Abstract
Objectives
The aim of this study was to characterize the mineral density parameters through natural enamel brown spot lesions (BSLs) and to visualize and map their mineral distribution pattern in comparison to enamel whitespot lesions (WSLs).
Methods
Study specimens included seventeen proximal WSLs (ICDAS 1, 2), seventeen proximal BSLs and seventeen sound proximal specimens (ICDAS 0) collected from The Oral Surgery Department at Sydney Dental Hospital, Sydney, Australia. Imaging was undertaken using a high resolution, desktop micro-computed tomography system. A calibration equation was used to transform the grey level values of the images into true mineral density values. The qualitative analysis and the quantification of the lesion parameters including the mineral density and the thickness of the enamel lesion surface layer were performed using mineral density profiles plotted in FIJI and the visualized mineral maps in MATLAB respectively.
Results
The maps of brownspot lesions revealed irregular demineralization patterns with faint boundaries and outlines. The regular triangular shape of proximal lesions was recognizable only in some parts of individual BSLs or was completely unrecognizable within the entire lesion. Scattered free-form areas of high density enamel were observed within or close to the surface of BSLs. A layer of high density enamel with a mineral density close to that of sound enamel was observed in all of the BSLs. The mean mineral density of the body of BSLs, including the scattered areas of high mineral density, was significantly higher than the corresponding values in white-spot lesions. The mean thickness of the surface layer in BSLs (79 ± 15 μm) was also significantly higher than white-spot lesions (51 ± 11 μm) (p < 0.05).
Conclusion
This study demonstrated that the mineralization parameters such as density and the thickness of the surface layer as well as distribution patterns through natural enamel brown spot lesions (BSLs) are different from enamel white-spot lesions (WSLs). The higher mineral density of the body of the lesion and the increased thickness of the surface layer in brown spot enamel lesions may suggest possible subsurface remineralization in the majority of naturally arrested BSLs.
1
Introduction
Enamel caries is the result of the disruption of the microbial homeostasis of dental plaque and the consequent breakdown of the mineral equilibrium between enamel and the biofilm fluid. Enamel lesions have been classified using a range of criteria based on their activity, staging and location . Staging criteria such as International Caries Detection and Assessment System (ICDAS) not only are utilized for the classification of caries, but also provide the basis for the choice of an adequate therapy. Among various activity criteria such as color, luster, hardness, roughness and overlying microbiota , the color of the lesion represents a simple and clinically perceived index for the classification of non-cavitated enamel caries. Accordingly, dull and chalky white enamel lesions are considered as active demineralization areas , whereas brown-spot lesions are assumed to reflect naturally arrested or hardened enamel caries .
Brown-spot lesions (BSLs) are localized brown to black discoloration of enamel usually located gingival to the contact area of proximal tooth surfaces. These lesions are commonly formed at a previously stagnant and plaque retentive region which has turned into a highly cleansable free-smooth surface such as in the proximal surfaces adjacent to extraction sites which have not been restored for a long time . Due to the absence of the adjacent tooth in these cases, clinicians can perform direct visual and tactile examination of proximal BSLs and therefore directly recognize the possible arrest of the carious process and the hardening of the demineralized enamel in these lesions.
Some literature has mentioned the faint radiographic evidence of remineralization in brown-spot lesions and the subsequent challenges for the radiographic diagnosis of these lesions . Several studies have also shown the increased mineralization and resistance of the external surface of BSLs . Despite these observations, the internal structure and mineral distribution patterns of brown-spot lesions have not been investigated extensively or with high resolution methods.
In fact there is a knowledge gap regarding the mineral structure of the ‘hardened surface layer’ and the supposed ‘remineralized subsurface area’ in these lesions. Concerning the current interest in the remineralization and non-invasive management of dental caries , understanding the mineral content and microstructural characteristics of naturally arrested enamel lesions can provide valuable insights about caries arrest and remineralization processes in vivo. Therefore, the aim of this study is to characterize the mineral density parameters through natural enamel BSLs and to visualize and map their mineral distribution pattern in comparison to enamel white-spot lesions (WSLs).
