Extracted human premolar and molar teeth were collected from adolescent and young patients (10–25 years) to carry out in vitro bonding procedures after acquiring ethical approval from the National Research Ethics Service Committee London-Riverside (REC Reference 14/LO/0123). Informed consent for study participation was obtained from each participant, and from a parent and/or legal guardian for participants under the age of 18 years and all methods were performed in accordance with the relevant guidelines and regulations. After extraction, the teeth were cleaned in running water to remove any blood and soft tissue debris, then stored in a 1% chloramine-T trihydrate bacteriostatic/bactericidal solution for a maximum of one week and thereafter stored in distilled water (ISO/TS 11405:2015)56. Criteria for tooth selection included: intact buccal enamel surface without cracks and caries (examined under stereomicroscope x10 magnification), with no history of previous endodontic, orthodontic or bleaching treatments57.
Beta-tricalcium phosphate (β-TCP) and monocalcium phosphate monohydrate (MCPM) powders (Sigma-Aldrich, UK) were used in equimolar amounts as principal constituents of the solid phase for preparing acidic CaP etchant pastes (capable of forming brushite on enamel) using 37% PA [BPA] and 5 M citric acid (CA; Acros Organics™, Fisher Scientific, UK) [BCA] as a liquid phase, in a powder-to-liquid ratio of 0.8:1 and 3:1 respectively. The assigned powder and liquid were mixed using a stainless steel spatula on a glass slab for 30 s until a homogenous workable paste was obtained. The 30 s pH of the resulting paste was measured by a flat-end electrode using a digital pH meter (Oakton, Singapore). All formulations were prepared under ambient conditions (20–23 °C and 50–60% humidity). A conventional 37% PA gel (Orthotechnology, USA) was used as the control etchant, whilst Transbond XT light-cure primer and adhesive (3 M Unitek, Monrovia, California, USA) were used for all brackets bonding. Two types of pre-adjusted upper premolar brackets were used: metal (Pinnacle, stainless steel, MBT, slot 0.022 × 0.028 inch, Orthotechnology, USA) and ceramic (NeoCrystal, monocrystalline sapphire, MBT, slot 0.022 × 0.028 inch, Henry Schein, USA).
Sample preparation for bracket bonding
Premolar teeth were mounted in acrylic blocks using rubber moulds (14 mm length ×14 mm width ×17 mm depth). First, soft sticky wax was used at the tooth root apex inside the mould to align the tooth in such a way that the middle third of the buccal surface was parallel to the analyzing rod of a surveyor, to ensure a debonding force running parallel to the bonded bracket base. Then self-cure clear acrylic (Oracryl, Bracon, UK) was poured around the tooth up to about 1 mm apical to the level of cemento-enamel junction. The teeth were subsequently stored in distilled water at lab temperature until bonding.
Enamel conditioning and bracket bonding procedure
The mounted teeth were randomly allocated into two main groups to be bonded with 120 metal (stainless steel) and 120 ceramic (sapphire) brackets. Each main group was further divided into three subgroups according to the type of etchant used: 37% PA gel [control, C] (Resilience, Ortho Technology, Inc. Tampa, Florida, USA), CaP paste made of 37% PA solution [BPA], and CaP paste made of 5 M CA [BCA]. The conventional etch-and-rinse protocol5 was followed to bond all brackets. The buccal surface of each tooth was polished (10 s) with pumice slurry using rotary rubber cups, followed by water irrigation (10 s) and oil-free air dryness (10 s). This was followed by application of the etchant on the middle third of the buccal surface for 30 s, then irrigation with water for 20 s and dryness for 20 s. A thin layer of bonding primer was applied onto the etched enamel and spread by air-jet (3 s). The bracket base was loaded with the adhesive composite and placed onto the middle third of buccal surface with a pressing force of 300 g (Correx force gauge, Bern, Switzerland) for 10 seconds to ensure an even adhesive thickness. Excess composite was removed by a dental probe. LED Light curing (3 M ESPE, Elipar DeepCure-S, USA, 1470 mW/cm2 light intensity) was applied for 20 s (10 s on each mesial and distal side) according to the manufacturer instructions.
