2.1

Enamel subsurface lesion preparation

Extracted human third molars were obtained from the Melbourne Dental School, The University of Melbourne after informed patient consent. The study was approved by the University of Melbourne’s Human Research Ethics Committee (number 1136929). The teeth were first washed thoroughly in distilled deionised water (DDW) then sterilised with 4.1 kGy of gamma radiation. After sterilisation, any soft tissues were removed from the teeth and sound relatively planar buccal and lingual surfaces free of cracking, staining and fluorosis (as viewed under a dissecting microscope) were selected. The outer enamel surfaces were polished wet to a mirror finish using Soflex™ discs on a slow speed contra-angle dental handpiece. Each polished surface was cut from the tooth as an approximately 8 x 4 mm block, using a water-cooled diamond blade saw and the whole block was then covered with acid-resistant nail varnish except for two (occlusal and gingival) mesiodistal windows (approximately 1 x 8 mm each) separated from each other by 1 mm. The blocks were treated to create lesions in the enamel windows by suspending each block in 40 mL of unagitated demineralisation buffer, consisting of 80 mL/L Noverite K-702 polyacrylate solution (Lubrizol Corporation, Wickliffe, OH), 500 mg/L hydroxyapatite (Bio-Gel ^® HTP, Bio Rad Laboratories, Richmond CL), and 0.1 mol/L lactic acid (Ajax Chemicals, Auburn NSW) pH 4.8, for 4 days at 37 °C . A change of solution was made after two days at which time the blocks were removed from the solution, rinsed thrice with DDW, blotted dry and placed into fresh demineralisation buffer. The blocks were similarly rinsed with DDW and dried after four days of demineralisation. This demineralisation procedure produces consistent subsurface lesions of 100 μm depths with intact surface layers, as evaluated by contact microradiography of sections of the lesions. After demineralisation, the enamel blocks were cut perpendicular to the windows into two 4 × 4 mm half-blocks and the cut surface of each block was covered with nail varnish. One of the half-blocks was retained as the demineralisation control and stored in a labeled 1.5 mL microcentrifuge tube together with a drop of DDW, thereby creating a humidified environment. The other half-block (test half-block) was used for remineralisation (see below).

2.2

Remineralisation protocol

A total of ten solutions were tested. One solution contained only artificial saliva (AS). AS consisted of 50 mM NaCl, 0.5 mM CaCl ₂ and 0.5 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ pH 7.0. Four AS solutions contained 12.6% (w/v) of one of the following four polyols: xylitol, sorbitol, maltitol, or mannitol. This concentration of polyol was used as it is the maximum concentration attained in saliva while chewing with commercially-available gum (two pieces of gum) containing these compounds at the upper end of the normal range 0.5–1.0 g per piece of gum. The concentration was determined as the maximum concentration achieved in saliva while chewing two pieces of a high dose xylitol gum using stimulated saliva flow rates and release kinetics data . Five AS solutions contained 0.376% (w/v) CPP-ACP and four of these also contained 12.6% (w/v) of one of the following four polyols: xylitol; sorbitol; maltitol; or mannitol.

Each test half enamel block was placed into 5 mL of one of the ten AS solutions and incubated at 37 °C for seven days. Each day the 5 mL AS solution was replaced with fresh 5 mL AS solution. Six half-blocks each containing two demineralised subsurface lesions were used for each of the ten AS remineralisation solutions.

