remain devices coated by biofilm in the patient body, because the resultant infection disease will occur. To solve a problem of biofilm formation on biomaterials surface, application of antibacterial agents is the first choice. Silver (Ag) ion is known as one of the most effective antibacterial agents. Therefore, the surface treatment which enables to modify the implants to incorporate Ag should be developed for realizing antibacterial property. We focus on the micro-arc oxidation (MAO) treatment which has been already applied to medical devices consisting of Ti because of its efficacy on improvement of hard-tissue compatibility. The MAO treatment was also expected to be available for Ag-introducing method onto Ti surface. Thus, in this study, MAO treatment with the Ag-containing electrolyte was performed. The purpose of this work was to control and optimize the Ag ion release from the modified layer by MAO treatment condition. Surface analyses were performed to characterize the composition, morphology, and crystal structure of the MAO-treated Ti, followed by a bacterial adhesion test.
<Materials and Methods>
Commercially pure Ti (Grade 2) disks were ground by SiC abrasive papers up to #800 grit. The composition of the electrolyte for MAO treatment was 100-mM calcium glycerophosphate, 150-mM calcium acetate, and 0 to 2.5-mM silver nitrate (AgNO3). After pouring the electrolyte into the electrochemical cell, a Ti specimen as anode and a stainless steel plate as cathode were connected to a DC power supply (PL-650-0.1, Matsusada Precision Inc., Japan). A positive voltage, with the adjustment of the constant current density condition of 251 Am-2 was applied for 10 min. After MAO treatment, Ti specimens were rinsed in ultra-pure water for 3 min. The surface morphologies and the chemical compositions of the specimens were analyzed using a scanning electron microscope with energy dispersive X-ray spectrometer (SEM/EDS, S-3400NX, Hitachi High-Technologies Corp., Japan). The Ag-ion release from the MAO-treated specimens into a physiological saline (0.9mass% NaCl aq.) was measured by an inductively coupled plasma atomic spectrometer (ICP-AES, ICPS-7000 ver.2, Shimadzu Corp., Japan). The saline had been exchanged for the fresh one every 7 d. To evaluate the antibacterial property of the MAO-treated specimen, the bacterial adhesion test was performed with anaerobic gram-negative bacterium (Escherichia coli, NBRC3972, NITE, Japan) in accordance with the standard test method (ISO22196:2011). The bacteria were seeded on the specimen surface and then incubated at 35 °C for 24 h. The colony forming units (CFU) mL-1 of the living bacteria dispersed into the saline was measured using the culture medium sheet for E. coli (JNC Corp., Japan).
＜Results and Disccution＞
The MAO-treated specimens in the electrolyte containing Ag showed a typical structure of porous oxide layer as reported in previous studies. Thus, it is supposed that the presence of Ag ions in the electrolyte does not influence the formation of the porous oxide layer during MAO treatment. The incorporations of Ag as well as Ca and P from the electrolyte in the oxide layer during MAO treatment were confirmed by EDS analysis. From the results of ICP-AES measurement, the release rate of Ag ion from the oxide layer into a physiological saline was unstable until 21 d after the immersion. However, the Ag ion release was stabilized after 28 d and it had kept for at least 140 d. The specimens treated in the above 0.5-mM AgNO3 showed no viable bacterium on their surface even after the immersion in a physiological saline for 28 d. Thus, it was confirmed that the incorporated Ag in the porous oxide layer by MAO was effective against bacterial adhesion. In addition, we also found that the more Ag was incorporated when the potential reached higher value. According to the above results, we have finally developed the two-step MAO treatments which can control the Ag-ion release during immersion in a physiological saline by altering the electrolyte composition or the maximum voltage setting in the middle period of the treatment.