Following bracket bonding, the bonded teeth were stored in distilled water at 37 °C for 24 h. Then half of the samples (n = 20 per subgroup) were debonded, while the other half subjected to a thermo-cycling regimen of 5000 cycles between cold and hot water baths of 5 °C and 55 °C with a dwell time of 30 s in each bath and transfer time of 5 s (ISO/TS 11405:2015, testing of adhesion to tooth structure), followed by bracket debonding.
Bracket debonding for SBS, Adhesive Remnant Index (ARI) and enamel damage assessment
The SBS test was conducted using a chisel on a universal testing machine (Instron, Model 5569 A, USA) with an occluso-gingival load applied vertically at the bracket base at a crosshead speed14 of 0.5 mm/min and SBS values were calculated in MPa by dividing the load at failure by the bracket base surface area. The debonded teeth were examined under x10 magnification (Stereomicroscope (MEIJI, EMZ-TR, Japan) for the amount of remnant adhesive left and scored according to the ARI system58. Enamel damage was also examined under x10 magnification, and two random samples of each subgroup were further sputter-coated with gold palladium (15 nm) and examined using SEM machine (Hitachi High Technologies, S-3500N) at an accelerating voltage of 10 kV.
Sample preparation for X-Ray diffraction (XRD)
Calcium phosphate phase determination of BCA and BPA formulations was carried out using XRD. Three pastes of each formulation were prepared, each was kept in a glass vial for 24 h and ground with a mortar and pestle after complete setting to get CaP powder. The powder samples were examined using a Bruker D4 Diffractometer in a Flat-plate geometry using Cu Kα12, 40 kV and 30 mA X-ray radiation.
Scanning Electron Microscopy (SEM) of enamel before bonding and after bracket debonding
In order to examine the etch-patterns produced, CaP precipitation potential and enamel damage, SEM examination of the etched buccal enamel surface with 37% PA gel and BPA paste was conducted before bracket bonding and after bracket removal.
SEM analysis of enamel before bonding
Nine extracted premolars were used in this analysis and divided into three groups: three teeth were kept intact and the others randomly etched with either of the two aforementioned etchants. The crown of each tooth was sectioned mesio-distally through the occlusal central fossae using a diamond wafering blade to obtain nine buccal halves. The etchant was applied on the buccal enamel surface for 30 s, followed by irrigation with water for 20 s and then dried for 20 s. All samples were kept dry at ambient laboratory conditions for 24 h, then sputter-coated with gold palladium (15 nm) and examined using SEM machine (Hitachi High Technologies, S-3500N) at an accelerating voltage of 10 kV.
The quality of enamel etch pattern produced was assessed and scored according to the etch-pattern scale59:
Type (1): Ideal etch, preferential dissolution of the prism cores resulting in a honeycomb-like appearance.
Type (2): Ideal etch, preferential dissolution of the prism peripheries resulting in a reverse honeycomb or cobblestone-like appearance.
Type (3): A mixture of type 1 and 2 patterns.
Type (4): Pitted, roughened enamel surface. Structures look like unfinished maps or networks, enamel prisms not evident.
Type (5): Flat smooth surface, no apparent etch.
SEM analysis of enamel post debonding of brackets
The samples for this analysis were obtained from bracket-bonded premolars previously subjected to metal and ceramic bracket debonding following 24 h water storage at 37 °C and 5000 cycles thermo-cycling. Three metal and three ceramic bracket-debonded premolars of each of the PA gel- and BPA paste-etched subgroups were randomly collected, the crown of each tooth was sectioned mesio-distally through the occlusal central fossae using a diamond wafering blade to obtain the buccal bracket-debonded half, sputter-coated with gold palladium and examined with SEM. The debonded enamel surface was assessed according to the Enamel Damage Index60 which includes the following categories:
Grade (0): Smooth surface without scratches, and perikymata might be visible.
Grade (1): Acceptable surface, with fine scattered scratches.
Grade (2): Rough surface, with numerous coarse scratches or slight grooves visible.
Grade (3): Surface with coarse scratches, wide grooves, and enamel damage visible to the naked eye.