2
Materials and methods
2.1
Study specimens
Extracted molar and premolar teeth were collected from The Oral Surgery Department at Sydney Dental Hospital, The University of Sydney (Ethics approval protocol No X12-0065 & HREC/12/RPAH/106). The majority of the teeth with white spot enamel lesions were extracted for orthodontic purposes from young individuals aged 14–18. Teeth with brown spot enamel lesions were extracted for periodontal or periapical disease from mature aged patients. Following extraction, teeth were partially sterilized in MILTON anti-bacterial solution (0.95% (w/w) sodium hypochlorite, Milton Australia PTY LTD.) for 15 min and then brushed clean under running water to eliminate any plaque or removable stain. After decontamination, the samples were stored in Hanks Balanced Salt Solution (HBSS) at 4 °C prior to use. Thymol granules (Sigma Aldrich, Australia) were added to the solution for disinfection and prevention of fungal growth.
Two calibrated clinicians visually assessed the specimens and seventeen proximal WSLs (ICDAS 1, 2) , seventeen proximal BSLs and seventeen sound proximal areas (ICDAS 0) were chosen as study and control groups. The inclusion criteria for brown spot enamel lesions, included being non-cavitated with localized dark brown discoloration which is intrinsic to enamel and is not an external surface stain ( Fig. 1 ).
Three hydroxyapatite discs with known low (1.240 g/cm 3 ), medium (1.406 g/cm 3 ) and high (1.666 g/cm 3 ) mineral densities were used for grey level calibration and determination of the mineral density values. Hydroxyapatite discs were located and fixed on top of each specimen before imaging. Details for the fabrication process and the materials used for constructing the phantoms are described elsewhere .
2.2
Parameters for x-ray micro-tomography scanning and tomographic reconstruction
Imaging of the teeth and phantoms was performed using a high resolution fourth generation micro-computed tomography system (Skyscan 1172, Skyscan, N.V, Aartsellar, Belgium) with an accelerating source voltage of 100 keV, a source current of 100 μA and an exposure time of 885 ms. Before the scan, teeth were stored in Hanks Balanced Salt Solution (HBSS) and during scan they were stabilized in a plastic tube using polystyrene foam, preventing dehydration of the specimens. Low energy x-rays were eliminated using an inbuilt filter equal to 1.0 mm thickness of aluminum and 0.05 mm of copper to restrict spectral bandwidth of the polychromatic radiation. The equivalent monochromatic energy spectrum of filtered x-ray had an effective mean energy of 60 keV. The long axes of the teeth were parallel with the center of rotation of the mounting device. During the scanning process, the samples were rotated over 360° at angular increments of 0.14° generating 2570 two-dimensional shadow projections with an image matrix of 2000 pixels × 1048 pixels. These images were saved as 16 bit Tagged Image File Format (TIFF) and consequently exported to a 3-D cone beam reconstruction program (NRecon software, version 1.4.4; SkyScan) for the reconstruction of the 3-D object. The tomographic reconstruction produced a dataset of slice views in 16 bit TIFF format, which were perpendicular to the specimen rotation axis and had a voxel size resolution of 8.82 μm. The reconstructed volumes of the whole image stacks were vertically re-sliced in Fiji (W.S. Rasband, U. S. National Institutes of Health, Bethesda, Md, USA) to produce vertical tomographic images.
2.3
De-noising and mineral mapping
The produced tomographic images were consequently imported into MATLAB (MatLab R2012b 8.0.0.783, Mathworks, Natick, MA, USA) for the de-noising process and for increasing the signal to noise ratio of micro-CT images. De-noising was performed using a method based on total variation regularization , with the following parameters:
Following de-noising, lesions were color-coded for improved visualization and mapping of mineral density using colormapeditor command in MATLAB by choosing Jet color map with fixed RGB (Red, Green, Blue) index values for all of the colorized images. The color codes were based on the grey level values and corresponding mineral densities of each specimen, producing calibrated mineral maps.
The calibration of the mineral density was implemented by measuring and averaging the grey level values of 10 points on selected images of each hydroxyapatite phantom, followed by plotting the obtained grey level values against the mineral density value of that phantom. Based on the plotted values, the calibration equation was calculated and used to transform the grey level values of the images into true mineral density values.
2.4
Quantitative analysis
For each specimen, a total of 6 slices corresponding to the beginning, middle and the end of the lesion were selected from the image stack. For calculating the mineral density of each region of interest (ROI) including the surface layer and the body of the lesion, the values of all pixels in the specific region were measured and averaged. In the sound specimens, the ROIs were selected at the outer layer as well as the inner layer of enamel.
The thickness of the surface layer of the carious lesions was measured using line scans of the mineral content profile across the surface layer of the lesions ( Fig. 2 B and D). The beginning of the surface layer was defined on the external enamel surface and the end of the surface layer was defined as the position where the inner slope of the mineral curve of the surface layer undergoes a radical change. This definition is in accordance with the method of Groeneveld and Arends .
