Adhesion of Streptococcus mitis and Actinomyces oris in co-culture to machined and anodized titanium surfaces as affected by atmosphere and pH

Abstract

Background: With the rising demand for osseointegrated titanium implants for replacing missing teeth, often in patients with a history of periodontitis, implant-related infections have become an issue of growing concern. Novel methods for treating and preventing implant-associated infections are urgently needed. The aim of this study was to investigate if different pH, atmosphere and surface properties could restrict bacterial adhesion to titanium surfaces used in dental implants.

Methods: Titanium discs with machined or anodized (TiUnite™) surface were incubated with a co-culture of Streptococcus mitis and Actinomyces oris (early colonizers of oral surfaces) at pH 5.0, 7.0 and 9.0 at aerobic or anaerobic atmosphere. The adhesion was analysed by counting colony forming (CFU) units on agar and by confocal laser scanning microscopy (CLSM).

Results: The CFU analysis showed that a pH of 5.0 was found to significantly decrease the adhesion of S. mitis, and an aerobic atmosphere, the adhesion of A. oris. S. mitis was found in significantly less amounts on the anodized surface than the machined surface, while A. oris was found in equal amounts on both surfaces. The CLSM analysis confirmed the results from the CFU count and provided additional information on how the two oral commensal species adhered to the surfaces: mainly in dispersed clusters oriented with the groves of the machined surface and the pores of the anodized surface.

Conclusions: Bacterial adhesion by S. mitis and A. oris can be restricted by acidic pH and aerobic atmosphere. The anodized surface reduced the adhesion of S. mitis compared to the machined surface; while A. oris adhered equally well to the pores of the anodized surface and to the grooves of the machined surface. It is difficult to transfer these results directly into a clinical situation. However, it is worth further investigating these findings from an in vitro perspective, as well as clinically, to gain more knowledge of the effects acid pH and aerobic atmosphere have on initial bacterial adhesion.

Figure 1

Scanning electron microscopy (SEM) images of machined (a) and anodized (b) titanium surfaces, provided by Nobel Biocare AB. The machined surface is relatively smooth with distinct orientation of the surface irregularities (anisotropic) while the anodized surface is rough with a homogenous structure (isotropic).

Figure 2

Effects of environmental pH (5.0, 7.0 and 9.0) and aerobic or anaerobic conditions on the adhesion of S. mitis and A. oris in co-culture after 2.0 hours’ incubation. Statistically significant less adhered S. mitis was found after incubation at pH 5.0 than pH 7.0 and of A. oris after incubation at aerobic environment compared to anaerobic environment.

Figure 3

Effect of surface properties on the adhesion of S. mitis and A. oris in co-culture to titanium after 2.0 hours of incubation. Statistically significant less adhered S. mitis was found on the anodized surface than the machined titanium surface.

Figure 4

Interactions between surface, pH and atmosphere affects tha bacterial adhesion. The first figure (a) illustrates the effect of pH and surface properties on the adhesion of S. mitis in co-culture with A. oris. The adhesion of S. mitis is less on both surfaces after incubation at pH 5.0 than 7.0 the reduction is however statistically significant larger for the adhesion to machined titanium than anodized titanium. Figure (b) illustrates the effect of surface properties and aerobic or anaerobic environment on the adhesion of S. mitis in co-culture with A. oris. The adhesion to the anodized surface was found to be reduced by aerobic incubation while the opposite was found for the adherence to machined titanium, this difference is however not statistically significant.

Figure 5

Confocal laser scanning microscopy images of machined titanium (a,c) and anodized titanium (b,d) surfaces incubated with a co-culture of A. oris and S. mitis. The bacteria are labeled with LIVE/DEAD® (Molecular Probes) thus green color indicates live cells and red color indicates dead cells. Surfaces a-c were incubated anaerobic and d aerobic. The two bacteria were found in equal amounts on the machined titanium surface after anaerobic incubation at pH 7 (a) while mostly A. oris could be found on the anodized surface, after incubation at the same conditions (b). The adhesion of S. mitis to both surfaces was further reduced after incubation at pH 5 irrespective of atmosphere (c,d). The number of non-viable cells (red) of A.oris was found to be greater after aerobic incubation than anaerobic (d).

Figure 6

Confocal laser scanning microscopy images of A. oris (small distinct dots and clusters, indicated by white arrows) and S. mitis (chains) adhered to machined (a) and anodized (b) titanium surfaces, after aerobic incubation at pH 7. The bacteria are labelled with LIVE/DEAD® (Molecular Probes) visualizing live cells in green and dead cells in read. Red arrows indicate partly detached chains of S. mitis .