1. Introduction
The interaction of S. aureus with polyurethane is studied with electron and ion microscopy instrumentation (SEM, FIB and TEM). Biodestruction is a corrosion process of natural and artificial materials operated by microorganisms; it is remarkable for the polymeric materials commonly used in medicine, especially in orthopaedic stomatology where polyurethane occupies a special position [1-3].
Polyurethane dental prostheses have advantage over acrylic ones which provoke inflammatory reactions and intolerance when colonized by aggressive bacteria or fungi. In fact prostheses in the oral cavity are constantly attacked by microorganisms and their colonization often concurs in the destruction of the artificial materials. This can lead to the release of allergenic substances, toxic to the human body [2,4-6].
A first approach to the investigation of polyurethane biodestruction was to understand how bacteria interact with the artificial material. Scanning Electron Microscope (SEM) was used to morphologically evaluate the damage induced by microorganisms (adherence, formation of microcolonies and biofilm) at different stages, depending on the exposure time of the material to the bacteria and on the state of the polyurethane surface. SEM observation revealed significant morphological changes of polymer surface due to bacteria activity: higher porosity, cracks and particles formation. Transmission Electron Microscope (TEM) was a complementary technique, useful to investigate the samples’ inner structure and composition and to trace the pathway of polyurethane small particles (actually nanoparticles) while interacting with bacteria.
S. aureus is a permanent component of the microbial population in the oral cavity and it is considered a pathogen agent with biodestruction capacity. In fact staphylococci can adhere to the titanium and polymeric materials’ (polyethylene) surfaces forming a mucoid matrix made up to 90% of water called biofilm [7,8].
Staphylococci cause acute and chronic inflammation of soft tissues of the oral cavity (periodontal disease, sialodenity, gingivitis, etc) and they also influence the development of caries [9,10]. This may exacerbate chronic infectious processes and it can lead to the destruction of the prosthetic materials. In the presence of negative processes for the survival of microorganisms such as nutrients depletion, threat of drying, impact of chemical and physical factors in the external environment, bacteria can form biofilms [11]. The ability of bacteria to form biofilms is directly related to the increase of the biodestruction capacity [12].
2. Materials and Methods
Objects of the research were samples of polyurethane (Dentalur Russia). Various types of plastic surfaces (smooth, rough, surfaces resulted from sawing and splitting operations) were investigated.
Samples were analyzed in dual beam Focused Ion Beam/Scanning Electron Microscope (FIB/SEM) Quanta 200 3D (FEI Company, USA) in both high and low vacuum, mostly at 5 kV electron beam acceleration [13-15].
The focused ion beam operated at low beam currents is used for imaging, at high beam currents is used for site specific in situ milling. The FIB/SEM investigation can be applied to bulk samples prepared for SEM analysis or to bulk resin-embedded specimens prepared for TEM observation. One of the FIB/SEM major advantages is that the operator may select his favourite site of investigation with less bonds than in TEM analysis.
TEM and STEM (Scanning Transmission Electron Microscope) images were acquired on a FEI TECNAI F20 X-TWIN, operated at 200 kV, equipped with a HAADF (High Angle Annular Dark Field) detector: STEM HAADF technique was preferred for the improved contrast and direct interpretation of the images.
Standard TEM images (Figure 2(a)) together with SEM ones show outside and inside the bacterial cells small dark particles that have higher density/mass than the S. aureus cell components and it is supposed that they are polyurethane nanoparticles. STEM (in TEM) images were produced by scanning the electron beam, focused in a very small spot, over the area of interest (like in a SEM) and collecting the electrons that came across the sample. One or more detectors collect the transmitted electrons, according to their scattering angle. A detector collects the very low angle electrons to form the so called Bright Field (BF) images, whereas an annular detector collects the electrons scattered at higher angle to form Dark Field (DF) images. A third annular detector collects the electrons scattered at angles typically higher than 50 mrad to form HAADF images. This last method allows direct interpretation of the images, being an imaging technique where the specimens’ denser/heavier areas appear brighter in the final image.
The culture of S. aureus was isolated from a patient with a periodontal disease and incubated in broth with polyurethane. Control samples were a polyurethane slice with a smooth surface in broth and a broth with S. aureus without polyurethane. pH was checked after specimens incubation.
The incubation procedure is described in detail in ref.1 as well as samples preparation for TEM and SEM. Polyurethane samples and bacteria were incubated from 1 to 45 days at 37˚C. After centrifugation (6000 rev/min for 10 min) the bacterial pellet was placed on a silicon substrate for the FIB/SEM observation and prepared for the TEM analysis.
In samples preparation chemical-physical methods of dehydration were not applied in accordance to the traditional techniques, because standard drying operations leads to structural changes of biofilms and cells.
3. Results
Biocorrosion is a process that involves several steps from the material colonization to the formation of biofilms as discussed in ref.1.
Images obtained with FIB/SEM show that polyurethane undergoes biocorrosion by S. aureus (Figure 1).
Figure 1. FIB/SEM electron image shows small particles of polyurethane (circles) on biofilm surface; this confirms that polyurethane undergoes biocorrosion by S. aureus.
S. aureus is able to corrode surfaces of polyurethane generating particles of different size ranging from micrometers to nanometers as shown in Figure 2(a) (TEM image) and Figure 2(b) (STEM image). The same im-
(a)
(b)
Figure 2. (a) TEM image of S. aureus after incubation with polyurethane. Polyurethane particles can be observed on the cell wall (black ↑), inside the cell surrounded by membranes (white ↑) and in the external environment, in the proximity of the cell wall (black dashed ↑); (b) STEM image of the same sample of (a) (rotated by 180˚) in comparison to the TEM image. Polyurethane particles have higher electron density than the cell biological components, so they appear darker than the surrounding medium in TEM images and brighter in STEM ones. This image gives a better view of particles internalized into the cell (↑).
ages show that S. aureus internalizes polyurethane nanometer sized particles.
Figure 3 (STEMHAADFimage) shows circular membranous structures similar to vesicles inside S. aureus.