Preparation of titanium surfaces
Commercially pure turned titanium discs (grade 4), with a diameter of 8 mm and a central hole, were divided into four groups. The original turned discs served as controls (TU) and the other groups were each modified in one of three different ways: sol-gel treatment to create a nanoporous TiO2 coat (SG), heat-treated in a similar way to the sol-gel treated discs (HT), or anodically oxidized and calcium treated (OC).
For the sol-gel treatment (SG), discs were cleaned in a basic hydrogen peroxide solution (H2O; 30% H2O2, and 25% NH4OH in the ratio 5:1:1) at 85°C for five minutes. After extensive rinsing in distilled water, discs were dried in flowing N2. The sol was prepared by mixing solution 1 [10.22 g tetraisopropylorthotitanate, (Merck, Hohenbrunn, Germany) dissolved in 15 ml ethanol] with solution 2 [15 ml of ethanol, 170 μl H2O and 840 μl HNO3]. After mixing for one hour, 100 μl PEG 400 (Merck, Hohenbrunn, Germany) was added and the solution stirred vigorously. The clear sol was kept at room temperature during aging and the dip-coating process. Dip-coating was performed with using a computer-controlled stepper motor stage with a dipping speed of 30 mm/min, and the discs were sintered in an oven at 500°C (air) for 30 minutes. After heating the discs were ultrasonically cleaned in ethanol for four minutes and finally dried in flowing N2. The heat-treated surfaces (HT) which served as controls for the SG surfaces were sintered at 500°C (air) as described above. The anodically oxidized and calcium incorporated (OC) surfaces were prepared by anodic oxidation with an electrolyte consisting of sodium glycerophosphate hydrate (C3H6(OH)2PO4Na2 × H2O) and calcium acetate (Ca(CH3COO)2) [20, 21].
Characterization of titanium surfaces
To investigate surface roughness on the micrometer level, three discs of each surface were investigated at ten sites using an optical interferometer (MicroXamTM, PhaseShift, Tucson, USA). Each measurement was performed over a 200 × 260 μm area. A high-pass Gaussian filter (50 × 50 μm) was used to separate roughness from errors of form and waviness [22]. The evaluation was performed with the Surfascan software and the images were produced using SPIP™ (Scanning Probe Image Processor, Image Metrology, Denmark). Three different three-dimensional parameters were used to characterize the surface: average height deviation [Sa(μm)], a spatial parameter - density of summits [Sds(1/μm2)] and a hybrid parameter including variation in height and spatial direction [Sdr (%)].
The topography of model silica surfaces, dip-coated as for the SG surfaces, was characterized using atomic force microscopy (AFM 3100, Nanoscope III, Digital Instruments). To characterize the topography on the nanometer level of resolution, the two-dimensional surface parameter, average height deviation [Ra (nm)], was applied. Thickness of the SG coating was measured with null ellipsometry at λ = 632 nm (Auto-El-III, Rudolph Research, USA). The assumed refractive index of TiO2 in anastase crystal structure was n = 2.49.
Bacterial strains and culture
For biofilm assays the oral type strain Streptococcus sanguinis ATCC 10556 and Actinomyces naeslundii isolated from dental plaque [23] were used. All strains were routinely maintained on blood agar or in Todd-Hewitt broth (TH) at 37°C in 5% CO2.
Assay for adhesion and early biofilm formation
Immediately prior to bacterial inoculation, discs were cleaned in an ultrasonic bath with Extran MA01® (Merck, Darmstadt, Germany) diluted 1:40 in distilled water, treated with ethanol, and placed in polystyrene 6-well (flat-bottomed) titer plates (MULTIWELL™, Becton Dickinson, Franklin Lakes, NJ, USA). Overnight broth cultures were diluted 1:50 in fresh, pre-warmed Todd Hewitt broth at 37°C in 5% CO2 and grown to the mid-exponential growth phase (OD600 nm≈0.6). Cultures were then diluted to give final concentrations of approximately 1 × 108 cells/ml for S. sanguinis and 1 × 107 cells/ml for A. naeslundii. 1.5 ml of S. sanguinis and 4.5 ml of A. naeslundii suspensions were then inoculated into the wells. The microtiter plate was sealed with paraffin tape and incubated at 37°C on a rotary shaker at 300 rpm in 5% CO2. Following incubation for 2 and 14 h, the surfaces were rinsed three times with 10 mM potassium phosphate buffer, pH 7.5 (PBS) to remove loosely bound cells. The adherent bacteria were fixed in 4% paraformaldehyde for 16S rRNA hybridization. All biofilm experiments were carried out using independent bacterial cultures three times for each surface type.
