*2.4. Establishing the Biofilm*

To breed the biofilm on the implants, the required bacterial samples were thawed and bred with 300 mL nutrition solution in a beaker at 37 ◦C for 24 h. The carrier parts of the sterilized implants were masked to reduce bacterial contamination and fixed on a base plate. The experimental set-up was then immersed in the beaker with the culture medium to initiate formation of the biofilm. The beaker was placed in the rocking incubator for 14 days at a temperature of 37 ◦C. Previous tests with daily checks with a scanning electron micrograph (SEM) had demonstrated that 14 days were necessary to achieve a mature, multilayer biofilm (Figure 2). Every two days, the bacterial culture medium was replaced with freshly prepared nutritional solution.

**Figure 2.** Progredient formation of biofilms on different surfaces.

#### *2.5. Electrolytic Cleaning (Test Group)*

The implants in groups 1, 3, and 5 were cleaned by an electrolytic process. Figure 3 shows the construction of the electrolytic chamber. After demasking of the biofilm-coated test implant with a customized ejector, the implant was mounted on the bottom of the electrolytic chamber with a customized connector. The test implant served as the cathode. The connector had an inner titanium sleeve (titanium grade 4) covered by an insulating polyetheretherketone (PEEK) carrier. The inner titanium sleeve was connected to the electric source, and the PEEK carrier masked the carrier part of the test implant for only three quarters. This design was chosen to guarantee that it was possible to separate the test specimen using a customized tool, without touching the PEEK carrier, which was not been cleaned during the electrolytic procedure. The connector was mounted in a polytetrafluoroethylene (PTFE) socket to ensure stability of the electrolytic chamber.

After the cathode was fixed, the glass cylinder of the chamber was mounted, and 35 mL of electrolyte (sodium iodide (200 g/L), potassium iodide (200 g/L), L(+)-lactic acid (20 g/L), and water (800 g/L)) was poured into the chamber. Fresh electrolyte was used for each test.

The anode (titanium grade 4) was coated with platinum to prevent passivation of its surface. The anode was fixed in a hollow PTFE tube to ensure direct contact of the platinum-plated part with the electrolyte (∅ 5.0 mm/L = 4.0 mm). After the other end of the PTFE tube had been mounted in the cap of the chamber, the anode and cathode were connected to a voltage source (PeakTech 6060, PeakTech, Ahrensburg, Germany).

A voltage of 6 V was applied for 5 minutes, resulting in a current of up to 1100 mA.

After the cleaning process was finished, the cap of the chamber was removed, and the used electrolyte was poured into a beaker. The biofilm floated as a visible layer on the fluid in the beaker, and was pipetted with some fluid to prove the vitality of the collected bacteria (waste from electrolytic cleaning).

**Figure 3.** The assembly of the powder-water spray device. PEEK = polyetheretherketone, FPM = fluorine rubber, PTFE = polytetrafluorothgylene.

#### *2.6. Air-Powder-Water Spray (PSS) Cleaning (Control Group)*

The implants in groups 2, 4, and 6 were treated by a PSS (BA 8000, Mectron GmbH, Cologne, Germany). To ensure a reproducible and comparable cleaning effect, a test apparatus was assembled, as shown in Figure 4. The aim was to reproduce the working angle and distance according to the manufacturer´s operating manual (45◦ and 10 mm). The test implants were mounted on a sterile PEEK carrier (Figure 4, in orange), which masked three quarters of the carrier part and was then fixed in a PTFE connecting bar (Figure 4). The holder provided the same working angle and distance for all

the tested implants. The holder and its fixed handpiece were moved manually along the implant axis while the test implant rotated, being driven by a motor around its axis (40 rpm) to ensure proper and repeatable cleaning of the implants (Figure 4). Sterile water (Aqua ad injectabilia, Braun, Melsungen, Germany) and aminoacetic acid powder (glycine powder, Mectron GmbH, Cologne, Germany) were used with an air pressure of 2 bar being applied for 60 s. The waste liquid produced during the cleaning process was collected in a beaker to prove the vitality of the removed biofilm (waste from air-powder-water cleaning).

**Figure 4.** The structure of the electrolytic chamber.

#### *2.7. Rinsing and Test Sample Incubation*

After cleaning, the test and control implants were rinsed with air-water spray for 5 seconds (distance of 5 cm) and dried with oil-free air. The test parts were then broken off at the predetermined breaking point in a sterile manner, transferred to Eppendorf tubes filled with 1.5 mL nutritional solution, and incubated at 37 ◦C for 24 h. Ehrensberger et al. demonstrated that no additional colonies grew when incubation time was extended 18 h. [30]
