Bioactive glass air-abrasion was used to pre-condition enamel white spot lesion.
Pre-conditioning increased the average surface roughness of the lesion.
An ultra-thin, clinically insignificant layer was removed from the lesion surface.
Pre-conditioning enhanced subsequent remineralization using bioactive glass 45S5.
To evaluate the effect of pre-conditioning enamel white spot lesion (WSL) surfaces using bioactive glass (BAG) air-abrasion prior to remineralization therapy.
Ninety human enamel samples with artificial WSLs were assigned to three WSL surface pre-conditioning groups ( n = 30): (a) air-abrasion with BAG-polyacrylic acid (PAA-BAG) powder, (b) acid-etching using 37% phosphoric acid gel (positive control) and (c) unconditioned (negative control). Each group was further divided into three subgroups according to the following remineralization therapy ( n = 10): (I) BAG paste (36 wt.% BAG), (II) BAG slurry (100 wt.% BAG) and (III) de-ionized water (negative control). The average surface roughness and the lesion step height compared to intra-specimen sound enamel reference points were analyzed using non-contact profilometry. Optical changes within the lesion subsurface compared to baseline scans were assessed using optical coherence tomography (OCT). Knoop microhardness evaluated the WSLs’ mechanical properties. Raman micro-spectroscopy measured the v -(CO 3 ) 2− / v 1 -(PO 4 ) 3− ratio. Structural changes in the lesion were observed using confocal laser scanning microscopy (CLSM) and scanning electron microscopy–energy dispersive X-ray spectrometry (SEM–EDX). All comparisons were considered statistically significant if p < 0.05.
PAA-BAG air-abrasion removed 5.1 ± 0.6 μm from the lesion surface, increasing the WSL surface roughness. Pre-conditioning WSL surfaces with PAA-BAG air-abrasion reduced subsurface light scattering, increased the Knoop microhardness and the mineral content of the remineralized lesions ( p < 0.05). SEM–EDX revealed mineral depositions covering the lesion surface. BAG slurry resulted in a superior remineralization outcome, when compared to BAG paste.
Pre-conditioning WSL surfaces with PAA-BAG air-abrasion modified the lesion surface physically and enhanced remineralization using BAG 45S5 therapy.
The optimal goal of minimal invasive (MI) dental caries management is to prevent and “heal” the incipient lesion by inhibiting the demineralization process, preventing any further mineral loss and enhancing the remineralization repair process . The enamel white spot lesion (WSL) results from the physical changes occurring in enamel due to the caries process before it has reached the enamel–dentin junction (EDJ) . Different protocols have been described to remineralize enamel WSLs including bioactive glass (BAG) 45S5 . BAG 45S5 is an inorganic amorphous, calcium, sodium phosphosilicate material which contains fivefold ratio of Ca/P . It interacts with aqueous solutions such as saliva to form a hydroxycarbonate apatite (HCA) layer, attached chemically to the treated surfaces . In vitro and in vivo studies demonstrated that BAG 45S5 allowed close contact of the living cells at its surface and did not contain leachables which produce inflammation . It has been shown that BAG 45S5 mixture prepared to treat dentin hypersensitivity and incipient enamel caries is a biocompatible material concluded by assessing the viability and the morphological alternations of pulp cells .
The remineralization of enamel WSL is a complex physico-chemical process where the remaining mineral crystals are less reactive, covered by salivary proteins and the limited diffusion of ions lessens the net mineral gain . In order to promote the WSL remineralization process, an additional stage of pre-conditioning the lesion surface using phosphoric acid prior to the remineralization therapy has been described and shown to increase the remineralization of WSLs . In the present study, air-abrasion with PAA-BAG powder was used to pre-condition the lesion surface as opposed to cutting it, in order to promote remineralization using different topical therapies including mixtures of BAG 45S5. Polyacrylic acid (PAA) altered the hydroxycarbonate apatite (HCA) induced by BAG 45S5, with smaller structures deposited on the surfaces of enamel WSLs, remineralized using PAA-BAG slurry and assessed using Raman micro-spectroscopy and SEM . The ( COOH) functional group of PAA may bind the calcium and phosphate ions to form nano-precursors small enough to penetrate the carious lesion more effectively .
