Abstract
Objectives
To investigate the effects of a 5% NovaMin containing dentifrice on dentine tubule patency and surface roughness at 100 g and 400 g tooth brush abrasion forces.
Methods
75 polished human dentine samples were prepared and randomly allocated into one of five groups; control (1), Na 2 PFO 3 100 g abrasion force (2), NovaMin 100 g (3), Na 2 PFO 3 400 g (4) and NovaMin 400 g (5). The control group underwent two 2-min cycles of artificial saliva (AS), one 2-min erosion cycle; the rest underwent two toothbrush abrasion cycles in an AS/dentifrice slurry and one 2-min erosion cycle. All samples were imaged at baseline and post intervention using Tandem Scanning Microscopy and Profilometry to analyse tubule patency and roughness.
Results
Mean tubule patency increased significantly between baseline and post intervention in groups 1,2 and 4 and decreased significantly post intervention in groups 3 and 5 (p < 0.01). Post intervention, there were statistically significant differences in mean patent tubules between NovaMin and the Na 2 PFO 3 and control groups (p < 0.001). Surface roughness increased for all groups between baseline and post interventions (P < 0.001); mean (SD) roughness increases for groups 1, 2, 3, 4 and 5 were 0.14 (0.05) μm, 0.18 (0.04) μm, 0.16 (0.06) μm, 0.19 (0.07) μm and 0.21 (0.02) μm respectively. Differences between group 1 and 5 were significant (p < 0.01).
Conclusions
Brushing with NovaMin resulted in significant dentine tubule occlusion at 100 g and 400 g, but brushing with Na 2 PFO 3 resulted in increased tubule patency. Surface roughness increased significantly at 400 g brushing with NovaMin. There was no correlation between tubule patency and surface roughness.
Clinical significance
A NovaMin desensitising dentifrice resulted in tubule occlusion even at high brushing forces. There was minimal increase in surface roughness at the lower (100 g) brushing force.
1
Introduction
Dentine hypersensitivity is defined as a short duration, sharp dental pain response to stimuli in the absence of any other pathology . The generally accepted mechanism behind dentine hypersensitivity is the hydrodynamic theory . This involves the rapid transmission of fluid through dentinal tubules triggering neuroreceptors located in the pulp in response to stimuli such as cold and air . The condition is very prevalent in Europe (42%) and especially the UK . Diet as well as tooth-brushing are important aetiologies.
In order for the fluid flow in the hydrodynamic theory to be possible, the dentinal tubules must be patent (open). Studies have identified that teeth diagnosed with dentine hypersensitivity possess a greater number of patent tubules on the dentine surface . It is therefore not surprising that the number of patent tubules on the dentine are used to assess the efficacy of desensitising (tubule occluding) products . Olley et al. developed a robust reproducible method to quantify patent tubules in dentine samples. Dentine samples were scanned using Tandem Scanning Microscopy (TSM) and the images analysed using a software program to quantify the number of patent dentinal tubules . This method was used in a further study investigating the effects of three toothbrush abrasion forces on tubule patency using a standard Na 2 PFO 3 toothpaste . This study reported an association between increased tubule patency and increased abrasion after one erosion-abrasion cycle, with significant differences at the 100 g and 400 g abrasion forces . However, the effects of a densensitising (tubule occluding) dentifrice at these brushing forces (100 g and 400 g) and whether the higher brushing force could increase tubule patency despite the dentifrice are unknown. This study investigates if there is a protective effect (reduced tubule patency) of a desensitising dentifrice (5% NovaMin, GlaxoSmithKline Consumer Healthcare, Brentford, UK) at both 100 g and 400 g toothbrush abrasion forces in an erosion/abrasion regime. NovaMin is a bioactive glass with calcium sodium phosphosilicate as the active ingredient. It is reputed that this can react in the oral environment to form a hydroxy-carbonate apatite (HCA) over time and is similar to the natural tooth mineral composition . It has been previously shown when comparing two NovaMin containing dentifrices, control and water, that the NovaMin results in dentine tubule occlusion .
