Strain and growth conditions
Conidia suspensions of A. fumigatus NCPF 7367, obtained from Public Health England Culture Collections, were prepared as described by Mowat et al.  Briefly, the strain was streaked out from glycerol stocks stored at − 80 °C and incubated at 37 °C for 3 days. Conidia were harvested by flooding the surface of the plates with 5 ml of phosphate-buffered saline (PBS) containing 0.025% (v/v) Tween-20 (Sigma Aldrich, St. Louis, MO, USA) and rocking gently, washed in the same buffer, counted using a Burker hemocytometer, and diluted to a density of 105 spores/ml in 3-(N-morpholino) propanesulfonic acid (MOPS)-buffered Roswell Park Memorial Institute medium (RPMI) 1640 (Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FCS). This suspension was used as inoculum.
Five different bone substitutes, commonly used in sinus floor augmentation procedures, were selected for the experiment. The biomaterials included one allograft (demineralized bone matrix, Puros, Zimmer Biomet, Palm Beach Gardens, FL, US), one xenograft (anorganic bovine bone, BioOss, Geistlich, Wolhusen, Switzerland), and three alloplastic materials (ß-tricalcium phosphate, ßTCP, RTR, Septodont, Saint Maur des Fossés, France; synthetic nano-hydroxyapatite, NHA, Fisiograft Bone, Ghimas, Casalecchio di Reno, Italy; synthetic hydroxyapatite, HA Idrossilapatite, Centro di Odontoiatria Operativa, Padova, Italy). About 50 mg of each bone substitute was distributed in a 24-well microtiter plate, inoculated with 0.5 ml of the conidia suspension, and incubated at 37 °C with orbital shaking (130 rpm) for 4 and 8 h, to allow the attachment of the fungal cells. After these contact periods, the materials were washed to remove the unbound cells, and 1 ml of fresh RPMI medium was added to allow the growth of the biofilm for further 20 h. Finally, samples were thoroughly washed three times with PBS.
Confocal microscopy analysis
Biofilm bound to the different materials was stained for polysaccharides with 1 ml of 25 μg/ml concanavalin A-Texas Red conjugated (CATR) (Molecular Probes, Eugene, OR, US) for 30 min in static conditions, gently washed once with water and examined by confocal microscopy (CLSM) using a Nikon C1-SI confocal microscope (Nikon Instruments Europe BV, Amsterdam, Netherlands). Excitation and emission wavelength were set to 561 and 600 nm, respectively. Z-stacks were acquired with a 20x/0.5 air objective at 2 μm intervals. The image stacks collected by CSLM were analyzed with the EZ-C1 Free Viewer (Nikon Corporation, Tokyo, Japan) and the Image J 1.47 (Wayne Resband, National Institutes of Health, Bethesda, MD, USA) softwares.
Twelve images, randomly acquired from three independent experiments for each time of adhesion, were analyzed by the COMSTAT software package (www.comstat.dk) to evaluate the amount of the biomass .
Scanning electron microscope analysis
Specimens were fixed at 60 °C for 2 h, then mounted on aluminum stubs covered with two-sided conductive carbon adhesive tape. Subsequently, the samples were sputtered with gold (Sputter Coater K550X, Emitech, Quorum Technologies, Lewes, UK) and immediately analyzed by means of a scanning electron microscope (SEM) (Quanta250 SEM, FEI, Hillsboro, OR, US) operated in secondary electron detection mode. The working distance was adjusted in order to obtain the suitable magnification; the accelerating voltage was set to 30 kV.
Statistical analysis was performed by means of SPSS 15.0 software (SPSS, Chicago, IL, US). Data for descriptive statistics were expressed as mean ± SD. The normality of the distribution and the equality of variance were assessed with Kolmogorov–Smirnov and Levene test, respectively. Data were then analyzed with Kruskal–Wallis test, and statistical significance was pre-set at p < 0.05.