2.3

Sectioning and transverse microradiography

Each demineralised control enamel half-block and remineralised test enamel half-block were rinsed in ethanol to remove the nail varnish and washed thoroughly in DDW. Each test half-block was paired with its corresponding control half-block, coded (blinded) and placed into freshly poured transparent cold curing methacrylate resin with the lesion windows parallel. The resin vial was marked at the top corner to identify the test and control half-blocks and the resin was allowed to set at room temperature overnight. Sections approximately 200 μm thick was cut from embedded blocks perpendicular to the lesion surface through the midline of both half-lesions using an internal annulus saw microtome. The sections were lapped down to 95± 5 μm using a RotoPol/RotoForce lapping instrument with 1200 and 2400 grit lapping paper. The lapped sections were removed from the lapping instrument with absolute ethanol and rinsed in deionised water, blotted dry and stored on soft tissue between glass slides. Each section, which contained the remineralised lesions and the demineralised control lesions from the same enamel block, was radiographed along with an aluminium stepwedge of 37.5 x 7 μm thick increments using Microchrome High Resolution glass plates (1 × 3 x 0.06 in., Microchrome, USA) and nickel filtered copper Ka radiation at 20 kV, 30 mA for 8 min using a microradiography unit (XMR, Diffraction Technology Pty Ltd) with a PANalytical glass XRD fine focus tube with a copper target (Spectris Australia Pty Ltd) powered by a DF3 generator (Spellman High Voltage Electronics Corporation). Each glass plate was developed in Microchrome Developer D5 for 5 min, placed into glacial acetic acid stop bath for 30 s and then fixed in Microchrome Fixer F4 for 5 min.

2.4

Microdensitometry

Radiographic images of the lesions were viewed via transmitted light through a microscope. The images were acquired by a digital camera and analysed using imaging software Image Pro Plus Version 7.0. Images of the lesions and the neighboring areas of sound enamel were scanned using the programs line luminance function that gave readings in gray values. An area free of artifacts or cracks was selected for analysis. Each scan comprised 200 readings taken from the tooth surface through the lesion to sound enamel. The aluminum stepwedge image on each slide was scanned and the averaged step gray value readings were plotted against aluminum thickness. The readings of the tooth section image were within the linear portion of the stepwedge curve and linear regression was used to convert the gray value data into values of equivalent thickness of aluminum. The section thickness was measured and the % volume mineral (% vol min) data computed using the equation of Angmar and the linear absorption coefficients of aluminum, organic matter plus water and apatitic mineral (131.5, 11.3 and 260.5 respectively). The image of the median strip of sound enamel between the two lesions was scanned six times and averaged to give a control sound-enamel densitometric profile. The lesion images (remineralisation windows and demineralised control windows) to the gingival and occlusal side of the median strip were similarly scanned, as close as possible to the median strip but avoiding any irregularities commonly found at the lesion edges, and the % vol min profiles computed.

2.5

Remineralisation analysis

The % vol min profile of each enamel block’s demineralised and remineralised lesion were compared with the median sound enamel % vol min profile of the same section. The difference between the areas under the densitometric profile of the demineralised lesion and the median sound enamel, calculated by trapezoidal integration, is represented by ΔZd. The difference between the areas under the densitometric profile of the remineralised lesion and the median sound enamel, calculated by trapezoidal integration, is represented by ΔZr. These parameters were then converted to % change values after remineralisation; % remineralisation (%R) represents the % change in ΔZ values:

2.6

Statistical analysis

2.6.1

Sample size calculation

The number of enamel blocks used for this study was determined using IBM SamplePower package for two one-sided tests for equivalence assuming no difference between treatment means, α = 0.05, power (1−β) ≥ 0.85, equivalence threshold (δ) = 12 and a range of standard deviations (4–8). A sample size of n = 6 per treatment provided sufficient power against this range of SD values.

2.6.2

Test for homoscedasticity

Data were analysed with SPSS Version 21. The distributions of residuals for each of the parameters LDd, ΔZd and ΔZd-ΔZr and %R for all AS solutions were determined. Homoscedasticity across all treatments was assessed using Levene’s test .