For the saliva experiments, unstimulated whole saliva was collected from a healthy volunteer with good oral health, centrifuged for 10 minutes to pellet mucins and bacteria, and the supernatant filter-sterilized (pore size 0.22 μm). Aliquots of bacterial suspensions (1.5 ml of S. sanguinis and 4.5 ml of A. naeslundii) were centrifuged and the pellet resuspended in 6 ml of the sterile saliva. Bacterial suspensions (containing 107 colony-forming-units per ml as shown by culturing) were then added to the wells. Plates were shaken gently for 14 hours at 37°C in an atmosphere of 5% CO2. After this time, surfaces were rinsed three times with 3 ml PBS, fixed with 4% paraformaldehyde and incubated overnight at 4°C.
16S rRNA FISH and confocal laser scanning microscopy
Fixed bacteria on the discs were washed with cold, sterile PBS and subjected to cell membrane permeabilization with 100 μl lysozyme (Sigma, St Louis, MO, USA) [(70 U μl-1) in 100 mM Tris-HCl, pH 7.5 (Sigma, St Louis, MO, USA) containing 5 mM EDTA (Merck, Damstadt, Germany)] for 9 minutes at 37°C. After rinsing with ultra-pure water, the bacteria were dehydrated through a series of ethanol washes. Hybridization buffer [0.9 M NaCl, 20 mM Tris-HCl buffer, pH 7.5, with 0.01% sodium dodecyl sulfate (SDS) and 25% formamide] containing 20 ng of labeled oligonucleotide probe ml-1 was pipetted onto the titanium discs. The probe cocktail consisted of the streptococcal probe STR493 (5'-GTTAGCCGTCCCTTTCTGG-3') [24], fluorescently labeled green with ATTO-488 to assess the amount of S. sanguinis, and a red-labeled ATTO-565 probe EUB338 (5'-GCTGCCTCCCGTAGGAGT-3') [25] to assess total biofilm volume. Hybridization was carried out at 47°C in a humid chamber for 90 minutes. The surfaces were washed three times with 20 mM Tris-HCl (pH 7.5) containing 5 mM EDTA and 0.01% sodium dodecyl sulfate, and twice with 159 mM NaCl, for 30 and 15 minutes, at 47°C under gentle shaking. Finally, the titanium surfaces were washed with ice-cold ultra-pure water, mounted and glued onto glass slides for analysis using inverted confocal scanning laser microscopy (CSLM) (Eclipse TE2000, Nikon Corporation, Tokyo, Japan). Green fluorescence was provided by an Ar laser (488 nm laser excitation) and red fluorescence was given by a G-HeNe laser (543 nm laser excitation). CLSM images were acquired with an oil immersion objective (×60). Each stack had a substratum coverage field area of 215 × 215 μm, and the z-step was 2 μm. Images were obtained from 15 randomly selected sites per disc.
Image analysis
The image stacks were converted into TIFF format and analyzed using the bioImage_L software [26] to calculate the structural parameters of the biofilm. S. sanguinis takes up both the universal probe EUB338 (red) (Figure 1a) and the streptococcus specific STR493 probe (green) (Figure 1b) and in the images presented these cells appear yellow due to co-localization of the probes (Figure 1c). A. naeslundii, which takes up only the EUB338 probe, appears red (Figure 1c). Since the software used could not identify yellow cells, the biovolume of S. sanguinis was quantified using the numbers of green cells. The biofilm biovolume of A. naeslundii was then calculated indirectly by subtracting the S. sanguinis (green) biofilm biovolume from the total.
Statistical analysis
The non-parametric Mann-Whitney U test was used to analyze differences in biofilm biovolume between the test and control surfaces and p-values < 0.05 were considered significant.