## 2 Materials and Methods  
A total of 120 single-rooted extracted human premolars with straight, fully formed roots, a single canal and no previous endodontic treatment were used. Caries absence, linear roots and mature apex without pathological modifications also represented inclusion criteria. The teeth were prepared to working length using an F2 ProTaper instrument (Dentsply Maillefer) in continuous rotation. They were randomly divided into 4 groups as presented in Figure 1.  
Teeth were extracted due to orthodontic or periodontal indication with maximum 4 weeks prior to study beginning. Digital x-rays under two incidences were performed for group uniformity, all teeth with internal root resorbtions, calcifications, fractures and teeth with more than one canal per root being eliminated from the study. _Mechanical-antiseptic preparation of the root canals._ The teeth were prepared by a single operator at working length using ProTaper(r) system (Dentsply Maillefer) and X-SmartTM endodontic micromotor Motor(r) (Dentsply, UK). After each instrument, the canals were irrigated using 5 mL of 2.5% NaOCl solution. _The root canals oburation._ Each tooth was endodontically obutrated using gutta-percha in combination with one of the four sealers. The oburation method used was warm vertical condensation technique in apical third and injection of gutta-percha in the coronal two-thirds. After complete setting, the samples were placed in resin blocks and then sectioned using a microtome (Buehler-IsoMet 1000) in slices with 1 mm thickness. Four samples from the apical third were selected for each tooth (two apical and two median) and were randomly distributed for SEM, AFM and push-out analysis. _Scanning electronic microscopy (SEM)._ All samples prepared for SEM analysis were immersed in 2.5% sodium hypochlorite for 1 min, 17% EDTA for 2 min and finally three times distilled water for 2 min (R. Espinar-type ultrasound bath (RAYPA)). A single examiner measured the highest gap width as the highest distance between root canal dentin and the sealer with a magnification of \(\times\)2000. _The push-out test._ Each slice of tooth was subjected to a constant force, by means of a cylinder-piston of 0.70 mm thick attached to a universal mechanical testing machine (Lloyd Instruments--LR5k plus). On the surface of the filler material, a constant load of 0.5 mm/min was maintained until failure. Failure forces were recorded for each section of the tooth. The retention strength S (MPa) of the \"monoblock system\" of the tested tooth slice was calculated. Each tooth slice was numbered and examined on both sides, before and after the push-out test under the stereomicroscope (STEMI 2000 C Zeiss, Jena, Germany), at a magnification of 20\(\times\). The bond strength values for all sealers were analyzed and compared using ANOVA statistical test and SPSS Statistic Software. _Atomical force microscopy (AFM)._ The images were obtained with a commercial Ntegra Spectra microscope (NT-MDT, Russia). After acquisition, the images were processed using Nova v1.1.0.1837 (NT-MDT). For each sample, the measurements were performed at the contact area, dentin-sealant. For the analysis of the interface, researchers used statistical  
Figure 1: Sample distribution for SEM, AFM and Push-out study. parameters calculated for the image on which was applied the correction \"fit lines\" by which a second-degree polynomial is found and extracted for each scan line. _FTIR Spectroscopy evaluation_. A Teflon mold was used to obtain 20 samples of the experimental materials (10 samples E1-DCR and 10 samples E2-HA) with the dimensions: diameter d = 15 \(\pm\) 1 mm and thickness h = 1 mm. The samples were immersed in blood (5 samples E1-DCR and 5 samples E2-HA) and hemoglobin (5 samples E1-DCR and 5 samples E2-HA) for 42 days at 40 \({}^{\circ}\)C and then incorporated in the resin so that they could be sliced. IR spectra were recorded using an FT-IR Spectrum BX spectrometer using an attenuated reflection (ATR) device. Statistical analysis.