Ninety healing abutments removed from patients at the Dental Implant Clinic, Dental Hospital, Tokyo Medical and Dental University, were used. As this clinical study is an in vitro experimental study, the university ethical committee decided that ethical approval was not necessary.

These healing abutments were at least for 4 weeks up to 6 weeks in patients’ oral cavities. All the healing abutments were for the implants of Nobel Biocare. The following two solutions were prepared: 1% sodium dodecyl sulfate solution (SDS solution, Fujifilm Wako Pure Chemical Corp., Tokyo, Japan) and 7.5% sodium bicarbonate solution (NaHCO₃, LEC Chemical Corp., Tokyo, Japan).

Electrochemical cleaning

Three different electrochemical apparatuses with the two-electrode electrochemical cell was set up as follows: group I, two stainless steel (TS 200, Iwata Manufacturing Co. Ltd., Seki, Gifu) electrodes; group II, a copper electrode (Pure Copper Type 26784, Shimomura Corp., Kowloon, Hong Kong) as a working electrode and a carbon electrode (Carbon Rod, Sano Factory, Tokyo, Japan) as a counter electrode; and group III, a carbon electrode (Carbon plate, Sano Factory, Tokyo, Japan) as a working electrode and a carbon electrode (Carbon Rod, Sano Factory, Tokyo, Japan) as counter electrode. All the electrodes were immersed in an electrolyte solution and the samples were placed on the flat electrode placed at the bottom of the electrolytic chamber with a customized connector at room temperature. Electrochemical tests were conducted in all cell types. The samples served as anode or the cathode in all chambers used in this study for electrolysis. The glass chamber was mounted and 600 ml of electrolyte of 7.5% NaHCO₃ was poured into the chamber. The fresh electrolyte was used at each electrolysis. In group I, one stainless electrode was a working electrode, where the samples were placed, and another stainless-steel electrode as a counter electrode. In group II, the copper electrode was used as a working electrode and carbon electrode was used as a counter electrode. In group III, carbon electrodes were used as a working electrode and a counter electrode. Voltage was applied with an electric power supply (ATTO Crosspower 500, ATTO Corp., Tokyo, Japan). Applied voltage was constant at 10 V and three different direct currents of 0.5 A, 1 A, and 1.5 A were applied for 5 min to induce the electrochemical (oxidation/reduction) reactions. These reactions can generate the oxidative species that remove and inactivate bacteria.

The major reaction at the cathode is the reduction of water or the reduction of oxygen.

Oxidation 2H₂O + 2e- = H₂ + OH-

Reduction O₂ + 4e- + 2H₂O = 4OH-

Immediately after removal from the patient’s mouth, the contaminated healing abutments were placed in 1% SDS solution into 20 min and randomly divided for cleaning with the three different electrolytic groups: group I, group II, and group III. Keeping a confidence level “alpha” 0.05 and power of the study 90%, the sample size was estimated to be 30 in each group. The result of the clinical pilot study was used for the sample size calculation. Samples were randomly divided into three groups using a computer-generated simple randomization method. Thirty contaminated healing abutments were used for each group.

After electrochemical cleaning, the healing abutment was rinsed with distilled water and placed in an individual plastic tube (Centrifuge tube 12-6265, SANSYO, Tokyo, Japan) containing 2 ml phloxine B peptide-staining solution (Phloxine B fluorescent dye, Sigma Aldrich, Tokyo, Japan). After staining, the healing abutment was rinsed again with distilled water and air-dried.

Analysis methods

The following methods were used to analyze the chemical and morphological properties of contaminated surface and surface composition after electrolysis.

Analysis of the electrolytes before and after electrolysis

The electrolytic solutions (catholyte and anolyte) were analyzed for pH before and after electrolysis by pH meter (LAQUA D-71 pH meter, Horiba, Kyoto, Japan).

Evaluation of the amount of stained area (contamination) of the healing abutments

The healing abutments were photographed using a light microscope and digital capture system at × 2 magnification (SMZ800, Nikon, Tokyo, Japan). Three images were captured for each healing abutment: two images from the body of the healing abutments rotated at 180° and one image from the top. The captured photographs were digitally analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) to measure the stained (and contaminated) surface areas. The color threshold value was manually adjusted within the range of 0 to 250 to detect all the stained debris, while a dark background color was established in order to standardize all the measurements. This manual threshold manipulation allowed for the selection of a stained area in which the number of pixels was calculated. Surface area contamination was expressed as a fraction (%) of the total surface area by dividing the number of pixels within the selected area by the total number of pixels comprising the image.

Scanning electron microscopy (SEM) analysis and visual assessment

The healing abutments were evaluated using scanning electron microscopy (SEM; S-4500, Hitachi, Japan) in order to quantitatively analyze the possible surface changes caused by electrolysis. For holding the samples, standard aluminum SEM specimen mounting stubs with specially designed sample holders were used. SEM was used with a setting of 10 kV accelerating voltage and the magnification of × 1000. The samples were handled with sterilized titanium tweezers to prevent surface contamination. Left side of upper part of the body of healing abutments located below the identification letter was used as area of interest to be examined. Only images of sign of surface changes were saved, which resulted in large number of SEM pictures for each different charges and currents. Four representative images that showed the most obvious visual surface changes were selected irrespective of the number of healing abutments per electrolysis of different charges and currents. This resulted in a total of 32 (× 1000).

In order to objectively rate the surface changes, images were coded and then blindly and independently scored by three examiners (T.H., K.K., N.K.). This means that each assessor was unaware from which electrolysis treatment each image came from except for the image of the unused (control) healing abutment. The following categorical rating score proposed by Bain [13] was used: (1) smoother (less rough) than the control, (2) same as new untreated control, (3) rougher than the control, and (4) much rougher than the control.

Kendall’s coefficient of concordance was used to investigate the inter-examiner reliability in the evaluation of SEM images. There was no significant difference between the three examiners (w = 0.815, P < 0.01).

Energy-dispersive X-ray spectroscopy (EDS) analysis

For quantitative analysis of titanium surfaces, EDS analysis was used. The spectroscopy of the emitted X-ray photons was performed by energy-resolved X-ray analyzer (Horiba EMAX-7000, Japan) at 15 kV for 100 s with the working distance of 15 mm in the vacuum condition without conductive coating. Three different measuring areas in same size were randomly selected on the surface of each sample to examine the atomic percentage of titanium and carbon.

Statistical analysis

The residual areas on the contaminated surfaces of the healing abutments following three different electrochemical treatments were calculated, compared, and analyzed using one-way ANOVA test. The mean roughness scores with standard deviations were calculated for each individual examiner and selected electrolytic cleaning treatment. Pairwise comparisons were used to compare the mean results of the surface roughness scores of all examiners between the different electrolytic treatments. P values less than 0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 21.0 (IBM, Armonk, NY, USA).