2.1

Preparation of HA nanorods for treatment of dentine specimens

HA nanorods structure were synthetized using ultrasonication with precipitation method [ ]. Briefly, solution of 0.19 M potassium phosphate was added dropwise to 0.32 M solution of calcium nitrate for 60 min with regular stirring at 600 rpm (80 °C). pH (∼9) was maintained using ammonia solution followed by ultrasonication in an ultrasonic bath (40 ± 3 kHz) for 30 min at 50 °C under cooling water circulation. The formed colloidal solution was centrifuged at 2200 rpm for 15 min to produce precipitation. Precipitates were washed in deionized water, dried at 110 °C for 5 h before grounding and sieving. The HA powders were calcinated at 900 °C for 3 h at heating rate of 20 °C/min. To prepare the HA treatment solution for dentin re-mineralization, the HA nanorods were added to distilled water at 15% (w/v) concentrations [ ].

2.2

Experimental set-up and dentine blocks exposure

Dentine blocks/specimens were subdivided into three HA/HIFU experimental groups and a positive control group received HA application without HIFU exposure. Dentine disc-shaped blocks/specimens (∼5-mm height) were cut from the mid-coronal dentine using diamond-blades under water coolant (Buehler, Lake Bluff, IL, USA) and pulp-spaces were cleaned from any soft tissues remnant by excavation followed by saline irrigation. The exposed coronal dentine surfaces were wet-polished with 600 grit-size SiC papers, to simulate clinically formed smear layer during conventional tooth drilling followed by sonication in distilled water for 15 min. to remove cutting debris.

The HIFU experimental setup and exposure parameters were adopted from a previous protocol [ ]. Briefly, 15 cm (length) × 15 cm (width) × 25 (depth) cm water tank was used. Tightly sealed degassed distilled water was stored at room temperature (20 °C). A bowl-shaped piezo ceramic transducer with driving circuit consisted of a linear voltage-amplifier and a signal generator, was used with water as a medium to closely approximating ultrasound beam attenuation on dentine surface ( Fig. 1 ). Continuous sinusoidal standard waveforms were provided by an arbitrary wave form generator (20 MHz Wave Form Generator, Agilent Technologies, Santa Clara, CA, USA) connected to a power amplifier and transducer. Signal from function generator was boosted by using a linear voltage amplifier (AG 1021, AG Series Amplifier, T & C Power Conversion, Rochester, NY, USA; amplifier output impedance matched to transducer by manufacturer of source transducer). Piezo-ceramic, bowl-shaped, transducer (64 mm diameter; H- 115, Sonic Concepts, Bothell, WA, USA) [resonance frequency 250 kHz] was submerged in water tank and placed on a brass backing geometrically focused with focal depths of 59.97 mm and 50.65 mm. Signal generator initiated signals amplified through amplifier and then sent to transducer with negative pressure measured using PVDF needle (184 mV/MPa over the 1 kHz −3 MHz) hydrophone (RP-Acoustic, Germany) via hydrophone system connected to a TDS420 A digitizing oscilloscope (Tektronix, Beaverton, OR, USA). Conical shaped stream of cavitation bubbles was due to axis and focusing effect of the transducer. Stream was narrowest (10 bar) at strongest ultrasonic pressure.

Schematic diagram of the experimental setup used for the treatment of dentine specimens.

Dentine-surfaces were demineralized with 35% phosphoric acid for 15 s to simulate clinical condition, and rinsed for 20 s to remove acid traces followed by gently dried by blotting in non HIFU groups. HIFU exposure was conducted on each dentine block/specimen, such that each specimen was fixed to a glass-support and placed inside petri-dish, filled with 15 mL of HA nanorods treatment solution to exposed for 10 min, connected to an in-vitro device to simulate clinical pulpal hydrostatic pressure effect [ ]. Dentine specimens were fully immersed in sealed petri-dishes. Then, for HIFU exposed groups, each petri-dish was placed in the water filled tank at the HIFU focal point. HIFU was generated at the pre-determined exposure times of 5 s, 10 s or 20 s ( Fig. 1 ) . Each petri-dish was attached to a metal supporting-plate using cyanoacrylate-adhesive located within focal-point of the HIFU-transducer. Specimens were positioned at center of the focal point of HIFU driven at 120 v using a sinusoidal wave with a peak-to-peak amplitude of 2 v and 250 kHz. Demineralized dentine specimens of the negative control group were placed in petri-dishes containing 15 mL distilled water for 10 min as previously described. Specimens of all groups were retrieved from the petri-dishes and transferred separately to polystyrene containers to be stored inside 50 mL of artificial saliva (AS) at 37 °C for one-month without renewal of storage solution with regular pH (7.2) monitoring. The AS solution was prepared as previously reported [ ]. After storage, dentine specimens were retrieved for subsequent characterization.