It should not be supposed from this study that the effect of a desensitising dentifrice might offset the detrimental effect to dentine at higher brushing forces and therefore enable this to occur. The 400 g brushing force is represented as an overzealous regime to investigate the effects on the dentine surface.
In addition, the measurement of surface topography is widely used within dental material science, with rapidly evolving developments . Surface roughness measurements are often used to identify changes in tooth structure following erosive wear and to investigate the efficacy of anti-erosion and remineralising products . Furthermore, tribology studies use roughness parameters to make associations between wear patterns and diet . To the authors’ knowledge a correlation between surface roughness and tubule patency has not been investigated. It can be supposed that a change in the tubule patency of dentine, could effect the surface roughness of dentine due to the surface nature of dentine hypersensitivity . Therefore surface roughness may prove a useful indicator of tubule patency.
The overall aim of this study was to investigate the effects of a 5% NovaMin containing dentifrice on dentine tubule patency and surface roughness at two abrasion forces (100 g and 400 g). The null hypothesis was that there would be no difference in tubule patency and surface roughness brushing with a NovaMin containing dentifrice at 100 g and 400 g tooth-brushing forces. Also, that tubule patency is not associated with surface roughness.
2
Methods
2.1
Sample preparation
Unrestored and caries free human molars were collected under ethical agreement (12/LO/1836) and sterilised in sodium hypochlorite for a minimum of 72 h. The roots were removed and the crowns sectioned using a circular diamond saw (XL 12205, Benetec Ltd., London, UK) to produce 75 sections, no samples were discounted during the study. The sections were embedded in bisacryl composite (Protemp4 3 M ESPE, Germany) using custom made trays to make samples. Sample size calculations were based on Sehmi and Olley et al., 2015. Samples underwent a standardised polishing regime using a series of carbide grits (320, 1200, 2400 and 4000) in a water cooled polishing machine (Meta-Serv 3000 Grinder-Polisher, Buehler, Lake Bluff, Illinois, USA) to produce areas of optically flat dentine with a flatness tolerance of 0.4 μm . This process created an artificial smear layer on the surface of dentine (based on the protocol from Sehmi and Olley et al., 2015). This layer was removed following the first brushing (in the next stage of the experiment) by immersing the samples in citric acid to create an etching effect.
2.2
Experimental design
The 75-dentine samples were randomly allocated into one of five groups, with 15 samples per group. Group 1 was the “control group”; these samples did not undergo any toothbrush abrasion or exposure to dentifrice. Control samples were immersed in artificial saliva (AS) for 2 min followed by immersion in 0.3% Citric Acid pH 2.6 for 2 min and completed by immersion in AS for a further 2 min. The remaining groups compared two dentifrice products, Colgate Cavity Protection (Colgate Oral Pharmaceuticals, New York, USA) (Na 2 PFO 3 ) and Sensodyne ® Repair & Protect (5% NovaMin) (Calcium Sodium Phosphosilicate). Each dentifrice was investigated at two abrasion forces; 100 g and 400 g.
The dentifrice slurries were made immediately before use and consisted of 1-part dentifrice (330 ml) to 2-parts AS (660 ml) and hand-mixed for 2 min. The AS was made used within 24 h following an established protocol and consisted of Calcium Chloride Dehydrate 0.7 mmol/l, Magnesium Chloride 0.2 mmol/l, Potassium Dihydrogen Phosphate 4.0 mmol/l, HEPES (Acid buffer) 20.0 mmol/l and Potassium Chloride 30.0 mmol/l and buffered to pH 7 . A reciprocating and automatic tooth brushing machine (Dentagen, Munich, Germany) with standardised toothbrushes (Sensodyne ® Search 3.5 with small head sizes) was used for the abrasion experiments. To achieve the desired abrasion force (100 g or 400 g) external calibrated weights (Voltcraft PS 500 Pocket scale, Oldenzaal, Netherlands) were attached and the force applied to the tip of each toothbrush to engage on the centre of each dentine sample. The dentine samples were fully immersed in the toothpaste/AS slurries in reservoir baths located in the tooth brushing machine, which was thoroughly cleaned between groups. The dentine samples were abraded in dentifrice slurry at their designated abrasion force for 2 min (120 strokes) using a soft bristled tooth brush, followed by immersion in 0.3% Citric Acid pH 2.6 agitated at room temperature for 2 min and followed by a further 2 min dentifrice abrasion (as per Sehmi and Olley ).
