Changes in mineral density and fluoridated apatite formation of artificial root caries using fluoridated toothpastes with or without bioactive glass

doi:10.21203/rs.3.rs-3193439/v1

Objectives

To explore the potential mineral exchange and fluoridated apatite formation within the artificial root carious lesions (ARCLs) subsurface using different toothpastes either containing 5,000ppm-F, 1,450ppm-F or bioactive glass (BG) with 540ppm-F.

Material and Methods

The crowns of three extracted sound teeth were removed. Subsequently, roots were divided into four parts (n = 12). Each sample was randomly allocated into one of four groups: Group-1(Deionised water); Group-2(BG with 540ppm-F); Group-3(1,450ppm-F) and Group-4(5,000ppm-F). ARCLs were developed using demineralisation solution (pH4.8). The 13-day pH-cycling for samples included demineralisation solution (6hrs) and remineralisation solution (pH7) for 16hrs. Standard tooth brushing twice a day with an assigned toothpaste was carried out during pH-cycling. XMT was performed for each sample at baseline, following ARCLs and after 13-day pH-cycling. Samples were then analysed using SEM/EDX and ¹⁹F-MAS-NMR.

Results

XMT showed an increase in mineral contents in the ARCL areas for each toothpaste group after 13-day pH-cycling, however there was mineral loss in subsurface for all groups. SEM showed the occlusion of dentinal tubules on the root surfaces in all toothpaste groups. ¹⁹F-MAS-NMR confirmed the partial/full fluoridated apatite formation in all groups, interestingly the presence of more fluorapatite was evident in the Groups-3 and 4.

Conclusion

All toothpastes were potentially effective to increase mineral density on surface while mineral loss in subsurface. The 5,000ppm-F toothpaste had a superior effect with respect to mineral density increase by promoting fluorapatite formation in comparison to the BG with 540ppm-F and 1,450ppm-F groups.

Clinical Relevance:

Toothpaste containing BG with 540ppm-F, 5000ppm-F and 1450ppm-F toothpastes are likely to manage root caries.

Demineralisation

remineralisation

toothpaste

fluoride

bioactive glass

mineral density

World Health Organisation recently reported that one in six people will be over 60 years old in 2030 and by 2050, this population will double (2.1 billion). Interestingly, individuals aged 80 years or older are expected to triple between 2020 and 2050 to reach 426 million [1].

Root caries is one of the major and preventable global oral health problems in ageing population [2]. Root surfaces are more susceptible to demineralisation in comparison to enamel in acidic conditions possibly due to the small hydroxyapatite crystallites in dentine. High levels of carbonate and magnesium with large surface area to crystallite volume ratio and less peritubular dentine lining can also result in high solubility in root dentine [3]. The conventional approach ’drilling and filling’ remains clinically challenging due to high organic content of root dentine, close proximity to gingivae and dental pulp, difficulty in obtaining direct access in some cases and moisture control [4]. Therefore, it is imperative to establish cost-effective and efficient management methods to control root caries [5]. The minimally invasive management for root caries has previously been proposed [6]. The caries preventive effect of fluoride is related to reducing demineralisation and promoting remineralisation by minimising the solubilisation of the crystals during periods of pH decrease and facilitating the remineralisation of partially dissolved crystals through pH cycling [7]. Toothpastes containing fluoride are regarded as effective fluoride delivery vehicle [4]. It was reported that over 95% of the population in developed countries use fluoridated toothpastes [8].

In this respect, Baysan et al., (2001) previously demonstrated that high fluoride toothpaste (5,000ppm-F) was capable of reversing 56.9% of leathery primary root carious lesions to hard lesions when compared to the toothpaste containing 1,100 ppm F (28.6%) for a period of six months [9]. Subsequently, Ekstrand et al. (2008) indicated that 5,000ppm fluoride toothpaste significantly improved the management of root caries in comparison to the 1,450 ppm F one [10]. It should be noted that fluoride levels in saliva and biofilms decrease significantly within 15mins following the use of fluoridated toothpaste and rinsing. It should be noted that the presence of fluoride reservoirs can be filled with the use of fluoridated toothpaste, and this would then slowly release fluoride ions [11]. Therefore, bioavailable fluoride is beneficial to provide a favourable impact on promoting remineralisation and inhibiting demineralisation [8, 12].

