2.1. Sample Preparations
The institutional review board of the Charité – Universitätsmedizin Berlin, approved the collection of extracted human teeth (EA4/102/14). The samples were prepared from 48 caries-free extracted human molars that had been stored in 0.5% Chloramine-T Solution (Carl Roth, Karlsruhe, Germany). From the crown of each tooth, a horizontal slice was cut off with a 0.2 mm band saw (EXAKT 300 CL, EXAKT, Advanced Technologies, Norderstedt, Germany). Each of the obtained specimens was trimmed to a thickness of 2.0 mm by using a grinding machine (LaboPol 25, Struers, Willich, Germany), together with SiC P1000 sandpaper (Buehler, Düsseldorf, Germany). Subsequently, the dentin slices were polished by utilizing SiC P2500 and P4000 sand papers (Buehler, Düsseldorf, Germany). For the demineralization, 24 randomly selected samples were transferred into Buskes solution (2.205 g CaCl2 x 2H2O; 2.041 g KH2PO4; 10.0 ml 100% methylene diphosphonic acid; and 14.3 ml 100% CH3COOH; these were all added to 4.5 L distilled water and then supplemented with 10 M KOH (Merck, Darmstadt, Germany) until the pH was 5.0). This solution was stored for 72 hours in an incubator/shaker at 37°C and at 50 rpm (ES-20 Orbital Shaker-Incubator, Biosan, Riga, Latvia). All of the specimens were then stored in distilled water before being further processed, as follows:
Initially, the slices were air-dried for 3 sec and then etched for 15 sec by using 37% phosphoric acid (Total Etch, Ivoclar Vivadent, Schaan, Liechtenstein). The samples were then rinsed with an air-water-mixture for 15 sec. Subsequently, the non-demineralized and the demineralized specimens were randomly assigned to one of the three treatment modalities in equal parts, so eight samples per group were further processed as follows:
I (CAP I): The plasma jet (kINPen® MED, neoplas tools GmbH, Greifswald, Germany) irradiation was statically administered to the samples’ center for 60 seconds, whereby the plasma source was kept in a perpendicular position and within a distance of 8.0 mm to the dentin surface. The jet was constantly fed with 4.3 sL*min-1 argon.
II (CAP II): A DBD plasma source (PlasmaDerm®, Cinogy, Duderstadt, Germany) was utilized in order to ignite the CAP in the ambient air between the devices’ electrodes and the specimens’ surfaces for 60 seconds, whereby a distance of 1.0 mm was kept between both. III (Control): No additional treatment was performed.
Following the respective treatments, the samples were rewetted with 0.05 ml of 2% Chlorhexidine (Chlorhexamed® Forte, GlaxoSmithKline, Bühl, Germany) for 15 sec by using a microbrush (Appli Tip, Medirel, Agno, Switzerland). According to the manufacturer's instructions, the use of a fourth generation adhesive (OptiBond™ FL Primer and Adhesive, Kerr, Rastatt, Germany) was applied and light-cured for 40 seconds (Bluephase®, Ivoclar Vivadent, Ellwangen, Germany). Consecutively, hollow acrylic glass cylinders (Plexiglas® Tube, Steiner-Technology, Rendsburg, Germany) with an internal diameter of 3.2 mm were filled with a light curing composite (Tetric® Ceram A 3.5, Ivoclar Vivadent, Schaan, Liechtenstein), and attached to the center of the pre-treated dentin surfaces. Any excess composite was carefully removed. Again, light curing was performed from the two sides for 80 sec in total. All of the specimens were transferred into distilled water and thermocycling was conducted for 5000 cycles. The temperature range was 0°C - 55°C and the dwell times and the transfer times were 20 sec and 10 sec, respectively. Before any further processing, the samples were again stored in distilled water at a temperature of 23°C.