The Bacterial Anti-Adhesive Activity of Double-Etched Titanium (DAE) as a Dental Implant Surface

Abstract

This work aimed to compare the capability of Streptococcus oralis to adhere to a novel surface, double-etched titanium (DAE), in respect to machined and single-etched titanium. The secondary outcome was to establish which topographical features could affect the interaction between the implant surface and bacteria. The samples' superficial features were characterized using scanning electron microscopy (SEM) and energy dispersive x-ray spectrometry (EDS), and the wetting properties were tested through sessile methods. The novel surface, the double-etched titanium (DAE), was also analyzed with atomic force microscopy (AFM). S. oralis was inoculated on discs previously incubated in saliva, and then the colony-forming units (CFUs), biomass, and cellular viability were measured at 24 and 48h. SEM observation showed that DAE was characterized by higher porosity and Oxygen (%) in the superficial layer and the measurement of the wetting properties showed higher hydrophilicity. AFM confirmed the presence of a higher superficial nano-roughness. Microbiological analysis showed that DAE discs, coated by pellicle's proteins, were characterized by significantly lower CFUs at 24 and 48 h with respect to the other two groups. In particular, a significant inverse relationship was shown between the CFUs at 48 h and the values of the wetted area and a direct correlation with the water contact angle. The biomass at 24 h was slightly lower on DAE, but results were not significant concerning the other groups, both at 24 and 48 h. The DAE treatment not only modifies the superficial topography and increased hydrophilicity, but it also increases the Oxygen percentage in the superficial layer, which could contribute to the inhibition of S. oralis adhesion. DAE can be considered a promising treatment for titanium implants to counteract a colonization pioneer microorganism, such as S. oralis.

Keywords: Streptococcus oralis; atomic force; biofilms; dental implants; electron; microscopy; peri-implantitis; scanning; titanium; wettability.

Figure 1

The superficial micro-topography observed at SEM of machined discs (A), single-etched discs (B), and double-etched (DAE) ones (C). The magnification is 480×.

Figure 2

SEM images of etched (A) and DAE (B) samples at 480× magnification after the process of binarization that permitted the measurement of the percentage of porosity by using ImageJ 1.52q for Mac OS X (USA). (C) Average values of the percentage of porosity of the samples (error bars = standard deviation), calculated on 10 different pictures for each group. * p-value < 0.01.

Figure 3

SEM images of titanium discs etched (group 2), 2500× (A), high magnification 20K× (B) and (group 3) DAE discs, 2500× (C) and high magnification 20K× (D). (E) 3D reconstruction of atomic force microscope (AFM) images (10um × 10um) of DAE discs (group 3).

Figure 4

Spectra of energy dispersive x-ray spectrometry (EDS) analysis at 5 kV of titanium discs machined, A, (group 1), etched, B, (group 2), and DAE, C, (group 3) with the relative picture of the surface analyzed at 1500×.

Figure 5

(A) Static contact angles with sessile drop method and (B) wetted area of groups 1, 2, and 3. (C) Average water contact angle. * p-value < 0.05. (D) Average wetted area (error bars = standard deviation) * p-value < 0.05.

Figure 6

Colony-forming units (log10)/mL (A) and biofilm biomass formation (B) of S. oralis on titanium discs machined (group 1), etched (group 2) and double-etched (group 3) at 24 and 48 h (error bars = standard deviation). *p-value<0.05.

Figure 7

(A) Live/dead images of S. oralis on controls and groups 1, 2, and 3 at 24 and 48 h (green: live cells; red: dead cells). (B) Percentage of living cells at live/dead observation. * p-value < 0.05.

Figure 8

Schematic diagram illustrating the workflow of this study.