The objectives of this study were to assess the physical effects of this WSL surface pre-conditioning and to study the impact of this modification on overall lesion remineralization. The physical changes were assessed using non-contact white light confocal profilometry and optical coherence tomography (OCT). The mineral content at the lesion surface following the application of the remineralization therapies was evaluated using Raman micro-spectroscopy used in a StreamLine™ scanning technique to map the lesion surface measuring the v -(CO 3 ) 2− / v 1 -(PO 4 ) 3− ratio of 2880 spectra per sample. The biomechanical properties of the WSLs were assessed using Knoop microhardness testing. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectrometry (EDX) were used to study the ultra-structural changes within remineralized artificial WSLs created using a bi-layer demineralization protocol. The two null hypotheses investigated in this study were that pre-conditioning the WSL using PAA-BAG air-abrasion had no effect on lesion surface characteristics and that this pre-conditioning had no effect on the following remineralization therapy using BAG 45S5.
Materials and methods
Caries-free human molars were collected using ethics approval reviewed by the East Central London Research Ethics Committee (Reference 10/H0721/55), stored in refrigerated de-ionized water and used within a month from the extraction. One buccal enamel slab from each tooth was sectioned using a diamond wafering blade (XL 12205, Benetec Ltd., London, UK). The slab’s initial surface integrity was inspected using a confocal tandem scanning microscope (TSM) (Noran Instruments, Middleton, WI, USA), with an 20× air objective in reflection scanning mode. Ninety samples were included in this study. The samples were included face down in acrylic resin using a hard-anodized aluminum and brass sample former (Syndicad Ingenieurbüro, München, Germany). The superficial enamel layer was removed to create more consistent, reproducible artificial enamel lesions , using a water-cooled rotating polishing machine (Meta-Serv 3000 Grinder-Polisher, Buehler, Lake Bluff, IL, USA) with a sequential standard polishing protocol started from 600- to 4000-grit silica carbide disks, followed by 3 min of ultrasonication to remove the smear layer. Dental wax was applied to protect part of the enamel leaving an exposed window of 3 mm × 1 mm in the central area of the exposed enamel slab. The specimens were submitted to a previously documented bi-layer demineralization protocol of 8% methylcellulose gel buffered with a layer of lactic acid solution (0.1 mol/L, pH 4.6) for 14 days at 37 °C, to create artificial WSLs with an average depth of 70–100 μm . The sound enamel areas around the WSL were covered with polyvinyl chloride tape throughout the experimental procedures, and removed at the end of the remineralization therapies. The samples were assigned into nine experimental groups according to the surface pre-conditioning and remineralization therapies ( n = 10; Table 1 ).
|Group ( n = 10)||Experimental procedures||Before surface pre-conditioning||After surface pre-conditioning||After remineralization therapy|
|Surface pre-conditioning a||Remineralization therapy b|
|1||PAA-BAG air-abrasion||BAG paste (36 wt.% BAG)||0.18 ± 0.02||0.71 ± 0.15||0.80 ± 0.13|
|2||PAA-BAG air-abrasion||BAG slurry (100 wt.% BAG)||0.17 ± 0.01||0.57 ± 0.15||0.60 ± 0.06|
|3||PAA-BAG air-abrasion||De-ionized water (−ve control)||0.16 ± 0.01||0.49 ± 0.09||0.59 ± 0.08|
|4||Acid-etching (+ve control)||BAG paste (36 wt.% BAG)||0.19 ± 0.01||0.90 ± 0.07||1.14 ± 0.10|
|5||Acid-etching (+ve control)||BAG slurry (100 wt.% BAG)||0.21 ± 0.02||0.70 ± 0.04||0.82 ± 0.05|
|6||Acid-etching (+ve control)||De-ionized water (−ve control)||0.20 ± 0.01||0.80 ± 0.07||0.82 ± 0.05|
|7||Unconditioned (−ve control)||BAG paste (36 wt.% BAG)||0.19 ± 0.02||0.20 ± 0.01||0.24 ± 0.02|
|8||Unconditioned (−ve control)||BAG slurry (100 wt.% BAG)||0.29 ± 0.07||0.25 ± 0.04||0.31 ± 0.06|
|9||Unconditioned (−ve control)||De-ionized water (−ve control)||0.16 ± 0.15||0.20 ± 0.02||0.25 ± 0.03|