2.6.3

Tests for equivalence

Equivalence across all five AS solutions and those five AS solutions containing CPP-ACP with respect to percent remineralisation (%R) was tested using an F-test for equivalence for more than two normal distributions using STATISTICA 12 and Excel 2013 software. Then 10 pair-wise comparisons for the five AS solutions with or without CPP-ACP using two one sided tests (TOST; ) of equivalence with a Bonferroni correction for multiple comparisons were performed to test equivalence of each pair of test AS solutions using QI Macros for Excel and Excel 2013 software.

2.6.4

Tests for differences

As the distribution of residuals for the ten AS solutions for LDd, ΔZd and ΔZd-ΔZr approximated a normal distribution and homoscedasticity was confirmed with Levene’s test, differences were measured using a one-way ANOVA with post hoc Dunnett’s tests for comparison with a control group. As the distribution of residuals for percent remineralisation (%R) values for the ten AS solutions was skewed, differences between the %R values were measured with a Kruskal-Wallis test and post hoc Wilcoxon Rank Sum tests with a Bonferroni correction for multiple pairwise comparisons.

2.1

Enamel subsurface lesion preparation

Extracted human third molars were obtained from the Melbourne Dental School, The University of Melbourne after informed patient consent. The study was approved by the University of Melbourne’s Human Research Ethics Committee (number 1136929). The teeth were first washed thoroughly in distilled deionised water (DDW) then sterilised with 4.1 kGy of gamma radiation. After sterilisation, any soft tissues were removed from the teeth and sound relatively planar buccal and lingual surfaces free of cracking, staining and fluorosis (as viewed under a dissecting microscope) were selected. The outer enamel surfaces were polished wet to a mirror finish using Soflex™ discs on a slow speed contra-angle dental handpiece. Each polished surface was cut from the tooth as an approximately 8 x 4 mm block, using a water-cooled diamond blade saw and the whole block was then covered with acid-resistant nail varnish except for two (occlusal and gingival) mesiodistal windows (approximately 1 x 8 mm each) separated from each other by 1 mm. The blocks were treated to create lesions in the enamel windows by suspending each block in 40 mL of unagitated demineralisation buffer, consisting of 80 mL/L Noverite K-702 polyacrylate solution (Lubrizol Corporation, Wickliffe, OH), 500 mg/L hydroxyapatite (Bio-Gel ^® HTP, Bio Rad Laboratories, Richmond CL), and 0.1 mol/L lactic acid (Ajax Chemicals, Auburn NSW) pH 4.8, for 4 days at 37 °C . A change of solution was made after two days at which time the blocks were removed from the solution, rinsed thrice with DDW, blotted dry and placed into fresh demineralisation buffer. The blocks were similarly rinsed with DDW and dried after four days of demineralisation. This demineralisation procedure produces consistent subsurface lesions of 100 μm depths with intact surface layers, as evaluated by contact microradiography of sections of the lesions. After demineralisation, the enamel blocks were cut perpendicular to the windows into two 4 × 4 mm half-blocks and the cut surface of each block was covered with nail varnish. One of the half-blocks was retained as the demineralisation control and stored in a labeled 1.5 mL microcentrifuge tube together with a drop of DDW, thereby creating a humidified environment. The other half-block (test half-block) was used for remineralisation (see below).

2.2

Remineralisation protocol

A total of ten solutions were tested. One solution contained only artificial saliva (AS). AS consisted of 50 mM NaCl, 0.5 mM CaCl ₂ and 0.5 mM Na ₂ HPO ₄ /NaH ₂ PO ₄ pH 7.0. Four AS solutions contained 12.6% (w/v) of one of the following four polyols: xylitol, sorbitol, maltitol, or mannitol. This concentration of polyol was used as it is the maximum concentration attained in saliva while chewing with commercially-available gum (two pieces of gum) containing these compounds at the upper end of the normal range 0.5–1.0 g per piece of gum. The concentration was determined as the maximum concentration achieved in saliva while chewing two pieces of a high dose xylitol gum using stimulated saliva flow rates and release kinetics data . Five AS solutions contained 0.376% (w/v) CPP-ACP and four of these also contained 12.6% (w/v) of one of the following four polyols: xylitol; sorbitol; maltitol; or mannitol.

