## 2 Materials and Methods
### Operative Procedures  
Each sample was sectioned with a sterile cylindrical diamond bur (coarse grain 2886-314-014, Komet Dental, Lemgo, Germany) with a high-speed handpiece and water spray cooling exposing a flat surface of dentin surrounded by enamel. Prior to performing enamel-dentinal adhesion, the surface was then lapped with decreasing grain sandpaper (120, 180, 400 and 600 \(\upmu\)m) under abundant cold-water irrigation. Samples were then rinsed with water and gently air-dried for 10 s. Selective etching was performed with 37% orthophosphoric acid (Total Etch, Ivoclar Vivadent Group, Vienna, Austria) for 30 s on enamel and 15 s on dentin. The samples were thoroughly rinsed with water for 10 s and air-dried for another 10 s.  
The samples were divided into the following groups, based on the type of adhesive used: Group 1: Universal Bond (Tokuyama Dental, Tokyo, Japan)--Sound tooth Group 2: All-Bond Universal (Bisco Inc., Schaumburg, IL, USA)--Sound tooth Group 3: Universal Bond (Tokuyama Dental, Tokyo, Japan)--Tooth with DI-II Group 4: All-Bond Universal (Bisco Inc., Schaumburg, IL, USA)--Tooth with DI-II Groups 1 and 3: following manufacturer indications, two drops of Tokuyama Universal Bond adhesive (one drop bottle A+ one drop bottle B) were mixed in a disposable miniwell and then were applied to the air-dried enamel and dentin surfaces evenly with a microbushfor 10 s and dried with mild air for 5 s. No light curing is needed since it is a self-curing system [20]. Groups 2 and 4: following manufacturer indications, Bisco All-bond Universal adhesive was applied in two subsequent coats [20] to the air-dried enamel and dentin surfaces scrubbing with a microbrush for 10-15 s per coat, and then dried with mild air for at least 10 s and light-cured for 10 s with LED light-curing (VALO lamp, Ultradent, South Jordan, UT, USA/\(\lambda\) 385-515 nm) (Table 1). After adhesion procedures, each sample was completed with a nanofilled composite resin restoration (Asteria A1B, Tokuyama Dental, Tokyo, Japan, batch: W03010) built by multiple incremental additions simulating a conventional direct restoration and covering the whale adhesive interface. Each incremental layer of composite resin was light-cured for 20 s using the same curing lamp (VALO lamp, Ultradent, UT, USA/\(\lambda\) 385-515 nm). All prepared samples were stored in sterile saline solution at room temperature for one week prior to SEM investigations. After one-week storage time, the samples were longitudinally sectioned into two parts with a 0.3 mm thick diamond disc (Edenta AG, Au, Switzerland) mounted on a slow speed handpiece and then lapped with decreasing grit abrasive paper (200, 400 and 1200 \(\upmu\)m) with copious supply of water. In order to perform SEM scans, samples were deprived of the roots by dissecting the tooth transversely with a diamond disc (about 0.3 mm thick) (Edenta AG, Au, Switzerland). From each sample, two sections were obtained for the evaluation of the adhesive interface to the SEM. Samples were processed for SEM observation going through dehydration and gold sputtering (Assing Scientific Instruments B7340). The dehydration procedure is necessary for gold sputtering and exposes the sample to mechanical stresses due to different content of water in enamel dentin and resin-based materials. To avoid that it is possible to scan the samples without gold sputtering with a lower image quality in Variable Pressure SEM devices, such as the one that we used but this leads to other artifacts during observation since the SEM will work in partial vacuum and dehydration happens during SEM scan. This strategy would be also less accurate since every observation alters the sample with poor repeatability than it was decided for the standard gold sputtering processing procedure.