To compare the efficacy of fluoride varnishes either casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) or bioglass particles on the severity index of root caries.
Visual-tactile assessments including lesion hardness was carried out to categorise the severity index of 80 extracted teeth with primary root caries. These teeth were randomly divided into four groups; CPP-ACP and fluoride, bioglass and fluoride, fluoride only, and no treatment. Standardised toothbrushing using a dentifrice containing 1450 ppm fluoride was carried out twice a day for all groups. All samples were stored in remineralising solution at 37°C for 45 days. Visual-tactile assessments were carried out at baseline, and after 45 days. Surface roughness measurements (Ra) were performed at baseline and after 7, 14, 30 and 45 days. X-ray Microtomography was performed at the baseline and after 45 days for three samples from each group to quantify the change in mineral contents in the lesion area.
The Visual-tactile assessment results showed a reduction in the severity index of root caries, being 20% in CPP-ACP and fluoride, 100% in bioglass and fluoride, 80% in fluoride only, and 60% in non-varnish (toothbrushing only). Non-significant change in surface roughness was observed in all groups. X-ray Microtomography assessment showed a highly significant increase in the mineral deposition in all cases (p > .001).
The combination of bioglass with fluoride has a potentially superior effect than either CPP-ACP with fluoride or fluoride only to reverse and arrest the root caries in a laboratory setting.
The combination of bioglass particles and fluoride formulation is likely to have a significant impact in reversing and arresting root caries in a minimally invasive approach. However, randomised controlled double-blinded clinical trials are required to translate these results into clinical practice.
The world population aged 60 and above is increasing and is set to rise from 11% in 2000 to 20% in year 2050 . A recent data revealed an increased risk of developing root caries in older population due to exposed root surfaces following gingival recession. This could be related to the aging process or due to a history of periodontal disease . Nowadays, it is clearly stated that older people retain most of their natural teeth . This brings a constant need for preventive and restorative dental services amongst older adults, especially for underserved populations such as those institutionalised.
Exposed root surface becomes susceptible to developing carious lesion as the demineralisation of dentine can occur at pH values of 6.0 . To manage these lesions using a minimally invasive approach, regular toothbrushing with a dentifrice having different fluoride concentration, ranging from 1100 to 5000 ppm, has been employed with a variation in the success rate . Following the introduction of dental varnishes, there is a potential benefit from increasing fluoride release through maximising contact time on the tooth surface . This offers an intraoral reservoir of fluoride ions to challenge the cariogenic process over a longer period of time which would contribute to the enhancement of the remineralisation process . The use of dental varnishes is considered safe, particularly for patients who are unable to use other fluoride delivery systems e.g. mouthwash . Ekstrand reported a significant improvement in the management of root caries following the application of a dental varnish containing fluoride (22.6 ppm) when compared with a dentifrice containing 1450 ppm fluoride. Furthermore, the use of dental varnish was found to perform only slightly better in comparison to a dentifrice containing 5000 ppm fluoride .
Several studies have been conducted to assess the efficacy of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) for the management of enamel caries. CPP-ACP has the ability to stabilise calcium phosphate, and further to maintain its potential availability during an acid challenge . Reynolds reported a considerable reduction in caries progression with the application of CPP-ACP with additional fluoride when compared with the use of either fluoride, or CPP-ACP alone.
Recently, calcium sodium phosphosilicate bioglass has been shown to play a key role in the hard tissue remineralisation of both enamel and dentine . Bioglass has the ability to release calcium, sodium, and phosphate ions into aqueous solution. This allows for an ionic exchange of sodium with H + or H 3 O + at the glass-liquid interface, permitting the formation of supersaturated ionic reservoir. The remaining silanols that result following bioglass dissolution act as a nucleation site on the tooth surface to form an apatite structure by attracting the released calcium and phosphate ions, forming a calcium phosphate rich layer . A recent systematic review by Fernando demonstrated that bioglass has the ability to boost remineralisation in dentine. However, this was not fully tested, and further studies are required.