2.3

TEM/SEM characterization of HA nanorods and dentine-substrates

HA nanorods were characterized by TEM for average-size and shape. In addition, TEM/SEM investigations were done to characterize the structural variations of dentine-substrates for all groups. Dentin beam-shaped specimens ( n = 5 ) of 1 × 1 mm were sectioned from dentin blocks. Specimens were buffered with 0.1 M sodium cacodylate for 1 h and treated with 1% osmium tetraoxide (OSO ₄ ) in phosphate buffered solution (1 h). The specimens were rinsed with distilled water and later dehydrated in ascending ethanol followed by infiltration with araldite resin. Diamond knife in an ultra-microtome (Ultracut E, Leica Microsystems, Bayreuth Germany) was used to cut ultra-thin sections (∼90 nm) and collected on grids stained with uranyl-acetate, UO ₂ (CH ₃ COO) ₂ ·2H ₂ O, for 10 min. Specimens were viewed using TEM at 100KV (JEOL-1010, Japan).

Dentine discs ( n = 5 ) having a diameter of 2 mm, were analysed for the structure of dentine collagen with or without HIFU treatment. Prior to application of the HIFU treatment, dentin discs were marked on the lateral and opposite sides with diamond saw to facilitate the SEM analysis. All specimens were dehydrated in ascending grades of ethanol and were immediately transferred to the pressure chamber of the critical point drying machine (CPD 30; Leica). After mounting the specimens on aluminium stubs (conductive tape; double sided carbon tape), the specimens were sputter coated with gold-palladium and viewed by SEM (Hitachi S-3400 N, USA) operated at an accelerating voltage of 10 kV at different magnifications. The analysis for each disc specimen was done from the center of each quadrant and images viewed on the monitor which were evaluated by two examiners.

2.4

AFM and nanoindentation characterization of dentine-substrates

The structural variations of dentine-surfaces ( n = 5 ) were investigated by AFM in tapping-mode (Multimode/AFM, NanoScope IV, Bruker) using a silicon-nitride probe (NP-S, Bruker) with a nominal tip radius of 10 nm at 18–24 KHz resonance frequency, and a 0.06–0.12 N/m spring constant. In addition, the variations in reduced-elastic modulus (Er) of dentine-surfaces ( n = 5 ) was characterized with a G200 Nano-indenter System (Agilent Technologies, Santa Clara, CA, USA) equipped with Berkovich diamond-indenter (40-nm) at a constant strain-rate of 0.05/s and 70-nm maximum indentation-depth of 70-nm, which corresponded to a load-range of 400–500 μN. Specimens for nanoindentation were tested immediately after treatment as baseline measurement and after 1-month storage in artificial saliva. Specimens were fixed to metal supports until the desired time point and upto 15 indentations were made within the intertubular dentine region with lateral spacing not less than 400 nm for each. The tip velocity for unloading and loading segments was averaged approx. 10 nm/s

2.5

Hydroxyproline assessment

The collagen content was determined using hydroxyproline assay kit (BioVision, CA, USA) according to manufacturer’s instructions. Retrieved dentine specimens were place in 100 μg ml ⁻¹ of bacterial collagenase Type-I in tricine buffer for dentine collagen dissolution ( n = 7 ) for 24 h and 7 days. The dentine specimens were exposed to 100 μg ml ⁻¹ of type 1 bacterial collagenase in tricine buffer for 24 h and 7 days. Amount of 100 μl of supernatant was collected and hydrolyzed for 3 h in 12 M HCl at 120 °C. Next, 10 μl of aliquots from each group were transferred to a 96-well plate and dried under vacuum for evaporation. Later, 100 μl of Chloramine-T buffer reagent was added to the experimental and standard formulations and further incubated for 5 min at room temperature. 100 μl of DMAB reagent (p–dimethylaminobenzaldehyde and perchloric acid, HClO4, 50 μl were added in each well and incubated further at 60 °C for 90 min as standard curves for HYP (0–1 g/ml per well) [0–1gml–1/well] were generated using a 96-well plate reader (Infinite 200 Tecan, Switzerland) spectrophotometer at 560 nm absorbance. The hydroxyproline content for each specimen was averaged from quadruplicate measurements of each specimen.

2.6

Single-point Raman spectroscopy and collagen-orientation analyses

To obtain molecular insights in binding mode of collagen fibers, Schrödinger small-molecule drug discovery suite 2018-2 software was used. Structures of collagen Type I were downloaded from Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) ( http://www.pdb.org ). Low energy conformations of all collagen molecules were docked and analyzed using binding site using extra precision (XP) mode which incorporates water desolation energy and protein-ligand structural motifs into binding free energy scoring function.

