Specimen preparation
In total, 20 human premolars, all extracted due to orthodontic reasons, were cleaned and stored in deionized water. Teeth were anonymized by securing that no tracking of the donor could be made through storage (all teeth were stored as they were obtained in a single source). The Ethics Committee of the School of Dentistry of National and Kapodistrian University of Athens has determined that under these conditions, no ethical approval is necessary for performing studies on extracted human teeth.
All selected teeth were free of fillings, surface fractures, or decays. The roots of the teeth were horizontally cut approximately 2~4 mm beneath the cemento-enamel junction with a diamond bur. Thereafter, the specimens were embedded in cylindrical molds (∅ 25 mm) in a self-curing epoxy resin (Caldofix, Struers, Ballerup, Denmark) with the apical flat-cut surface facing downwards. After curing for 1.5 h under heat (75 °C), the specimens were cut along their mesial-distal orientation. Then, the specimens were metallographic ground and polished in a grinding polishing machine (Dap-V, Struers). The specimens were ground employing SiC water coolant papers (220 to 4000 grit) and polished using a 0.04-μm colloidal silica suspension (OP-U, Struers). Then, the exposed horizontally tooth surfaces were bucco-orally separated into two zones with a sharp line cut (Fig. 1).
Simulated bonding and debonding procedures
In all specimens, one enamel zone remained untreated. The enamel zone of treated specimens was etched for 30 s with 37% phosphoric acid (Pegasus; Astek Innovations Ltd., Altrincham, UK) and consecutively sprayed with water for another 30 s. After drying the specimens with airflow, a bonding agent (OrthoSolo Universal Bonding Enhancer; Ormco Corp., Glendora, CA, USA) was applied for 30 s and blown carefully with air. At the same location, a flowable composite material (Enlight Light Cure Adhesive; Ormco Corp., Glendora, CA, USA) was applied and light-cured (radii pus LED curing light, 970 mW/cm2; SDI limited, Bayswater, Australia) for 30 s. Thereafter, the composite was cautiously removed with an adhesive removal 18 fluted bur (Renew finishing system; Reliance Orthodontic Products Inc., IL, USA) until the enamel was again exposed. Enamel was finally ground and polished by renew finishing system points and orthodontic prophy pumice.
Mechanical properties (instrumented indentation testing)
Instrumented indentation testing (IIT) measurements were carried out with a universal hardness-testing machine (ZHU0.2/Z2.5; Zwick Roell, Ulm, Germany). Force-indentation depth curves were monitored applying 0.2 N with a 2-s dwell time by a Vickers indenter. On each of the 20 specimens, four curves at each zone were taken almost 100 μm from the outer border of the embedded teeth. The mean value was used as representative for intact and debonded zone. Force-indentation depth curves were recorded (Fig. 2), and the Martens hardness (HM), the indentation modulus (EIT), and the percentage of the elastic part of indentation work (%), known as elastic index (ηIT), were measured according to the ISO 14577-1 [14]. The Poisson’s ratio values were set at 0.33 for enamel.
Low-vacuum scanning electron microscopy EDX
Of each group, five specimens were selected to analyze the morphology of the untreated and treated enamel zones by means of low-vacuum scanning electron microscopy (LV-SEM) imaging. Secondary electron images and backscattered electron images were acquired with a SEM unit (Quanta 200; FEI, Hillsboro, OR, USA), working under the following conditions: 25.0 kV accelerating voltage 90 μA beam current, approximately 1.0 Torr pressure and × 600 nominal magnification.
The elemental composition of the different zones was investigated using energy-dispersive X-ray (EDX) spectroscopic analysis. Three spectra per specimen were collected using an XFlash 6|10 silicon drift detector (Bruker, Berlin, Germany) with a slew window under the same operating conditions: a 210 x 210 μm sampling window and a 200-s acquisition time. The quantification was carried out in a standard less mode using atomic number, absorbance, and fluorescence correction factors with the dedicated software (ESPRIT version 1.9; Bruker) for the elements Cl, Ca, O, Na, P and Si.
Raman microspectroscopy
Five randomly selected teeth were further analyzed by Raman microspectroscopy to investigate possible chemical alterations on treated surfaces. One spectrum from each region was acquired with a Raman spectrometer (EZRaman-I, Enwave Optronics, Orange, CA, USA) attached to a microscope (Leica BME, Leica microsystems, Heerbrugg, Switzerland) under the following conditions: 785-nm excitation laser, 380-mW power, 1200–300 cm−1 wavelength range, 6 cm− 1 resolution. Spectra were acquired and baseline corrected by EZRaman Reader (ver 8.2.8) software (Enwave Optronics, USA).
Statistical analysis
After data of HM, EIT, and ηIT were checked for normality and found to be not normally distributed, descriptive statistics were calculated including medians and interquartile ranges (IQR). Mixed-effects analysis of variance was performed to compare between intact and treated (etched, bonded, and debonded) surfaces, with experimental group and tooth unit as the fixed- and random-effects terms respectively, calculating average differences and their 95% confidence intervals (CIs). A two-side P value of ≤ 0.05 was considered significant for all analyses, which were run in Stata SE 10.0 (StataCorp, College Station, TX).