Bioactive glass (BG) with low fluoride and high phosphate was recently introduced in a toothpaste. These amorphous silicate glasses degrade in aqueous solutions [13] and release calcium, phosphate and fluoride ions. This process can then promote fluorapatite formation [14]. Interestingly, studies reported that fluoride ions released from the fluoride containing BGs were retained up to one week [14, 15]. Naumova et al (2019) also reported that there was a positive effect on bioavailable fluoride following the use of toothpaste containing fluoride and BG [13]. These indicated the increased concentration of fluoride ions in oral cavity, which would contribute to the remineralisation process. In addition, Bakry et al (2018) demonstrated that the same toothpaste was effective in treating subsurface enamel lesions in vitro. However, there is lack of evidence for the remineralisation of root caries including the subsurface using fluoridated toothpastes with or without BG. Therefore, the aim of this study was to investigate the potential mineral exchange and fluoridated apatite formation within the subsurface of artificial root carious lesions (ARCLs) following the use of fluoridated toothpastes with or without bioglass.

Sample preparation

Three extracted sound teeth without dental caries, sealant, restorations and cracks were collected for the study from the Oral Surgery. Ethical approval was obtained prior to the study (QMREC 2011/99). Each tooth was stored in 0.1% thymol [16]. The teeth were then carefully cleaned and polished using non-fluoridated prophylaxis paste (NUPRO Dentsply, USA). The crowns of each tooth were removed 1mm below the cemento-enamel junction by cutting the root surfaces using a 0.3mm thick diamond disc under running water at 3,000 rpm speed (Struers, Copenhagen, Denmark) and divided into four sections in order to reduce any potential tooth to tooth variations. Subsequently, these four samples were allocated into each group.

Preparation of Artificial Root Carious Lesions

Lesions were formed in the 1.5mmol/L CaCl2, 0.9mmol/L KH2PO4, 50mmol/L acetic acid and 1M KOH adjusted to pH 4.8 for five days. In addition, 5.0 mmol/L NaN3 was added to the solution to prevent microbial growth. Each sample was separately placed in 10ml of the solution with new solution changed every single day. During this period, these samples were kept in a shaking incubator (Staufen, Germany) at 37°C. In the meantime, the procedure avoided any possible desiccation to prevent the shrinkage of artificial root carious lesions. [17, 18].

The pH-cycling conditions

The rationale of the pH-cycling process was to simulate the dynamics between mineral loss and uptake as seen in the formation of dental caries [19]. This process contained exposure of dentine to alternate demineralisation and remineralisation. The demineralisation solution composition was introduced in the preparation of artificial root carious lesions. The remineralisation solution contained 1.5mmol/L CaCl₂, 0.9mmol/L KH₂PO₄, 20mmol/L HEPES and 130mmol/L KCl. 5 mmol/L NaN₃ was added to the solution to prevent microbial growth. The pH was adjusted at 7.0 using 1M KOH. All chemical reagents were obtained from Sigma Aldrich, UK. Each sample was immersed in 10mL of the demineralisation solution for six hours followed by immersion in 10mL of the remineralisation solution for 16 hours. Fresh solutions were used for each cycle. This procedure was performed for a period of 13 days at 37°C [18, 20].