Raman spectroscopy (Horiba-Scientific Xplora, Villeneuve d’Ascq, France) was used for the chemical analysis of dentine-surfaces along with collagen-orientation mapping. Raman peaks from dentine specimens ( n = 5 ) were taken for consideration of v1 v3 phosphate vibration, with three sub-bands (1060, 1030 and 960 cm ⁻¹ ) used at 552 and 604 cm ⁻¹ for v4 PO ₄ vibration, hydroxyproline/proline at 850−870 cm ⁻¹ and pyridinium ring vibration at 1032 cm ⁻¹ . For single point Raman spectra analysis, the laser was focused using Raman wavenumber AX100/0.90 NA air objective. Raman signal was acquired using a 600-lines/mm grating with the spectrometer equipped with CCD detector (DR-324B-FI-327, Andor Technology LTD, UK). Calibration was performed using LabSpec 6.3 analysis software as all measurements were performed under same excitation laser power and same measurement conditions. Confocal hole was set at 300 μm with a slit size at 110 μm to perform microspectroscopy at a spectral resolution of the system was 6.25 cm ⁻¹ . Total scans of 50 individual Raman were performed over a 30 × 6.5 μm sample surface with each spectrum measured using 10 s acquisition time with 10 accumulations and baseline correction performed using fifth degree polynomial function. At this point, the Raman spectroscopic peaks of both mineral and organic components found in dentine were assessed. Mineral components 960 cm ⁻¹ linked with PO ₄ ³⁻ non aromatic mode v ₁ [ ]; carbonated calcium phosphate around 430 and 451 cm ⁻¹ ( v ₂ ) [ ]; mono-hydrogen phosphate v ₁ ∼1003 cm ⁻¹ vibration [ ] indicating areas of active changes before or after HIFU treatment — Organic components peaks at 855 and 871 cm ⁻¹ indicates CC stretch of proline and hydroxyproline. Raman signal was measured using a near-infrared laser spot (diode; 0.5 μm ² ) operated at 785 nm detecting between 400–3200 cm ⁻¹ (100 mW sample surface power) [ ].

For collagen orientation analysis, Raman spectroscopy (Horiba Scientific Xplora, Villeneuve d’Ascq, France) was used to analyze dentine surfaces with or without HIFU assisted remineralization with a water immersed 60x objective. Laser power was set to 30 mW with a focal spot of ∼1 μm and ∼4 μm in lateral and axial direction using a CCD detector (DR-324B-FI-327, Andor Technology LTD, UK). The parameters used were fit in xy scan position of all collagen orientation maps performed as the parameters were color coded for pixel, orientation and length. In addition, the focal domain of Raman focus was modelled as a three-dimensional Gaussian function in x and y-directions of 1.5 μm. Sample dentine surface was scanned in a mapping mode using 1 μm steps (amide I band spectral region used was 1600–1700 cm ⁻¹ ). Steps used to scan at different polarization angles include Δ β = 15 °, from β = 90° to β = −90°. For calculation, the following equation was fitted in Matlab 7.5 (MathWorks Inc., Natick, MA, USA).

I is intensity response of Amide I; Amide I average intensity of all scans done on dentine surface; b fitting curve amplitude; β laser polarization angle; c phase shift [ ].

2.7

Crystallinity index and mineral maturity

Every individual spectrum was curve fitted for position and height and area measured underneath curves. Peaks corresponding to ν ₁ and v ₃ PO ₄ (900−1100 cm ⁻¹ ) and v ₄ PO ₄ (500−650 cm ⁻¹ ) were analyzed for parameters: (1) mineral maturity (1030/1110 cm ⁻¹ area ratio) and (2) mineral crystallinity index by inverse proportion of 604 cm ⁻¹ as peak corresponded to apatite. This was also a direct indication to crystallinity index of the specimen due to good resolution. As the peak was narrower, crystallinity index was considered higher. In order to validate using of the width at half-height of 604 cm ⁻¹ as an index, correlation between crystallinity index and maturity were also measured [ ].

2.8

Binding capacity of HA-nanorods to demineralized dentine and EDS analysis

HA-nanorods were examined using ATR-FTIR (NICOLET iS10; Thermo Scientific, USA) for standard spectrum information. Experimental and control dentine specimens ( n = 5 ) were characterized for ATR-FTIR spectra. EDS analysis of dentine specimens was used to semi-quantitatively characterize the minerals (Phosphorus/calcium) contents at three different location of dentine specimens ( n = 5) and all mean values were expressed in percentages.

2.9

Energy dispersive X-ray spectroscopy characterization

Dentine specimens (n = 5) were prepared as described for SEM for EDS analysis, however, specimens were carbon coated prior to the analysis. For each specimen measurements were taken from three different locations from the center towards forward. Voltage set was 15 kV and spot size set at 3.0 as one surface area measured was 900 μm ² with a scan duration of 180 s standardized for all specimens using FEI SEM with EDS (Field Electron and Iron Company (FEI), Hillsboro, OR, USA) detector and data analyzed using 5.21 EDAX software.

2.10

Statistical analysis

All data are presented as mean ± standard deviation and were analyzed using ANOVA followed by Tukey’s post hoc test. P < 0.05 were considered statistically significant.