, 1999; Decker et al., 2003a, b, 2008; Hauser-Gerspach et al., 2007; Meier et al., 2008; Vig Slenters et al., 2008); thus only

brief descriptions of its main parts are given here. The system consists of an anaerobic flow chamber (Minucells, Bad Abbach, Germany) with (1) a test specimen mounted with its test surface not facing the flow direction; (2) a Teflon® dispenser (Multimed GmbH, Kirchheim unter Teck, Germany) containing the bacterial suspension; and (3) a peristaltic pump click here (Spetec GmbH, Erding, Germany) with an integrated speed controller. In this study, the system was modified to mimic conditions related to peri-implantitis, namely an anaerobic atmosphere and a slow-flowing, nutrient-poor environment containing three different strains of peri-implantitis-related bacteria. Specifically, the circulating bacteria were allowed to adhere to the protein-coated titanium specimens under anaerobic conditions (MACS MG; Don Whitley Scientific Ltd; atmosphere of 80% N2, 10% H2 and 10% CO2) at 37 °C for 72 h. Sterile polished disks of commercially pure titanium (Grade 2, ASTM F-67), 5 mm diameter and 1 mm thickness, with a mean surface roughness of 120 nm (Straumann AG, Basel, Switzerland), were sterilized by steam autoclaving and gamma irradiation and used as substrates. The disks were placed for 15 min in freshly mixed serum/saliva Lenvatinib mixture (1 : 10) prior

to each experiment in order to allow protein pellicle formation (Hauser-Gerspach et al., 2007). Fasting stimulated saliva of three healthy

volunteers was homogenized, filtered through a 70-μm filter (Cell Strainer; Becton Dickinson), and centrifuged at 22 000 g for 45 min at 4 °C. The supernatant was filter-sterilized (45 and 0.22 μm; Millex-HV and Millex-GV respectively; Millipore, Switzerland) and mixed with pooled serum (Blutspendezentrum, Basel, Switzerland). The protein-coated substrates were placed in the anaerobic flow chamber, 0.2% glucose was added to the bacterial suspension, and the suspension was circulated at 0.8 mL min−1 for 72 h. To compensate for the decrease in pH of the bacterial suspension (7.26 ± 0.07 to 4.84 ± 0.21), it was renewed Exoribonuclease in 24-h intervals. After 72 h, the biofilm-coated titanium disks were evaluated using SEM, CLSM, and IMC. The biofilms were fixed overnight in 2% glutaraldehyde solution (Sigma, Buchs, Switzerland), washed once with PBS, and dehydrated in stepwise increasing concentrations of ethanol – 30%, 50%, 70%, 90%, 2 × 100% for 10 min each. The samples (n = 3) were then critical-point-dried and coated with 10 nm of gold and examined (Fei Nova NanoSEM 230®, Eindhoven, the Netherlands). Oligonucleotide DNA probes, labeled at the 5′-end with Cy3 and Cy5 or with 6-carboxyfluorescein (FAM) and additionally labeled at the 3′-end (Microsynth AG, Balgach, Switzerland), are listed with their sequences and specificities in Table 1.