The surface pre-conditioning was conducted once before initiating the remineralization therapy. PAA-BAG abrasive powder (2–8–17 μm) was prepared by mixing 60 wt.% BAG 45S5 powder with 40 wt.% PAA powder (MW: 1500, Sigma Chemicals, Gillingham, Dorset, UK) for 5 min at 300 rpm. The homogeneity of the mixed powders was validated using Fourier-transform infrared spectroscopy (FTIR) (Perkin-Elmer, Beaconsfield, UK) to detect the vibration of C O (PAA) at 1710 cm −1 and of Si O (BAG) at 1030 cm −1 . An Aquacut™ (Velopex, Harlesden, UK) dental air-abrasion unit was used to pre-condition the lesion surface using the following operating parameters: air pressure, 20 psi; powder flow rate dial, 1 g/min; nozzle angle, 90°; nozzle-lesion distance, 5 mm and the internal nozzle diameter, 900 μm . The air-abrasion was conducted for 10 s in wet abrasion mode fulfilled by shrouding the air stream with a curtain of de-ionized water. For acid-etching treatment, 35% phosphoric acid gel (3 M ESPE Dental Products, St Paul, MN, USA) was applied onto the lesion surface for 30 s followed by 1 min rinsing with de-ionized water and 5 s drying with a gentle oil-free air stream using the three in one syringe of a dental unit. The negative control samples in the surface pre-conditioned groups remained untreated ( Table 1 ).
Post-conditioning remineralization therapy
The remineralization therapy included the application of a BAG paste or a BAG slurry twice daily (5 min per application) for 21 days. The remineralization agent was applied onto the exposed WSL surface using a microbrush with hand-agitation by a single operator blinded to the surface pre-conditioning procedure. Following each application, the samples were rinsed thoroughly with de-ionized water and incubated at 37 °C in de-ionized water, which was refreshed at each application for all of the nine experimental groups. The BAG slurry was prepared using de-ionized water (L/P ratio of 1 g/ml) prior to the treatment using BAG 45S5 particles (2–6–12 μm) . The BAG paste was prepared using the following formula: 36 wt.% BAG 45S5 powder, 25.70 wt.% chalk (CaCO 3 ) and 38.3 wt.% glycerine and stabilizers. In order to prevent any premature BAG chemical reaction, the paste did not include water in its composition. Therefore, the de-ionized water was introduced into the paste at the lesion surface to initiate the reaction kinetics of BAG particles. The negative control of the remineralization agent was de-ionized water, while the positive control was a BAG slurry (100 wt.% BAG particles) relying upon the results of a previous study where this formula was shown to remineralize enamel WSLs when compared to a “standard” remineralization solution .
The samples’ surface topography was scanned three times: before surface pre-conditioning, after the surface pre-conditioning and finally, post-remineralization therapy, using non-contact white light confocal profilometry (Xyris™ 4000 WL, TaiCaan™ Technologies Ltd., Southampton, UK) with a 10 μm step-over distance and 10 nm vertical resolution. A standard scan area of (3 mm × 2 mm) was chosen over the center of the surface including the lesion in the middle surrounded by sound enamel from each side acting as internal sample reference levels. The resulting 3D images were analyzed using a manufacturer’s software supplied by leveling the reference sound enamel areas to a “zero” plane. The step height of the lesion surface in relation to the sound enamel level was obtained by averaging five measurements within each sample. In order to obtain the average roughness ( S a ) of the lesion surface, further three areas of 250 μm × 250 μm were scanned within the lesion surface and analyzed for each sample.
Optical coherence tomography (OCT)
The specimens were scanned prior to surface pre-conditioning and again after remineralization therapy was complete using OCT (VivoSight, Kent, UK) operating at 1305 nm central wavelength, 10 kHz rate and 15 mW energy power. This system uses XY mirror sets with multi-beam Z technology in order to maintain the axial resolution over an extended range of depth . The lateral resolution is 7.5 μm and the axial resolution is 10 μm in air which corresponds to 6.1 μm in enamel assuming a refractive index ( n ) of 1.63. The OCT beam was oriented perpendicularly and set at a fixed distance over the sample surface by means of a moving stage. For each sample, 50 b -scan images were acquired at 5 μm intervals covering an area of 3000 μm × 250 μm with a pixel size of 3.36 μm × 4.06 μm. The multi-beam scan creates a scan depth of approximately 2 mm.