Flat-surface enamel specimen preparation for Confocal Laser Scanning Microscopy (CLSM) and raman spectroscopy
The buccal crown half of each molar tooth was sectioned mesio-distally through the occlusal central fossae using a diamond wafering blade (XL 12205, Extec Ltd., UK). The buccal enamel halves were mounted face down in cold-cure acrylic resin using silicon molds (8 × 21 × 24 mm). The outer enamel layer was removed using a water-cooled rotating polishing machine (Meta-Serv 3000 Grinder-Polisher, Buehler, Lake Bluff, Illinois, USA) and a sequential polishing protocol with silicon carbide abrasive disks (Versocit, Struers A/S, Copenhagen, Denmark) at a speed of 200 rpm: 600-grit for 10 s, 1200-grit for 20 s, 2500-grit for 30 s, and 4000-grit for 60 s. Each was followed by 1 min of water bath ultra-sonication to remove the smear layer at the enamel surface, excepting the 4000-grit which was followed by 3 min ultra-sonication. This standardized polishing protocol permits the removal of approximately 400 µm from the outer enamel layer and results in a flat, smooth and highly polished enamel surface36 (Fig. 9A). The surface of each enamel sample was divided into 3 areas using adhesive tapes: one untreated enamel, and the other two etched with either 37% PA gel or BPA paste according to the aforementioned etch-and-rinse protocol used in the bracket bonding procedure. This was followed by replacing the adhesive tapes with a black permanent marker line (Fig. 9B). The samples were kept dry at ambient laboratory conditions for 24 h before examination.
A representative image of a flat, highly polished molar buccal enamel surface (circled) embedded in an acrylic block (A), and divided into 3 zones: one untreated enamel and two etched with 37% PA gel or BPA paste before examination (B).
Confocal Laser Scanning Microscopy (CLSM) samples
CLSM was conducted to compare the etch-patterns produced by the BPA etchant paste and 37% PA gel. Before enamel etching, 0.1 wt.% Rhodamine B dye61 (Sigma–Aldrich, UK) was added to the control 37% PA gel and the liquid phase (37% PA solution) of the BPA paste to obtain fluorescent etchants. Three samples were used for CLSM examination. The microscopy examination was performed using a CLSM (Leica SP2 CLSM; Leica, Heidelberg, Germany) equipped with a 63x magnification/1.4 NA (numerical aperture) oil-immersion lens, and a laser illumination setting of 568-nm krypton (rhodamine excitation). Representative images of the most common distinguishing characteristics detected in each specimen were captured. All images were further reconstructed with Image J software.
Raman spectroscopy samples
Ten enamel samples were used. A Renishaw inVia Raman microscope (Renishaw Plc, Wotton-under-Edge, UK) running in a Streamline scanning mode was used to scan each area using a 785-nm diode laser (100% laser power) focused using a 20×/0.45 NA air objective. The signal was acquired using a 600 lines/mm diffraction grating centered at 750 cm−1 and a CCD (charge-coupled device) exposure time of 2 s. The microscope was calibrated using an internal silicon sample with a characteristic band at 520 cm−1. For each control and treated area, a Raman map was recorded at the middle part covering an area of 200 × 300 µm2 acquired with a 2.7 µm resolution. Raman maps were exported into an in-house curve-fitting software to fit the spectra and obtain the intensity mean of four phosphate peaks, in addition to the phosphate peak ratio (v1/v4)53.
Statistical methods
Sample size determination was based on one-way analysis of variance (ANOVA) comparing the SBS of three different subgroups. A sample of 15 specimens per subgroup is required to detect a significant difference with an effect size of 0.35 and 80% power 2-tailed test at 5% level of significance. Sample size calculation was achieved using G-power version 3.1.7 (Franz Faul, Uni Kiel, Germany). Analysis was conducted using SPSS statistical software (version 22, SPSS Inc., IBM, Chicago, USA). Data were tested for normality using Histogram/Q-Q plots/Shapiro-Wilk tests. One-way ANOVA was conducted for parametric data analysis (SBS, Raman peak intensity), followed by Tukey HSD post hoc multiple comparisons. Kruskal-Wallis and Mann-Whitney tests were carried out for non-parametric data analysis (ARI score). All statistical analyses were conducted at level of significance p< 0.05. The statistic value of level of significance for multiple Mann-Whitney comparisons, when conducted after Kruskal-Wallis test, was 0.008 instead of 0.05; calculated according to Bonferroni correction.