The qualitative analysis and the quantification of the lesion parameters including the mineral density and the thickness of the surface layer, were performed using visualized mineral maps in MATLAB and mineral density profiles plotted in Fiji respectively.
2.5
Statistical analysis
The value of mineral density and the thickness of the surface layer for each sample were calculated by averaging the measured values in several slices from various representative locations of the lesion. The measurements were checked for normal distribution. Statistical analysis was performed using statistical software GraphPad Prism (Graphpad Software.San Diego, CA) to test for differences between the means. The results of mineral density quantification in different groups (WSL, BSL and sound enamel) were analyzed by One-way analysis of variance (ANOVA). Multiple comparisons between groups were performed by posthoc Tukey test. Comparison of the thickness of the surface layer between white and brown-spot lesions was performed using unpaired Student’s t -test. P-values less than 0.05 were considered to be statistically significant.
2
Materials and methods
2.1
Study specimens
Extracted molar and premolar teeth were collected from The Oral Surgery Department at Sydney Dental Hospital, The University of Sydney (Ethics approval protocol No X12-0065 & HREC/12/RPAH/106). The majority of the teeth with white spot enamel lesions were extracted for orthodontic purposes from young individuals aged 14–18. Teeth with brown spot enamel lesions were extracted for periodontal or periapical disease from mature aged patients. Following extraction, teeth were partially sterilized in MILTON anti-bacterial solution (0.95% (w/w) sodium hypochlorite, Milton Australia PTY LTD.) for 15 min and then brushed clean under running water to eliminate any plaque or removable stain. After decontamination, the samples were stored in Hanks Balanced Salt Solution (HBSS) at 4 °C prior to use. Thymol granules (Sigma Aldrich, Australia) were added to the solution for disinfection and prevention of fungal growth.
Two calibrated clinicians visually assessed the specimens and seventeen proximal WSLs (ICDAS 1, 2) , seventeen proximal BSLs and seventeen sound proximal areas (ICDAS 0) were chosen as study and control groups. The inclusion criteria for brown spot enamel lesions, included being non-cavitated with localized dark brown discoloration which is intrinsic to enamel and is not an external surface stain ( Fig. 1 ).
Three hydroxyapatite discs with known low (1.240 g/cm 3 ), medium (1.406 g/cm 3 ) and high (1.666 g/cm 3 ) mineral densities were used for grey level calibration and determination of the mineral density values. Hydroxyapatite discs were located and fixed on top of each specimen before imaging. Details for the fabrication process and the materials used for constructing the phantoms are described elsewhere .
2.2
Parameters for x-ray micro-tomography scanning and tomographic reconstruction
Imaging of the teeth and phantoms was performed using a high resolution fourth generation micro-computed tomography system (Skyscan 1172, Skyscan, N.V, Aartsellar, Belgium) with an accelerating source voltage of 100 keV, a source current of 100 μA and an exposure time of 885 ms. Before the scan, teeth were stored in Hanks Balanced Salt Solution (HBSS) and during scan they were stabilized in a plastic tube using polystyrene foam, preventing dehydration of the specimens. Low energy x-rays were eliminated using an inbuilt filter equal to 1.0 mm thickness of aluminum and 0.05 mm of copper to restrict spectral bandwidth of the polychromatic radiation. The equivalent monochromatic energy spectrum of filtered x-ray had an effective mean energy of 60 keV. The long axes of the teeth were parallel with the center of rotation of the mounting device. During the scanning process, the samples were rotated over 360° at angular increments of 0.14° generating 2570 two-dimensional shadow projections with an image matrix of 2000 pixels × 1048 pixels. These images were saved as 16 bit Tagged Image File Format (TIFF) and consequently exported to a 3-D cone beam reconstruction program (NRecon software, version 1.4.4; SkyScan) for the reconstruction of the 3-D object. The tomographic reconstruction produced a dataset of slice views in 16 bit TIFF format, which were perpendicular to the specimen rotation axis and had a voxel size resolution of 8.82 μm. The reconstructed volumes of the whole image stacks were vertically re-sliced in Fiji (W.S. Rasband, U. S. National Institutes of Health, Bethesda, Md, USA) to produce vertical tomographic images.
2.3
De-noising and mineral mapping
The produced tomographic images were consequently imported into MATLAB (MatLab R2012b 8.0.0.783, Mathworks, Natick, MA, USA) for the de-noising process and for increasing the signal to noise ratio of micro-CT images. De-noising was performed using a method based on total variation regularization , with the following parameters:
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