Therefore, both CPP-ACP and bioglass formulations, combined with fluoride, have an effect on the remineralisation process within root carious lesions. This could be particularly useful for older populations for whom routine oral hygiene activities become challenging due to the deterioration in mobility, resulting in compromised oral health as a result of insufficient salivary function and impaired plaque control. Thus, the aim of this ex vi vo study was to compare the efficacy of a fluoride varnish with CPP-ACP, with a novel varnish containing fluoride and bioglass and with regular toothbrushing (control group) for a period of 45 days on root caries, using visual-tactile assessments for lesion hardness, non-contact optical profilometry to assess the changes in surface roughness, and finally using X-ray microtomography to investigate the changes in mineral concentration.
As both CPP-ACP and bioglass could be a source of calcium and phosphorous to which enhances the remineralisation process within the root carious lesion. The hypothesis of this study was to confirm that there are no differences in effectiveness of CPP-ACP with fluoride, and bioglass with fluoride on root caries.
Materials and methods
Root caries sample collection
80 extracted teeth with primary active root caries were collected from the Dental Emergency Clinics at the Institute of Dentistry, Bart’s and the London, Queen Mary University of London. Ethical approval was obtained from the Office for Research Ethics Committees Northern Ireland (ORECNI, 16/NI/0101) prior to the study. The teeth were stored in 1% thymol prior to sample preparation. They were then cleansed and polished using a slow speed handpiece and polishing cup with a non-fluoridated prophylaxis paste (NUPRO DENTSPLY, USA). The study flow chart is illustrated in Fig. 1 .
Four different treatment strategies were tested with a total of 80 samples. Each group (n = 20) received one of the allocated treatments as described in Table 1 . The samples were dried using a three-in-one syringe, and a thin and uniform layer of dental varnish was applied to each root caries according to manufacturer’s instructions. The area was then gently wetted with deionised water to accelerate varnish set.
|Groups||Varnish active ingredients||Other treatment|
|Test A (MI varnish, GC, Japan)||CPP-ACP and 5%NaF||Toothbrushing 1450 ppm F|
|Test B (Experimental varnish, DENTSPLY, USA)||Bioglass and 5%NaF||Toothbrushing 1450 ppm F|
|Positive control (NUPRO, DENTSPLY, USA)||5%NaF||Toothbrushing 1450 ppm F|
|Negative control (Non-varnish)||–||Toothbrushing 1450 ppm F|
Remineralisation buffer solution
A remineralisation solution was prepared as previously described by Ten Cate using 1.5 mmol/L CaCl 2 , 0.9 mmol/L KH 2 PO 4, 20 mmol/L HEPES and 130 mmol/L KCl. 1.5 mmol/L NaN 3 was added to the solution to prevent microbial growth. The pH was adjusted at 7 using 0.5 M KOH . All chemical reagents were obtained from Sigma–Aldrich, UK.
The prepared specimens of each group were immersed in 300 mL of remineralising solution, and the solution was kept in an incubator at 37°C for 45 days. The remineralising solution was replaced every two days.
The mechanical brushing procedure was set to simulate the clinical setting were the recommended brushing time was two minutes for the entire mouth . The first brushing process started after 12 h following the application of the dental varnish. Toothbrushing was simulated with a medium bristle toothbrush (Oral-B) with 1,450 ppm fluoride dentifrice (Aquafresh GSK, UK) twice a day for 45 days. The procedure was performed using an electrically powered toothbrushing machine (Weybridge Equipment, UK) designed to produce a constant reciprocal movement in a linear pattern. For each specimen the force applied to the brush bristle was 150 gm , the brushing process was performed using 1 mL of a 1:3 slurry of dentifrice and deionized water, for 10 s . The specimens were then left for two minutes before washing with deionised water.
Visual-tactile assessment for root caries
Visual-tactile examinations were carried out to categorise the severity index of root caries with regards to lesion hardness. This index is based on a score system from 0 to 4 as illustrated in Table 2 .
|Severity index Score||Lesion surface hardness|
|1||Leathery to hard|
|3||Leathery with local softening|
The hardness of each lesion was assessed using an Ash No.6 blunt probe with a pressure of around 100 g. Soft lesions were easily penetrated by the probe whilst leathery lesions had some resistance to withdrawal and hard lesions failed to have any penetration . The selected lesions in this study at baseline presented leathery types in hardness with severity indices between 1, 2 and 3. Lesions that scored 4 were excluded as they require a restorative approach.
