Preventive effect of remineralizing materials on dental erosion lesions by speckle technique: An in vitro analysis
Journal Pre-proof PREVENTIVE EFFECT OF THE REMINERALIZING MATERIALS ON DENTAL EROSION LESIONS BY SPECKLE TECHNIQUE: AN IN VITRO ANALYSIS Silvia Regina...
Journal Pre-proof PREVENTIVE EFFECT OF THE REMINERALIZING MATERIALS ON DENTAL EROSION LESIONS BY SPECKLE TECHNIQUE: AN IN VITRO ANALYSIS Silvia Regina Garcia Olivan, Ravana Angelini Sfalcin, Kristianne Porta Santos Fernandes, Raquel Agnelli Mesquita Ferrari, Anna Carolina Ratto Tempestini Horliana, Lara Jansiski Motta, Silvana Machado Ortega, Marcelo Mendes Pinto, Alessandro Melo Deana, Sandra Kalil Bussadori
PII:
S1572-1000(20)30007-7
DOI:
https://doi.org/10.1016/j.pdpdt.2020.101655
Reference:
PDPDT 101655
To appear in:
Photodiagnosis and Photodynamic Therapy
Received Date:
29 October 2019
Revised Date:
24 December 2019
Accepted Date:
6 January 2020
Please cite this article as: Olivan SRG, Sfalcin RA, Fernandes KPS, Ferrari RAM, Horliana ACRT, Motta LJ, Ortega SM, Pinto MM, Deana AM, Bussadori SK, PREVENTIVE EFFECT OF THE REMINERALIZING MATERIALS ON DENTAL EROSION LESIONS BY SPECKLE TECHNIQUE: AN IN VITRO ANALYSIS, Photodiagnosis and Photodynamic Therapy (2020), doi: https://doi.org/10.1016/j.pdpdt.2020.101655
Prof. Dr. Sandra Kalil Bussadori Biophotonics Program, Nove de Julho University (UNINOVE), São Paulo, SP, Brazil Vergueiro St, 235/249 – Liberdade – São Paulo – Brazil – ZIP: 01504-001 Phone: 0055-11-33859222 e-mail: [email protected]
Highlights:
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Laser speckle imaging was tested to the diagnosis and at evaluating the enamel surface after treatments with preventive materials Three different available commercially preventive materials were compared to a control group (without previous treatment) Treatment with different preventive materials showed promising results under a laser speckle imaging diagnosis
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Abstract
The aim of this in vitro study was to evaluate the preventive effect of different materials
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on dental erosion lesions by speckle coherent light scattering analysis. Fourty bovine teeth were divided in the following groups (n = 10): 1) DURA- Preventive Treatment
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with Fluoride Varnish (Duraphat, Colgate-Palmolive); 2) ELX- Preventive treatment with Elmex® Erosion Protection Toothpaste (GABA International AG, Therwil,
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Switzerland); 3) MP- Preventive treatment with MI Paste® (GC America); and 4) REGPreventive Treatment with Regenerate Enamel Science™ (Unilever). For all groups,
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each sample was divided into 3 areas: non-treatment (control); preventive treatment + erosive challenge; non-treatment + erosive challenge. The erosive challenge was carried out using Sprite® Zero soft drink (pH 2.58). After the erosive challenge, the samples were evaluated by speckle coherent light scattering method in the eroded area compared to the sound area. The results showed that there was a statistically significant difference between eroded area with and without preventive treatment, however, there was no
statistically significant difference among the different preventive materials tested. It could be conclude the efficacy of all the preventive materials tested on erosion prevention. Keywords: Dental erosion, prevention, laser, optical diagnosis, speckle
1. INTRODUCTION Dental erosion is a multifactorial condition which is influenced by a number of
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chemical, biological and behavioural factors. The erosive potential of erosive agents like acidic drinks or foodstuffs depends on chemical factors such as pH, mineral content, etc [1].
