## 2 Materials and Methods  
27 cylindrical blocks (height 4 mm and diameter 6 mm) of supra-nano composite col. A1 (Estelite Sigma Quick, Tokuyama Dental, Japan) were made using a silicone mold matrix.  
Considering a power level of 85% with a type-I error = 0.05, for parameter roughness (Ra), three samples for each independent group (surface-treated and independent control) were necessary.  
The technical characteristics of the composite resin provided by the manufacturer were: a resin matrix (bisphenol A-glycidyl methacrylate (Bis-GMA) and triethylene glycol dimethacrylate (TEGDMA)) and particles of reinforcement (71% of the total volume) formed by spherical particles of silica and zirconia, with sizes ranging from 0.1 to 0.3 \(\upmu\)m (average size of 0.2 \(\upmu\)m).  
The cylindrical blocks were obtained through two vertical composite increments of 2 mm inside the silicone matrix. Using a LED curing lamp (Valo, Ultradent, South Jordan, UT, USA) the composite layers were light-cured for 20\" at a distance of 1 mm at 3.200 mW/cm\({}^{2}\). In order to prevent the inhibition of polymerization due to the presence of oxygen, and to create a homogeneous surface, the last layer of composite was covered by a glass plate before light-curing.  
To make the samples' surface uniform, all the composite blocks were polished under 4\(\times\) magnification (EyeMag Pro S, Zeiss, Oberkochen, Germania), using Soft-Lex (3M ESPE, St Paul,Minnesota) coarse-grained, medium, fine and superfine discs for 10 s each. After each step the samples were washed with distilled water and air dried.  
The samples were randomly divided into 9 groups (n = 3). The surface of the samples from each group was treated with different roughening protocols:  
A) Control group, no surface treatment.  
B) Etching for 30 s.  
C) Etching for 60 s.  
D) Roughening with diamond bur.  
E) Roughening with diamond bur and etching for 30 s.  
F) Roughening with diamond bur and etching for 60 s.  
G) Sandblasting.  
H) Sandblasting and etching for 30 s.  
I) Sandblasting and etching for 60 s.  
Etching was performed with 35% orthophosphoric acid (Ultra-Etch, Ultradent) for 30 s in groups B, E and H or 60 s in groups C, F and I. After etching, each sample was washed with distilled water and air dried.  
Milling was carried out with a diamond bur with granulometry 151 \(\upmu\)m (6837 KR Komet) mounted on a handpiece for 3 s (groups D, E and F)  
Sandblasting (20 s) was performed with an intraoral sandblaster using Al\({}_{2}\)O\({}_{3}\) 50 \(\upmu\)m (Dentoprep, Renvig Dental).  
The observational analysis of the treated surface of the samples was carried out with an SEM (Phenom G2, Phenom, Eindhoven, the Netherlands), at 2.100x magnification, with 5 kV voltage and secondary electrons (SE). This SEM does not require any treatment of the sample surfaces [24,25].  
The samples were then observed and analyzed with a confocal microscope (Leica DCM 3D, Leica Microsystems) in order to measure roughness. In each sample an area of 2.5 mm\({}^{2}\) was selected using a systematic random sampling protocol for stereological and morphometrical analysis and the roughness (Ra) was calculated using the following formula:  
\[Ra=\frac{1}{l}\int_{0}^{l}\bigl{|}Z(x)\bigr{|}dx\]  
For each experimental group the mean and quantitative parameter standard deviation (SD) were calculated.  
Data were analyzed using the Student's t-Test and differences of p \(<\) 0.05 were considered statistically significant.  
The statistical analysis was performed using the SPSS 17.0 for Windows package and the Prism software package (GraphPad, La Jolla, CA, USA).