The use of different toothpastes

A total of 12 samples with ARCLs were allocated into four different treatment groups. Each group (n=3) received one of the allocated treatments (Table 1). Each toothpaste was diluted with deionised water at a dilution of 1:3 to make a toothpaste slurry. Tooth brushing was simulated using a medium-bristle toothbrush (Colgate Palmolive, UK) twice a day for 13 days. The brushing process was carried out using an electrically-powered tooth brushing machine (Boston Gear, Braintree, MA) designed to produce constant reciprocal movements with 150g force for 10s [21]. The samples were left for two minutes prior to rinsing with deionised water [22].

X-ray Microtomography (XMT)

The assessment of mineral density was carried out by the MuCAT XMT scanner. The in-house developed system was designed by means of time-delay integration to provide high quality tomographic images. Each sample was separately placed in a polymethyl methacrylate (PMMA) mould. Subsequently, the mould with samples was put inside a clear plastic tube with a drop of deionised water to keep the specimen fully hydrated and avoid contamination during the long scanning procedure (around 24 hours). The prepared sample was then mounted onto the XMT movable kinematic stage, ensuring the long axis of the tooth was parallel to the XMT rotational axis. The XMT scanner was set at 15µm voxel size resolution (3D). The x-ray generator was operated at 90kV and 270µA. After each sample scan, a calibration was performed and the projection data was transformed to 30keV monochromatic energy equivalent [23]. The reconstructed linear attenuation coefficients (LACs) were converted to the level of mineral density [22]. The XMT scans were carried out at baseline, following the development of ARCLs, and after 13 days of pH-cycling for each sample.

XMT Image analysis

The 3D images from the baseline and treatment scans after 13 days of pH-cycling were aligned using an in-house developed software called ‘Tomview’. These 3D images were then subtracted by running the International Data Line (IDL, Exelis Visual Information Solutions, Boulder, Colorado, USA) software and subsequently mineral loss/gain was detected according to the increase in radiopacity/radiolucency in detected areas (Figure 1). Randomly selected 45 points of LACs in these detected areas after each ARCL scan were compared with the corresponding 45 points in the baseline scans, and also with the treatment scans after 13 days of pH-cycling [22]. Each line profile was also conducted using ImageJ (U. S. National Institutes of Health, Bethesda, Maryland, USA). A line profile was drawn through the low mineral density to the high mineral density parts. These line profiles were then recorded for the successive scans by clicking on the plugins and macros, then recording the sections. This process was carried out to ensure that the line profile, which was drawn, was exactly in the same position for all the successive scans for that section of each sample.

Scanning Electron Microscope (SEM)

Surface morphology of each sample was observed with scanning electron microscopy (SEM, FEI Inspect-F, Hillsboro, USA) after coating with carbon nanofilm (~30nm) using the sputter coating machine (SC 7620, Quorum, Laughton, UK) to maintain conductivity, while the samples were analysed at an accelerating voltage of 5 kV, with a spot size of 3.0 and variable working distances (around 10 mm) for secondary electrons (SE). Subsequently, the voltage increased by 20kV with a spot size of 5.0 for backscattered electron (BSE) imaging.

Energy Dispersive X-Ray Analysis (EDX)

One sample from each group were selected to measure elemental distributions using the EDX analysis. These three samples were cut from the vertical direction to expose the subsurface of ARCLs. Each sample was mounted on stubs using conductive glue. They were then carbon-coated to reduce electrostatic charging so that the samples can elementally be analysed using EDX (FEI Inspect-F, USA). The system was operated at a high voltage of 10.00kV and 500X magnification. Eight points for each line were chosen from the surface to the subsurface in the area of mineral density of each carious lesion.

¹⁹F Magic angle spinning nuclear magnetic resonance (MAS-NMR)

The ¹⁹F-MAS-NMR enables fluorapatite to be distinguished from hydroxyapatite. ¹⁹F-MAS-NMR were carried out using a 600MHz Bruker spectrometer with a magnetic field of 14.1Tesla. The ¹⁹F-MAS-NMR spectra were collected at the resonance frequency of 564.7MHz. The samples were run in a 2.5mm zirconia rotor spinning at 22kHz using the standard double resonance Bruker probe with low fluorine background. 1M aqueous solution of sodium fluoride producing a sharp signal – 120ppm was used to reference at the ¹⁹F chemical shift scale. This was run with a 30s recycle delay with 1us pulse at 100W.