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The prevalence of dental erosion has grown steadily due to the current intake of more acidic foods and beverages. Thus, it is important to detect the early diagnostic of the
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lesion and to suit preventive measures both in children and adults [2]. Moreover, it is
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necessary to know which risk factors are involved to the erosion process [3]. The population lifestyle has changed during the last years and the quantity and, as aforementioned, the frequency and the intake of acidic foods and beverages have also
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increased. In the last 20 years, the intake of soft drinks increased about 300%, among US adolescents [4]. Moreover, gastric acid and diseases related to gastric dysfunction
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(pH 1 to 1.5) such as gastroesophageal reflux, regurgitation, vomiting due to
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alcoholism, nervous anorexia and bulimia have also been considered as risk factors for dental erosion [5]. In this manner, the early diagnosis is very important, however, dentists usually ignore the initial stages (smooth and vitreous surface, color change, and presence of pits and cavitations on occlusal surfaces), considering the small loss of surface as normal and inevitable from daily life. Through a concise routine evaluation, it is possible to observe
dental structure only with evident changes and with exposed dentine where may occurs dental sensitivity [6]. The carbonate (CO3) content of the enamel is approximately 3% and of the dentine is about 5 to 6%, which makes the mineral of the dentine more acid soluble than the mineral of the enamel. Also, the water content of the enamel is sufficient to the exchange among the tooth and the external environment of acids, minerals (Ca and PO) and other components during the erosion process [7].
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The buffer effect of the saliva becomes the pH into neutral, where Ca and PO come from the saliva and other substances may cause remineralization; besides, when in contact with fluoride, a more acid-resistant mineral surface will form [1].
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The casein phosphopeptide (CPP) complex, a compound of Ca and P, associated with amorphous calcium phosphate (ACP) has been extensively studied in recent years [8-
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10]. Also, the casein phosphopeptide-amorphous phosphate (CPP-ACP) has been shown
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to have an anticariogenic activity [11, 12]. MI Paste® is marketed for professional use, and it is a topical water-based paste containing Recaldent® (CPP-ACP), a milk-derived casein product. The casein phosphate stabilizes calcium and phosphate, and it makes the
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formation of calcium phosphate compounds easier on the surface of the tooth which act as a source of minerals for the remineralization process (it quickly transforms into
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hydroxyapatite) [8-10].
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Fluoride is the main remineralizing agent indicated for prevention and controlling dental erosion, because it is able to reduce surface solubility and increase surface resistance from mineral recovery [13, 14]. Fluoride toothpastes and mouthwashes are frequent sources of exposure to fluoride but not effective at controlling tooth erosion due to the low concentration of fluoride in its compositions (0.1% to 0.15% and 0.05% to 0.20% respectively) [15]. Controlling dental erosion seems to be better performed by varnishes
(even with 0.1% concentration) and gels. The varnishes adhere to the tooth for longer periods (12 hours or more) and prevent the immediate loss of fluoride after application, thus acting as slow-releasing reservoirs of fluoride [16, 17]. Moreover, tin- and fluoride-containing solutions are effective anti-erosive agents, and tin from Sn/F solutions is retained on and in dentine to various extents, and that its affinity to mineral is higher than to organic structures [18]. Under acidic challenge, tin is incorporated into the outer layer of enamel in a positive amount in relation to the
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tissue loss, probably due to a complex demineralization and reprecipitation process that modifies the upper layers of the enamel causing higher acid resistance [19]. A previous
study demonstrated that the amine fluoride (AmF), sodium fluoride (NaF) and stannous
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chloride (SnCl2) combination (Elmex® Erosion Protection Enamel Toothpaste) showed a significant reduction in erosive and erosive/abrasive enamel loss compared to the
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untreated control. Its protective effect might be due to the precipitation of stannous salts
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(Sn20HPO4, Sn3F3PO4, Ca (SnF3)2) which act as a protective layer against acidic attacks [20].
When calcium minerals are added to fluoride dentifrice, the level of Ca increases both
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in plaque and saliva. Retention of calcium minerals in the mouth after toothbrushing may cause pH buffering to provide a degree of protection against subsequent acid
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challenges [21]. A previous study [22] showed that a calcium silicate and sodium
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phosphate-containing dentifrice can effectively deposit calcium silicate on the enamel surface and from the first toothbrushing, an increase in the mineral phase of the HPA continues about four weeks. A technology (NR-5 ™) combining calcium silicate, sodium phosphate and fluoride salts has been developed, which proposes to increase the natural mineralization processes of human saliva by supplying Ca and PO4 [22].
Regenerate Enamel Science ™ is a novel dentifrice which presents the NR-5 ™ technology. Both enamel and dentin present fluorescent characteriscts under microscopic analysis, however, the organic content of dentine is three-fold fluorescent than enamel [23]. Pictures taken under coherent light illumination present a unique discrete pattern of bright and dark spots (as opposed to the continuum patterns provided by non-coherent illumination) which contains information on the structure of the surface (or sub-surface)
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of the sample. Known as laser speckle images (LSI), such images are very sensitive to small changes in the surface roughness, allowing a precise assessment of any
phenomena that alter such characteristics [24]. The different patterns of the laser
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speckle on the enamel surface are related to ultra-structural changes, therefore, it allows to detect differences between a healthy and an eroded enamel area [24-30].
