In the fabrication of the specimens, standard procedures

were followed to assure the same final quality of the machined titanium and Zc substrates. Briefly, wax discs were fabricated using a metallic matrix. After that, the disc-sprue assemblies were invested and cast in pure titanium alloy (Dentaurum, Ispringen, Germany) using a voltaic-arc casting machine. A high-temperature phosphate-bonded investment (Rematitam Plus, Dentaurum, Ispringen, Germany) was used and the rings were burnt out according to the manufacturer’s instructions. After casting, all the cast disc specimens were sandblasted with 50-μm Al2O3 airborne particles for 10 s at a 2-cm distance, 2-bar pressure and approximately 45° of angulation to eliminate contamination. Specimens were cleaned with liquid detergent and tap water, placed in isopropyl alcohol for 15 min and then dried with absorbent paper towels. Afterwards, the

experimental surface of Epigenetic inhibitor cast specimens was polished with water-cooled sandpapers of decreasing abrasiveness (250, 400 and 600 grit). Aiming to correlate the micro-organism count with the type of material surface, a two-dimensional contact stylus profilometer (Mitutoyo SJ-201P, Kawasaki, Honshu, Japan) was used, before clinical evaluation, for measuring the surface roughness of all the tested specimens (n = 24 samples for each tested material). To determine the surface roughness for each surface material, three single measurements (−1 mm, 0 and +1 mm) CHIR 99021 with a measuring length of 5.6 mm using a cut of 0.8 mm were performed on all the specimens. After measuring, mean roughness (Ra) was calculated for each material. After exposure of intraoral splints,

disc specimens of each substrate were immediately stained with 1% neutral red to disclose the formed biofilm over the discs. The technique for the assessment of biofifilm coverage has already been described.21 and 22 Briefly, the experimental surfaces were disclosed by 1% neutral red solution and photographed (digital camera: Canon EOS Digital Rebel EF-S 18–55; and flash: Canon MR-14 EX, Canon Inc., Tokyo, Japan) with standard film–object distance and exposure time. The camera was fixed stand (CS-4 Copy Stand; Testrite Inst. Co., Inc., Newark, NJ, USA). Total surface area and areas corresponding to the PJ34 HCl stained region were measured using the image processing software Image Tool 3.0 (The University of Texas Health Science Center, San Antonio, TX, USA). Biofilm percentage was calculated using the relation between biofilm area multiplied by 100 and total surface area of the tested surface of specimens. Biofilm specimens were then evaluated by DNA checkerboard hybridisation, according to the procedures described by do Nascimento et al.23 Samples were assessed to identify and quantify the Candida species found colonising the oral biofilm formed on the tested substrates.