Statistical analysis

The percentages in mineral density changes were calculated. The paired t-test was then carried out to compare the differences in mineral contents for each sample in the intra-groups. The data from XMT mineral density in inter-groups comparison was analysed using the Kruskal-Wallis H with Mann-Whitney test nonparametric tests and one-way ANOVA with multiple comparisons due to normal distribution. A significance level of 0.05 was performed using IBM SPSS Statistics 28.0 (SPSS Inc., Chicago, IL, USA).

Mineral density by XMT

The quantitative mineral density surface measurements (Table 2) demonstrated an increase in mineral contents in the ARCL areas for each toothpaste group after 13 days of pH-cycling. The highest percentage increase was for Group 3 (30%), whilst the mineral density decreased by 7% in the Group 1.

Line profile of mineral density by XMT

A line profile through the same lesion area on the scan showed an evidence of mineral loss after the development of ARCLs compared to the baseline measurements. In addition, after 13 day of pH-cycling, there was an increase in the mineral density on the surface, whilst decreased mineral density was observed within the subsurface for all toothpaste groups (Figure 2).

The mineral density in the Group 1 after the pH-cycling decreased within 0.34mm from the surface to the subsurface. In Group 2, the mineral density increased 0.21mm in depth from the surface to the subsurface after the pH-cycling. However, mineral loss was noticeable between 0.21mm and 0.34mm.

In the Group 3, the total mineral density changes from the surface to subsurface was 0.52mm, and there was an increased mineral density within 0.32mm from the surface to subsurface after the pH-cycling. The loss of minerals also occurred in the deep subsurface from 0.32mm to 0.52mm.

Finally, the depth of increased mineral density after pH-cycling was 0.29mm in the Group 4, whilst mineral loss was seen between 0.29mm and 0.42mm.

The change of mineral density was calculated by the quantitative analysis associated with the above line profiles plotted in Table 3. The increased mineral density was high in the Groups 3 and 4 when compared to that in Group 2. The decreased mineral density was similar in the subsurface for Groups 3 and 4.

SEM analysis

The SEM analyses showed that the dentine tubules were aligned parallel within the subsurface structure of ARCLs. There were rough and irregular surfaces as marked in the following images (Figure 3). The mineral loss was observed on the ARCL surface in the Group 1 (Figure 3i-ii).

In addition, the mineral contents within the surface of the dentine tubules were high in comparison to the subsurface in all toothpaste groups. There was also evidence of dentine tubules being either partially or completely obliterated in the toothpaste groups.

EDX

Table 4 revealed that EDX element analysis compared the Ca/P ratio within the area of mineral density of each carious lesion from the surface to the subsurface between the Groups 1-4. The Ca/P ratio for was 1.16 for the Group 1 (deionised water as the control group). For the Groups 2, 3, 4 (toothpaste groups), the ratio was 1.23, 1.25 and 1.17 respectively, which means that mineral deposits were observed in all three toothpaste groups after pH-cycling. Line profiles of EDX analysis reported the calcium atomic change from surface to the subsurface within the specific area of mineral density for each lesion (Figure 4).

¹⁹F MAS-NMR

The spectra of Group 1 (-105.3ppm) and Group 2 (-105.1ppm) corresponded to a partial fluoride substituted apatites. An additional peak in the spectra for Group 2 at -108.6ppm was presented corresponding to the signal of fluoride in crystalline calcium fluoride (CaF₂). The spectra of Groups 3 and 4 indicated the dominant presence of the fluorine-19 signal at -103.6ppm and -103.2ppm respectively (Figure 5). The strong spinning sidebands were also observed in the ¹⁹F MAS-NMR spectra for Group 4.