2.3
TSM imaging
TSM imaging and analysis were carried out at baseline and post experimental intervention by the same operator. The samples were rehydrated for a minimum of 5 h in phosphate buffered (pH 7) distilled water prior to imaging with the TSM (Noran instruments, Middleton USA) using an M-plan 40x SLWD (Brightfield Objective x 40/0 m35 NA objective). Gently air dried samples were placed on a platform at the microscope and imaged on the TSM machine digitally using a mounted camera (Andor iXon 885, Andor Technology Ltd, Belfast, UK) with iAndor software. The TSM light source was positioned over the centre of the dentine samples; the adjacent composite in the mount was marked to reliably relocate the same area after the experimental intervention. A previously validated computer algorithm (Image J software, USA) was used to count the number of patent dentine tubules greater than 0.83 μm .
2.4
Surface roughness
All of the samples were imaged and analysed for surface roughness by the same operator who was randomised to active ingredient. Scanning was carried out using a non-contact profilometer (NCP) with a red laser light source (2 μm spot size; NCP, LT–9010 M, Keyence Corporation, Japan) and motion controlled stage (Xyris 2000, Taicaan, UK). MountainsMap (DigitalSurf, France) analysis software was used to extract Sa roughness (average roughness of a measured surface) following application of a 25 μm Gaussian filter. Five randomly selected areas (each 0.04 mm 2 ) within the centre of the dentine samples were imaged and analysed before and after experimental intervention.
2.5
Statistical analysis
The sample size for this study was based upon a power calculation used in previously published study and pilot work ( ) with an alpha level of 0.05, 80% power, mean patent dentine tubules 180 and standard deviation 50 . Shapiro-Wilk and Kolmogorov-Smirnov tests, along with histogram plots were used to determine the normality of the data. The data were found to be normally distributed. Levene’s tests were performed to assess homogeneity of variances; TSM data had equal variance therefore a two way ANOVA and post hoc Tukey test were used to determine inter and intra group significance. However, surface roughness data did not have equal variance and in this case a Welch ANOVA and post hoc Games-Howell test were used. Pearson correlation tests were used to examine the relationship between dentinal patency and surface roughness.
2
Methods
2.1
Sample preparation
Unrestored and caries free human molars were collected under ethical agreement (12/LO/1836) and sterilised in sodium hypochlorite for a minimum of 72 h. The roots were removed and the crowns sectioned using a circular diamond saw (XL 12205, Benetec Ltd., London, UK) to produce 75 sections, no samples were discounted during the study. The sections were embedded in bisacryl composite (Protemp4 3 M ESPE, Germany) using custom made trays to make samples. Sample size calculations were based on Sehmi and Olley et al., 2015. Samples underwent a standardised polishing regime using a series of carbide grits (320, 1200, 2400 and 4000) in a water cooled polishing machine (Meta-Serv 3000 Grinder-Polisher, Buehler, Lake Bluff, Illinois, USA) to produce areas of optically flat dentine with a flatness tolerance of 0.4 μm . This process created an artificial smear layer on the surface of dentine (based on the protocol from Sehmi and Olley et al., 2015). This layer was removed following the first brushing (in the next stage of the experiment) by immersing the samples in citric acid to create an etching effect.
