Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology

Abstract

The removal of biofilms from microstructured titanium used for dental implants is a still unresolved challenge. This experimental study investigated disinfection and removal of in situ formed biofilms from microstructured titanium using cold atmospheric plasma in combination with air/water spray. Titanium discs (roughness (Ra): 1.96 µm) were exposed to human oral cavities for 24 and 72 hours (n = 149 each) to produce biofilms. Biofilm thickness was determined using confocal laser scanning microscopy (n = 5 each). Plasma treatment of biofilms was carried out ex vivo using a microwave-driven pulsed plasma source working at temperatures from 39 to 43°C. Following plasma treatment, one group was air/water spray treated before re-treatment by second plasma pulses. Vital microorganisms on the titanium surfaces were identified by contact culture (Rodac agar plates). Biofilm presence and bacterial viability were quantified by fluorescence microscopy. Morphology of titanium surfaces and attached biofilms was visualized by scanning electron microscopy (SEM). Total protein amounts of biofilms were colorimetrically quantified. Untreated and air/water treated biofilms served as controls. Cold plasma treatment of native biofilms with a mean thickness of 19 µm (24 h) to 91 µm (72 h) covering the microstructure of the titanium surface caused inactivation of biofilm bacteria and significant reduction of protein amounts. Total removal of biofilms, however, required additional application of air/water spray, and a second series of plasma treatment. Importantly, the microstructure of the titanium discs was not altered by plasma treatment. The combination of atmospheric plasma and non-abrasive air/water spray is applicable for complete elimination of oral biofilms from microstructured titanium used for dental implants and may enable new routes for the therapy of periimplant disease.

Figure 1. SEM micrographs, fluorescence microscopy images and photographs of contact areas on Rodac plates of untreated (a–c) and plasma treated (d–i) 24-h biofilms ( Figure 1A ) and 72-h biofilms ( Figure 1B ) formed in situ on microstructured titanium surfaces (experimental treatment sequence I).

Plasma treatment of the biofilms was performed using either a mean plasma jet power of 3 W (d–f) or 5 W (g–i). The untreated titanium surfaces are covered by a dense bacterial biofilm. After plasma treatment biofilm residues are visible in SEM images on the microstructured titanium surfaces (arrow). Higher red fluorescence of biofilms appeared under FM (Magnification: SEM a, d, g: ×10,000; FM b, e, h: ×100). No microbial growth is detectable on Rodac plates after plasma treatment (c, f, i).

Figure 2. SEM micrographs, fluorescence microscopy images and photographs of contact areas on Rodac plates of 24-h ( Figure 2A ) and 72-h ( Figure 2B ) in situ biofilms treated with air/water spray (a–c) or treated with plasma and subsequent air/water spraying (d–i 2 bar, 5 s, 10 mm distance; treatment subgroups II).

Plasma treatment of the biofilms was performed using either a mean plasma jet power of 3 W (d–f) or 5 W (g–i). Air/water spraying does not cause biofilm removal (a–c), however air/water spraying after plasma pre-treatment resulted in nearly biofilm free titanium surfaces (d–i). Only sparse biofilm remnants are visible on the microstructured titanium surfaces (arrows). (Magnification: SEM a, d, g: ×10,000; FM b, e, h: ×5). Microbial growth is detectable on Rodac plates after the first cycle of plasma treatment at 3 W (2B-f).

Figure 3. SEM micrographs, fluorescence microscopy images and photographs of contact areas on Rodac plates of 24-h ( Figure 3A ) and 72-h ( Figure 3B ) in situ biofilms.

Biofilms were pre-treated with plasma ((either 3 W (a–c) or 5 W (d–f)), air/water sprayed, and treated again with plasma (experimental treatment sequence III). No biofilm remnants could be detected on the microstructured titanium surfaces after this sequential treatment. (Magnification: SEM a, d: ×10,000; FM b, e: ×5). No microbial growth is detectable on Rodac plates after plasma treatment (c, f).

Figure 4. SEM micrographs and photographs of contact areas on Rodac plates (24-h incubation) of untreated (a, b) and plasma treated (c–f) microstructured titanium surfaces without biofilm (experimental treatment sequence IV).

Plasma treatment of the titanium surfaces was performed using either a mean plasma jet power of 3 W (c, d) or 5 W (e, f). No surface alterations were detected after plasma treatment of microstructured titanium surfaces. (Magnification: SEM a, c, e: ×10,000). No microbial growth is detectable on Rodac plates after plasma treatment (b, d, f).