There is limited evidence on the effect of different toothpastes containing either sodium fluoride or low concentration fluoride in bioactive glasses on root caries. To simulate the caries process, artificial saliva rather than body fluid was provided in the study. The immersion solution was changed at each tooth brushing time point to simulate the effect of saliva exchange in in-vivo conditions. Furthermore, each sample for the study groups was derived from the same tooth. Interestingly, the amount of mineral density was different from the baseline to ARCLs in each group (Table 3). This means that there were variations in different parts of the same tooth.

The increase of mineral density at the surface of ARCLs in all toothpaste groups demonstrated the ability of toothpastes to precipitate minerals and potentially remineralise the surface of ARCL. This outcome directly corresponded to the surface mineral density changes in the line profiles. The mineral density increase was also observed within the ARCL surface in all three toothpaste groups. However, the mineral density decreased after treated with deionised water, this may be attributed to the lack of fluoride exposure. As fluoride is a well-known remineralising agent, it can be speculated that this could be related to the acid resistant and less reactive fluorapatite layer [24]. Therefore, the pH-cycling model was designed to evaluate the effect of fluoride in either reducing demineralisation or enhancing remineralisation [25].

The line profile of the mineral density after treatment with the toothpaste containing 5,000ppm are close to the mineral density of the sound root dentine (Fig. 2(h)). This outcome might indicate that more fluorapatite formation was observed with the use of this toothpaste when compared to the BG with 540ppm and 1,450ppm ones. This outcome was supported by ¹⁹F MAS-NMR which demonstrated the presence of fluorapatite. In addition, the dentinal tubules were occluded within the 5000ppm F samples as observed in the SEM and BSE images.

Fluoride-substituted apatite can be formed below 45ppm fluoride concentration [26]. In this study, the slurries were diluted with water using the toothpastes containing 5,000 ppm-F, 1,450 ppm-F and BG with 540ppm-F and rinsed out after treatment, it should be noted that some fluoride ions might have been removed by rinsing. In addition, the atomic fraction of calcium is high in comparison to fluoride, therefore the Ca/P ratio in hydroxyapatite or fluorapatite approaches to 1.67 [27]. Changes in the Ca/P ratio indicate alterations in the inorganic components of hydroxyapatite. The Ca/P ratio in both prepared demineralisation and remineralisation solutions was 2.15, which was high in comparison to Ca/P ratio of hydroxyapatite/fluorapatite (1.67), therefore this could have resulted in supersaturation. In addition, the Ca/P ratio was high in the toothpaste treatments group when compared to the Group 1. This might suggest the precipitation of apatite mineral onto the ARCL surface as a result of hydroxyl ions being substituted (at least partly) by fluoride. This could explain the fluoride-substituted apatite or fluorapatite formation. In the Group 1, there was no fluoride application therefore, the formation of fluoride-substituted apatite could be attributed to the cumulative effect of fluoride i.e., from tea, tooth brushing prior to extraction.

The ¹⁹F-MAS-NMR signal between − 101.0 and − 107.0ppm was used to identify the fluoride substitution in apatite formation. The − 105.3ppm chemical shift in Group 1 indicated the origin of fluoride from the tooth itself as this tooth did not receive any fluoride application. In addition, there was a double peak in the spectra for the BG with 540ppm group (Group 2). The ¹⁹F-MAS-NMR spectra obtained for Group 2 at -108.6ppm, which corresponds to CaF₂, whilst the spectra of -105.1ppm represented as fluoridated apatite. The formation of CaF₂ would not be expected with a low fluoride toothpaste content. Chen et al., (2023) reported less than 10ppm of fluoride concentration after brushing the toothpastes with rinsing [28]. Mohammed et al., (2013) indicated the formation of CaF₂ over 45ppm fluoride concentration. This would be more likely to be formed following the use of toothpaste containing high fluoride prior to teeth extraction providing a physical barrier on the root surface slowing the demineralisation and providing a reservoir for fluoride. However, the formation of CaF₂ has the potential to negatively impact the structural stability of tooth since this reduces the availability of Ca²⁺ required for the process of apatite formation. As a result, there is a loss of PO₄³⁻ which ultimately leads to a decrease in the mineral content within the tooth [26]. In this respect, Gao et al (2016) showed that ¹⁹F-MAS-NMR chemical shift plotted against the F percentage in the apatite. The 100% highly fluoridated apatite (fluorapatite) presents at -103.5ppm chemical shift, whilst − 105.0ppm chemical shift would indicate a 40% fluoridated apatite [29].