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Studies have demonstrated the sensitivity of speckle image contrast in relation to the
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changes in enamel surfaces [31, 32]. Therefore, this non-destructive and non-invasive techinique has been used as an alternative way to quantify the progression of erosion of dental enamel [26].
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In this way, the aim of this study was to evaluate the preventive effect of some materials regarding to acid erosion by speckle coherent light scattering analysis.
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The hypothesis tested in this study was that all the tested materials would act as a
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preventive material to the eroded enamel when compared to non-treated groups.
2. MATERIAL AND METHODS 2.1. Sample preparation Forty enamel blocks (5x5x2 mm³) were cut from the buccal surface of bovine incisors. These were immediately embedded in acrylic resin leaving the enamel surface exposed.
Each enamel specimen was polished using 400#, 600#, 1200# and 2000#- grits silicon carbide (SiC) abrasive paper (Buehler, UK) under continuous water irrigation (60 s each paper-step). A felt disk with a diamond paste (3M, USA) was used for final polishing (Figure 1). At this point, the enamel surface was evaluated by visual inspection under stereomicroscopy in order to verify defects; in such a case specimens were replaced with fresh ones.
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For the preventive treatment, the samples were divided into 4 different groups, as
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follows: 1) DURA- Preventive Treatment with Fluoride Varnish (Duraphat, Colgate-
Palmolive); 2) ELX- Preventive treatment with Elmex® Erosion Protection Toothpaste
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(GABA International AG, Therwil, Switzerland); 3) MP- Preventive treatment with MI
Science™ (Unilever).
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Paste® (GC America); and 4) REG- Preventive Treatment with Regenerate Enamel
For all groups, each specimen was divided into three areas: The areas 1 and 2 were
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protected with an adhesive tape (3M, USA) (Figure 1) and the preventive treatment was carried out in the area 3. A preventive material layer was applied with microbrush on
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the enamel surface and the specimens were then immersed into artificial saliva (pH 7.0 -
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30ml/ specimen) for 6 hours to simulate a clinical situation [33]. At the end of 6 hours, the preventive material was removed out of the specimens. To the erosive challenge, the adhesive tape protection was removed from the area 2. The area 1 (control) was kept protected. Then, all the specimens were submitted to the erosive challenge for 3 days (Sprite® Zero, Coca-Cola Company, Atlanta, Georgia, United States, pH 2.58, 30ml/specimen), during 5 minutes for 4 times daily at room
temperature [34]. After each erosive challenge, the sapecimens were rinsed with distilled water for 5 seconds under current water and kept in artificial saliva until the next erosive challenge. After the third day, the samples were kept in artificial saliva until the speckle images were analyzed.
2.2 Laser Speckle Imaging Because of the highly scattering nature of dental tissue, the laser speckle imaging has
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been used to image the scattering pattern of a coherent light source [30] (Figure 2).
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Each specimen was photographed under difuse and coherent light illumination from a
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HeNe laser (Uniphase, USA) emitting at λ = 633 nm with 40 mW. To cop with the
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Gaussian intensity distribution of the laser beam, it was expanded by a telescope to achieve a circular distance 3-4 times larger than the specimen. This ensures a more homogeneous illumination and, since the specimen has symmetry with respect to the
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sound/eroded area, the beam intensity profile will decrease symmetrically in all portions
profile.
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of the specimen, so the comparative analysis is not affected by the beam intensity
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The digital images were transferred to a computer and treated numerically by a software developed by Deana et al. (2013) [30]. The digital image is transformed into a numerical matrix, according to the intensity found in each pixel, where the collected data can be processed quantitatively. Each image captured by the image acquisition system is stored in the RGB standard. In the RGB model, the images represented consist of three image components, one for
each primary color: [(R, G, B)]. The colors of a pixel can be represented as an 8-bit integer ranging from 0 to 255, with 0 corresponding to the color black and 255 being white. For analysis purposes, the first integer value of the primary color, ie, red, was extracted from the RGB model. In this way, the speckle image was associated with the brightness intensity of the digital image and the image was transformed into a numerical matrix.
𝑠=
̅ 𝐼𝑒𝑟𝑜𝑑𝑒𝑑 ̅ 𝐼𝑠𝑜𝑢𝑛𝑑
(1)
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In whicth 𝐼 ̅ is the average intensity of the speckle image.