The 16-bit TIFF file b -scan images were imported into a purpose-written ImageJ macro (ImageJ, MD, USA) which analyzed individual o -scans using a polynomial fitting function. Both the air/lesion interface reflection and the average subsurface scattering, up to 40 μm in depth, were recorded for each X and Y sample positions ( Fig. 2 ). The subsurface light scattering was normalized to the surface reflection within each sample to be analyzed statistically, measuring the reduction (%) of the subsurface light scattering at the end of remineralization therapies in comparison with baseline scans.
Lesion Knoop microhardness
A Struers Duramin microhardness tester (Struers Ltd., Denmark) with a Knoop diamond indenter (50 g load applied for 10 s) produced elongated diamond-shaped indentations which were imaged with an 40× air objective and the Knoop values were calculated from measurements of each long-axis indentation, using the manufacturer’s software supplied. Three measurements, 500 μm apart, were recorded and then averaged to measure the lesion surface microhardness of each sample at the end of remineralization therapies. Prior to each measuring session, the instrument was calibrated using a calibrated transfer-standard block (N 0441, UKAS calibration, UK).
The lesion surfaces were scanned using a Renishaw inVia Raman microscope (Renishaw Plc, Wotton-under-Edge, UK), running in StreamLine™ scanning mode, using a 785-nm diode laser (100% laser power) focused into the samples through a 20× air objective. The signal was acquired using a 600 lines/mm diffraction grating centered at 800 cm −1 and a CCD exposure time of 2 s. The Raman map was set at the center of the lesion surface covering an area of 700 μm × 500 μm and including 2880 spectra acquired with 2.7 μm resolution across the lesion. The demineralized enamel produced a slight background autofluorescence (AF), and to take this slowly varying background signal into account, the resultant spectra were exported into an in-house curve-fitting software to fit the spectra using a linear combination of a Gaussian function and a first order polynomial using the following fitting function:
F ( X ) = A X + B + C Exp − ( X − D ) 2 ( 2 E 2 )
The intensity of the peak was given by the fitting parameter C from the above equation. The intensity of phosphate peak at 959 cm −1 and that of carbonate peak at 1085 cm −1 were measured to obtain the ratio of v -(CO 3 ) 2− / v 1 -(PO 4 ) 3− per spectrum. The measurements of 2880 spectra were averaged to obtain one value from each sample.
Imaging was conducted for three selected experimental groups including, air-abrasion + BAG paste, air-abrasion + BAG slurry and unconditioned + de-ionized water (negative control). For CLSM imaging, three samples from each experimental group were sectioned vertically across the lesion and ground with carborundum paper up to 1200 grit. The samples were soaked in a freshly prepared 0.1 mM Rhodamine-B solution (R6626-Sigma–Aldrich, Dorset, UK) for 24 h, without further rinsing . A confocal laser scanning microscope (CLSM) (Leica Microsystems, Heidelberg GmbH, Germany) was used to image the samples with an 63× oil objective lens in conjunction with 540 nm excitation wavelength and 625 nm emission wavelength for the Rhodamine-B. Samples were scanned between 10 and 50 μm below the sectioned surface to avoid the smear layer, created during the cutting procedures .
For SEM scanning, three samples from each group were fractured across the WSL to provide a sagittal view without surface grinding or polishing artifact, secured to aluminum stubs and sputter coated with gold (Emitech K550, UK). The fractured surfaces were imaged using an FEI Quanta 200F field emission scanning electron microscope (FEI Co. Ltd., Cambridge, UK), with accelerating voltage of 10 kV, working distance of 10 mm. For EDX mapping of the lesion top surface, further three samples from each group were carbon sputter-coated and scanned using energy-dispersive X-ray spectroscopy (EDAX Inc., 91 McKee Drive, Mahwah, NJ 07430, USA), with accelerating voltage of 10 kV and working distance of 12 mm.
Statistical analysis was carried out using Stata statistical package (Stata-CorpLP v 11.2, TX, USA) at p = 0.05 significance. Data were tested for normality, and the non-normal data were transformed into the normal using appropriate transformation prior to further analysis. The profilometry data were tested using two-way analysis of variance (ANOVA) and Tukey’s HSD as post hoc including significant interactions, while those of Raman micro-spectroscopy, microhardness and OCT were analyzed using linear modeling by including the interaction term for pre-conditioning and remineralization therapy.