There was an evidence of decreased mineral density in the subsurface for all toothpaste samples (Fig. 2). Previously, Ten Cate and Arends (1981) reported the blockage of surface layer pores as a result of fluoride-enhanced deposition in the surface [30]. Once the remineralisation occurred at the beginning of pH-cycling, the pores of root dentine surface might have been obliterated. Therefore, the subsurface lesions would have been failed to remineralise further since the formation of fluoride-substituted apatite was unable to pass through the surface to reach the lesion subsurface. This was supported by the BSE images for high minerals at the edge of dentinal tubules. However, the SEM and BSE images also showed that few irregular white particles were noticeable on the subsurface of each sample rather than embedded in the dentinal tubules. This could be related to the polishing paste during the cutting and polishing process for the SEM analysis.

Ekstrand et al. (2013) previously reported that the effect of 5,000ppm fluoride toothpaste was significantly more effective for arresting root carious lesion progression and promoting remineralisation compared to the 1,450ppm fluoride toothpaste. The mean numbers of hard lesions were 2.13 (1.68) in the 5,000ppm fluoride toothpaste and 0.61 (1.76) in the 1,450ppm fluoride toothpaste (p < 0.001). This current laboratory-based study also indicated the high mineral density in the toothpaste containing 5,000ppm fluoride when compared to the 1,450ppm one. Interestingly, both fluoride toothpastes can contribute to the fluorapatite formation as the NMR results showed the peak at -103ppm in 5,000ppm and 1,450ppm toothpastes, which can also indicate the fluorapatite formation.

The line profile of calcium atomic observed using the EDX analysis also represented the mineral density change. As the characteristic X-ray emission probability increases with a high atomic number, heavy elements are useful in EDX analysis [31]. In addition, for elements with atomic numbers below 15 (phosphorus), EDX is challenging to detect this due to the low relative intensity of the K-M peak [32]. Therefore, calcium atomic was selected as the representative element for the results, as shown in Fig. 4. The calcium atomic percent was consistent from the surface to the subsurface within 0.3mm for each group. It should be noted that EDX is a superficial technique which only detects the changes in a region of about 2µm in depth [33]. Therefore, this technique can be used by sectioning the samples to expose the subsurface. In comparison with the mineral density in the XMT, the calcium atomic percent from the EDX failed to match in evaluating the mineral density of each sample. XMT was in 15X15X15µm³ voxels, avoiding the errors in determining the sample thickness. In this respect, Davis et al. (2018) reported the XMT could be a useful method to evaluate demineralisation and remineralisation [34]. However, the XMT is unable to distinguish the mineral element to investigate the density change(s). In addition, there is still lack of evidence to prove the effect of calcium atomic% on demineralisation/remineralisation using the EDX. Therefore, it should be noted that each technique used in this study demonstrated some limitations however they also complemented and supported each other with respect to the mineral density changes from surface to subsurface after each toothpaste use.