The intra examiner reliability to assess severity index and hardness of root caries was performed twice within a one week interval for examiner 1 (AS). The assessments for root caries then were carried out independently by two examiners (AB&AS) at baseline before varnish application and after 45 days.
Surface roughness measurement (Ra)
Non-Contact Optical Profilometry (NCOP) has been used as a method to quantify the changes in surface roughness (Ra) of enamel following demineralisation . NCOP is a non-invasive and precise technology utilising the different refractive indices of the components of white light to measure sample topography.
The crowns and the apical parts of the teeth were removed using a 0.3 mm thickness diamond disc under running water at 3000 rpm speed (Struers, Germany) keeping the root carious lesions. The 20 samples for each group were fixed onto a customised aluminium tray using cold-cured acrylic resin (Pegasus Plus, England), keeping the lesion area and 2 mm of the surrounding sound dentine exposed. The kinematic tray was designed to relocate the samples during multiple repeated NCOP scans throughout the study.
NCOP was carried out using a Proscan 2000 (Scantron, UK) with a S13/1.2 sensor to measure the surface roughness (Ra) of each sample at various time points throughout the study. The specimens were dried for a period of 5 min at 37 °C prior to each NCOP scan. An initial area scan of 1.5 × 1.5 mm was carried out to ensure suitability of the selected area. Following this, three NCOP line scans were selected within this scanning area, for measurement of Ra of each sample. NCOP line scans with operational parameters (step size: 0.001 mm, number of steps: 1500) were carried out. A sampling rate of 30 Hz was used . Surface roughness measurements (Ra) of all samples were carried out at baseline before varnish application, and then after 7, 14, 30 and 45 days. A correlation test was performed to analyse the relation between the change in Ra measurement and severity index for each group.
X-ray microtomography (XMT)
The XMT assessment to measure the change in mineral concentration was carried out to three samples with score 2 severity index from each group, using the MuCAT 2 scanner designed at Queen Mary University of London . Each sample was separately located inside a clear plastic tube filled with deionised water to carry out the XMT baseline scan to keep the specimen fully hydrated during the long scanning procedure (around 12 h). The prepared sample was placed on a movable kinematic stage, ensuring the long axis of the tooth was parallel to the XMT rotational axis. The XMT scanner was set at 15 μm voxel size resolution (3D). The x-ray generator was operated at 90 kV and 180 μA. After each sample scan a calibration scan was performed and the projection data was transformed to 40 keV monochromatic energy equivalent . The reconstructed linear attenuation coefficient (LAC) was converted to mineral concentration using the following equation:
c = μ − μ o μ m − μ o ρ m
where μ is the measured linear attenuation coefficient (LAC),
μ o is the pure organic component LAC of the tooth structure (0.268 cm −1 ),
μ m is the pure sample material LAC which assumed a pure hydroxyapatite (3.12 cm −1 ), and
p m is the concentration of the pure hydroxyapatite (3.16 cm −3 ).
Note that a good approximation is given where water, plastic and soft tissue are all considered as part of the organic component. Likewise, although the mineral in dental hard tissue is not pure hydroxyapatite, this is a good approximation in terms of X-ray attenuation .
The XMT scan was performed to the three samples at the baseline before varnish application and after 45 days. The obtained 2D images (>1000 projections) from the XMT scans were reconstructed to create the 3D image. The 3D image from the baseline scan of each sample was aligned with the reconstructed 3D image from final scan using in-house developed alignment software running under IDL (Exelis Visual Information Solutions, Inc). The difference in mineral concentration was then visually detected by subtraction of the final image from the baseline one. The mineral change in the lesion region was calculated by comparing the mineral concentration of 15 randomly selected points from the baseline image with the final image.
The intra- and inter-examiner reproducibility for the severity index were assessed using the Intra-class correlation coefficient (ICC). The percentage change in the severity index was calculated, then followed by paired t -test to find the significant difference between the baseline and final assessments.
The data from the surface roughness (Ra) were analysed using repeated measures ANOVA and pairwise comparisons. Bivariate correlation was also used to investigate the correlation between the changes in the severity index, hardness and surface roughness (Ra).
The percentage in mineral concentration change was calculated then followed by paired t -test to compare the difference in mineral content in each sample. A significance level of 0.05 was performed using IBM SPSS Statistics 24.0 (SPSS Inc., Chicago, IL, USA).