2.2
Experimental design
The 75-dentine samples were randomly allocated into one of five groups, with 15 samples per group. Group 1 was the “control group”; these samples did not undergo any toothbrush abrasion or exposure to dentifrice. Control samples were immersed in artificial saliva (AS) for 2 min followed by immersion in 0.3% Citric Acid pH 2.6 for 2 min and completed by immersion in AS for a further 2 min. The remaining groups compared two dentifrice products, Colgate Cavity Protection (Colgate Oral Pharmaceuticals, New York, USA) (Na 2 PFO 3 ) and Sensodyne ® Repair & Protect (5% NovaMin) (Calcium Sodium Phosphosilicate). Each dentifrice was investigated at two abrasion forces; 100 g and 400 g.
The dentifrice slurries were made immediately before use and consisted of 1-part dentifrice (330 ml) to 2-parts AS (660 ml) and hand-mixed for 2 min. The AS was made used within 24 h following an established protocol and consisted of Calcium Chloride Dehydrate 0.7 mmol/l, Magnesium Chloride 0.2 mmol/l, Potassium Dihydrogen Phosphate 4.0 mmol/l, HEPES (Acid buffer) 20.0 mmol/l and Potassium Chloride 30.0 mmol/l and buffered to pH 7 . A reciprocating and automatic tooth brushing machine (Dentagen, Munich, Germany) with standardised toothbrushes (Sensodyne ® Search 3.5 with small head sizes) was used for the abrasion experiments. To achieve the desired abrasion force (100 g or 400 g) external calibrated weights (Voltcraft PS 500 Pocket scale, Oldenzaal, Netherlands) were attached and the force applied to the tip of each toothbrush to engage on the centre of each dentine sample. The dentine samples were fully immersed in the toothpaste/AS slurries in reservoir baths located in the tooth brushing machine, which was thoroughly cleaned between groups. The dentine samples were abraded in dentifrice slurry at their designated abrasion force for 2 min (120 strokes) using a soft bristled tooth brush, followed by immersion in 0.3% Citric Acid pH 2.6 agitated at room temperature for 2 min and followed by a further 2 min dentifrice abrasion (as per Sehmi and Olley ).
2.3
TSM imaging
TSM imaging and analysis were carried out at baseline and post experimental intervention by the same operator. The samples were rehydrated for a minimum of 5 h in phosphate buffered (pH 7) distilled water prior to imaging with the TSM (Noran instruments, Middleton USA) using an M-plan 40x SLWD (Brightfield Objective x 40/0 m35 NA objective). Gently air dried samples were placed on a platform at the microscope and imaged on the TSM machine digitally using a mounted camera (Andor iXon 885, Andor Technology Ltd, Belfast, UK) with iAndor software. The TSM light source was positioned over the centre of the dentine samples; the adjacent composite in the mount was marked to reliably relocate the same area after the experimental intervention. A previously validated computer algorithm (Image J software, USA) was used to count the number of patent dentine tubules greater than 0.83 μm .
2.4
Surface roughness
All of the samples were imaged and analysed for surface roughness by the same operator who was randomised to active ingredient. Scanning was carried out using a non-contact profilometer (NCP) with a red laser light source (2 μm spot size; NCP, LT–9010 M, Keyence Corporation, Japan) and motion controlled stage (Xyris 2000, Taicaan, UK). MountainsMap (DigitalSurf, France) analysis software was used to extract Sa roughness (average roughness of a measured surface) following application of a 25 μm Gaussian filter. Five randomly selected areas (each 0.04 mm 2 ) within the centre of the dentine samples were imaged and analysed before and after experimental intervention.
2.5
Statistical analysis
The sample size for this study was based upon a power calculation used in previously published study and pilot work ( ) with an alpha level of 0.05, 80% power, mean patent dentine tubules 180 and standard deviation 50 . Shapiro-Wilk and Kolmogorov-Smirnov tests, along with histogram plots were used to determine the normality of the data. The data were found to be normally distributed. Levene’s tests were performed to assess homogeneity of variances; TSM data had equal variance therefore a two way ANOVA and post hoc Tukey test were used to determine inter and intra group significance. However, surface roughness data did not have equal variance and in this case a Welch ANOVA and post hoc Games-Howell test were used. Pearson correlation tests were used to examine the relationship between dentinal patency and surface roughness.
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