Another limitation is that the ¹⁹F MAS-NMR can indicate the formation of fluoridated apatite or fluorapatite, however the technique only analyses the powdered samples. Therefore, the system fails to distinguish the fluorapatite formation between the surface and subsurface. In addition, this study compared the SEM images for each group, however, these images were not recorded at baseline and after the development of ARCLs due to the destructive nature of this technique. Theoretically, these samples would have been exposed to fluoride environment in the oral cavity prior to extraction, which might have caused the variation of fluoride ions within the study teeth.

In the future, unerupted extracted wisdom teeth without any exposure can be considered. In addition, the 19F-MAS-NMR would be used to assess the fluoride levels. Another laboratory-based study without rinsing would also be interesting to conduct in future since rinsing following tooth brushing is not recommended for the fluoride retention [28]. In addition, extracted teeth with root caries would also be considered to mimic real life situation.

Within the limitation of this laboratory-based study, all toothpastes were potentially effective to increase mineral density of artificial root caries on the surface, however there was an evidence of mineral loss in the subsurface for all groups. Interestingly, the 5,000ppm F toothpaste had superior effect with respect to increased mineral density and fluorapatite formation when compared to the other toothpastes (BG with 540ppm F and 1,450ppm F).

Author Contribution

Haoran Chen: Conceptualisation, Methodology, Data curation, Formal analysis, Writing- Original draft preparation, Visualisation, Investigation, Validation, Writing- Reviewing and Editing.

Jiaxin Zhang: Methodology, Formal analysis, Visualisation, Writing- Reviewing and Editing.

Robert Hill: Conceptualisation, Methodology, Resources, Visualisation, Investigation, Writing- Reviewing and Editing, Supervision.

Aylin Baysan: Conceptualization, Methodology, Data curation, Visualisation, Investigation, Writing- Reviewing and Editing, Supervision, Project administration.

Ethics Approval and Consent to Participate

Not Applicable

Funding

No funding was obtained for this study.

Conflict of Interests

Haoran Chen, Jiaxin Zhang and Aylin Baysan declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Robert Hill has small number of shares in Biomin Technologies Ltd.

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Table 1. The allocated study groups

Groups	Toothpastes	Name Company	Ingredients
Group-1	Deionised water	Avidity Science, UK
Group-2	BG with 540ppm F	BiominF, Biomin®, UK	Glycerin, Silica, PEG400, Fluoro Calcium Phospho Silicate, Sodium lauryl Sulphate, Titanium dioxide, Aroma, Carbomer, Potassium Acesulfame
Group-3	1,450ppm NaF	Colgate-Advanced White, Colgate, UK	Sodium Fluoride, Aqua, Hydrated Silica, Sorbitol, PEG-12, Sodium bicarbonate, Aroma, Sodium lauryl sulfate, Xanthan gum, Cellulose gum, Sodium saccharin, Limonene, CI 74160, CI 77891
Group-4	5000ppm NaF	Duraphat®, Colgate, UK	Sodium Fluoride, Liquid Sorbitol (Non-crystallising), Silica, Silica (precipitated), Macrogol 600, Tetrapotassium Pyrophosphate, Xanthan Gum, Sodium Benzoate, Sodium lauryl sulfate, Spearmint Oil, Peppermint Oil, Carvone, Menthol, Anethol, Lemon Oil, Saccharin Sodium, Brilliant Blue FCF, Purified Water

Table 2. The mineral density (g cm⁻³) in the lesion area at baseline, after the development of ARCLs, and following 13 days pH-cycling (±SD), mean differences and p-value, with the percentage of mineral change for all groups.