Each test half enamel block was placed into 5 mL of one of the ten AS solutions and incubated at 37 °C for seven days. Each day the 5 mL AS solution was replaced with fresh 5 mL AS solution. Six half-blocks each containing two demineralised subsurface lesions were used for each of the ten AS remineralisation solutions.

2.3

Sectioning and transverse microradiography

Each demineralised control enamel half-block and remineralised test enamel half-block were rinsed in ethanol to remove the nail varnish and washed thoroughly in DDW. Each test half-block was paired with its corresponding control half-block, coded (blinded) and placed into freshly poured transparent cold curing methacrylate resin with the lesion windows parallel. The resin vial was marked at the top corner to identify the test and control half-blocks and the resin was allowed to set at room temperature overnight. Sections approximately 200 μm thick was cut from embedded blocks perpendicular to the lesion surface through the midline of both half-lesions using an internal annulus saw microtome. The sections were lapped down to 95± 5 μm using a RotoPol/RotoForce lapping instrument with 1200 and 2400 grit lapping paper. The lapped sections were removed from the lapping instrument with absolute ethanol and rinsed in deionised water, blotted dry and stored on soft tissue between glass slides. Each section, which contained the remineralised lesions and the demineralised control lesions from the same enamel block, was radiographed along with an aluminium stepwedge of 37.5 x 7 μm thick increments using Microchrome High Resolution glass plates (1 × 3 x 0.06 in., Microchrome, USA) and nickel filtered copper Ka radiation at 20 kV, 30 mA for 8 min using a microradiography unit (XMR, Diffraction Technology Pty Ltd) with a PANalytical glass XRD fine focus tube with a copper target (Spectris Australia Pty Ltd) powered by a DF3 generator (Spellman High Voltage Electronics Corporation). Each glass plate was developed in Microchrome Developer D5 for 5 min, placed into glacial acetic acid stop bath for 30 s and then fixed in Microchrome Fixer F4 for 5 min.

2.4

Microdensitometry

Radiographic images of the lesions were viewed via transmitted light through a microscope. The images were acquired by a digital camera and analysed using imaging software Image Pro Plus Version 7.0. Images of the lesions and the neighboring areas of sound enamel were scanned using the programs line luminance function that gave readings in gray values. An area free of artifacts or cracks was selected for analysis. Each scan comprised 200 readings taken from the tooth surface through the lesion to sound enamel. The aluminum stepwedge image on each slide was scanned and the averaged step gray value readings were plotted against aluminum thickness. The readings of the tooth section image were within the linear portion of the stepwedge curve and linear regression was used to convert the gray value data into values of equivalent thickness of aluminum. The section thickness was measured and the % volume mineral (% vol min) data computed using the equation of Angmar and the linear absorption coefficients of aluminum, organic matter plus water and apatitic mineral (131.5, 11.3 and 260.5 respectively). The image of the median strip of sound enamel between the two lesions was scanned six times and averaged to give a control sound-enamel densitometric profile. The lesion images (remineralisation windows and demineralised control windows) to the gingival and occlusal side of the median strip were similarly scanned, as close as possible to the median strip but avoiding any irregularities commonly found at the lesion edges, and the % vol min profiles computed.

2.5

Remineralisation analysis

The % vol min profile of each enamel block’s demineralised and remineralised lesion were compared with the median sound enamel % vol min profile of the same section. The difference between the areas under the densitometric profile of the demineralised lesion and the median sound enamel, calculated by trapezoidal integration, is represented by ΔZd. The difference between the areas under the densitometric profile of the remineralised lesion and the median sound enamel, calculated by trapezoidal integration, is represented by ΔZr. These parameters were then converted to % change values after remineralisation; % remineralisation (%R) represents the % change in ΔZ values:

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