Groups	Root dentine (baseline)	After ARCLs	13 days of pH-cycling	Mean difference	p-value	Overall change	p-value (intergroups)
Group 1. Deionised water	1.04±0.34	0.75±0.30	0.63±0.18	-0.12	0.003	7% decrease	Kruskal-Wallis H, H(3)= 125.310, p<0.001, all intergroups comparison (p<0.001)
	1.48±0.19	0.51±0.15	0.42±0.13	-0.09	<0.001
	1.33±0.08	0.73±0.19	0.73±0.11	0	0.006
Group 2. BG with 540 ppm	1.32±0.10	0.85±0.24	0.80±0.13	-0.05	0.130	11% increase
	1.26±0.18	0.77±0.21	0.84±0.08	0.07	0.010
	1.20±0.18	0.66±0.16	0.96±0.12	0.30	<0.001
Group 3. 1,450 ppm	1.21±0.12	0.83±0.19	0.96±0.10	0.13	<0.001	30% increase
	1.38±0.11	0.45±0.13	0.86±0.24	0.41	<0.001
	1.33±0.07	0.64±0.06	0.99±0.07	0.35	<0.001
Group 4. 5,000 ppm	1.24±0.17	1.04±0.22	1.08±0.22	0.04	0.003	22% increase
	1.26±0.13	0.71±0.17	1.04±0.13	0.33	<0.001
	1.26±0.20	0.72±0.13	1.00±0.18	0.28	0.006

Table 3. The quantitative analysis of mineral density change was related to the plotted line profile in Figure 2(e) Group 1. Deionised water; Figure 2(f) Group 2. BG with 540 ppm F; Figure 2(g) Group 3. 1,450 ppm F; Figure 2(h) Group 4. 5,000ppm F

Groups	Baseline-after ARCLs (g cm^-3)		ARCLs-After 13 days of pH-cycling (g cm^-3)
	Decrease	p-value	Increase	p-value	Decrease	p-value	Overall changes
Group 1. Deionised water	7.26	Kruskal-Wallis H, H(3)= 1.472, p=0.689, Group1 and 2 (p=0.974), Group1 and 3 (p=0.990), Group1 and 4 (p=0.533), Group2 and 3 (p=0.677), Group2 and 4 (p=0.221), Group3 and 4 (p=0.345)	-	ANOVA (p=.032), Group2 and 3 (p=0.062), Group2 and 4 (p=0.010), Group3 and 4 (p=0.356)	3.55	Kruskal-Wallis H, H(3)= 5.471, p=0.140, Group1 and 2 (p=0.505), Group1 and 3 (p=0.132), Group1 and 4 (p=0.505), Group2 and 3 (p=0.019), Group2 and 4 (p=0.110), Group3 and 4 (p=0.435)	-3.55
Group 2. BG with 540 ppm F	7.76		1.09		1.53		-0.44
Group 3. 1,450 ppm F	9.21		3.63		0.79		2.84
Group 4. 5,000 ppm F	6.28		3.97		0.76		3.21

Table 4. Ca, P, F and Ca/P ratio (atomic%) in four groups for area of mineral density of caries lesions (500x)

Deionised water (Group 1)	Ca	11.00±1.14	One-way AVOVA, p=.006 Multiple comparison, Group 1 and Group 2 (p=.015), Group 1 and Group 3 (p=.004), Group 1 and Group 4 (p=.755), Group 2 and Group 3 (p=.621), Group 2 and Group 4 (p=.033), Group 3 and Group 4 (p=.010)
	P	9.48±0.28
	F	0.30±0.34
	Ca/P	1.16±0.11
BG with 540ppm F (Group 2)	Ca	19.58±4.50
	P	15.94±4.18
	F	0.32±0.38
	Ca/P	1.23±0.07
1450ppm F (Group 3)	Ca	18.53±2.42
	P	14.85±1.89
	F	0.44±0.61
	Ca/P	1.25±0.05
5000ppm F (Group 4)	Ca	14.23±0.78
	P	12.18±0.73
	F	0.45±0.59
	Ca/P	1.17±0.05

Competing interest reported. H. C, J. Z and A. B declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. R. H has small number of shares in Biomin Technologies Ltd.

Changes in mineral density and fluoridated apatite formation of artificial root caries using fluoridated toothpastes with or without bioactive glass

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Abstract

Figures

Introduction

Materials and Methods

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

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