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The speckle shift was then obtained according to (1)
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By analyzing the normalized speckle signal instead of the raw signal, it is possible to perform a proper specimen-to-specimen comparison, accounting for deviation in the
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photometric parameters of the image as well as for inherent natural occurring changes in
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the enamel of each individual sample.
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2.3 Statistical analysis
The obtained data from erosion (E) and preventive treatment (PE) were processed as a
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ratio with the sound area (S). The Shapiro – Wilk test was used to analyze the data distribution and since it presented an approximate normal distribution, Analysis of Covariance (ANCOVA) and the paired t-test were then performed. The statistical analysis was performed by the BioStat 5.3 program and the level of significance was kept at 95% (α = 0.05).
3. RESULTS All the images were averaged (n= 4x4) and a pseudo-color algorithm was applied for better visualization. The average intensity of the backscattered light values was obtained from a standardized area of the eroded, sound and treated tissues by ImageJ-win64 program. The eroded speckle contrast values of all groups statistically differs from the sound baseline (p < 0.0001 for all groups) as well as the treated speckle contrast (p < 0.0001 for all groups). All treated groups statistically differs from its eroded counterpart
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(p < 0.0001; p = 0.0004; p = 0.0235 and p = 0.0007, for the groups DURA, ELX, MP and REG, respectively), as shown in figure 4. Using the untreated surface value as an
independent variable and the treated as dependent variable, the analysis of covariance
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found lack of statistical differences among all the treatments proposed in this study (p =
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0.5581).
4. DISCUSSION
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Dental erosion has been widely growing, therefore, the diagnosis of erosion must be made as early as possible and preventive measures must be taken [1]. The early
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diagnosis of erosion is very important and usually, dentists ignore the initial stages
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considering the small surface loss as a normal and unavoidable occurrence of daily life. There is no device available for the specific detection of dental erosion in routine office practice [12]. The diagnosis of the erosion is generally performed through visual inspection To investigate the differences on the properties of light scattering between healthy and eroded teeth, a change in these properties by the LSI is detected [31]. The
intensity of the optical phenomenon depends on the microstructure of the tissue, the roughness, the biochemical components and the wavelength to be used [27]. Several methods based on the changes of the optical properties between sound and lesion teeth have been proposed for the detection and quantification of these dental alterations [31]. Most of the publications in the area of biospeckle focuses on the temporal domain of the scattering pattern which allows information on the movement of particles, cells and
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fluids in general. As dental enamel does not present a vascularized network, the greatest interest is in the spatial domain of the optical scattering pattern.
A few references regarding to the roughness and tension measurements on dental tissues
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using the speckle technique were reported [26, 30], however, some preliminary results
described in the literature showed the great potential of its application [26, 28, 30, 35].
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Erosion is visible for a well-trained dentist with the naked eye, however, by illuminating
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the area with a laser and using appropriate statistics, the contrast between the sound and lesion areas is enhanced and a distinction between light scattered by the lesion and sound region of the tooth can be drawn [26, 30].
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When a coherent light illuminates a surface roughness, it is possible to observe the formation of light and dark optical speckles, which is the main characteristic of the
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technique known as laser speckle imaging [32]. LSI is very sensitive to small changes
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on surface roughness, allowing accurate evaluation of phenomenon which modifies this characteristic [24]. In this manner, the laser is sensitive to ultra-structural changes, it detects differences between sound and lesion areas on the dental enamel and since these changes are proportional to the severity of the lesion, it is possible to detect and quantify them [30].
Koshoji et al. (2014) [26] demonstrated the sensitivity of LSI regarding the degree of dental erosion in the early stages. Everett et al. (2003) [32] also identified initial and carious lesions both by light polarization change and LSI, showing sensitivity of both for surface discrimination and mass dispersion. Nader et al. (2016) [31] also demonstrated the sensitivity of LSI in detecting the early stages of dental demineralization. Thus, our study corroborates with the aforementioned studies since it is possible to
invasive manner using the speckle standard analysis [26].
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quantify the progression of the erosion of the dental tissue in a non-destructive and non-
Regarding to the prevention of acid erosion, the benefits of fluoride dentifrices are
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higher when compared to non-fluoride ones [36]. Fluoride is the main remineralizing
agent indicated for prevention and control of dental erosion, reducing surface solubility
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and increasing surface resistance from mineral recovery [13, 16]. However, fluoride
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dentifrices and mouthrinsings are frequent sources of exposure to fluoride, but they do not show effectiveness in the control of dental erosion due to the low concentration of fluoride in their compositions [15].
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The fluorotherapy used to control the dental erosion seems to be better performed by using gels and varnishes even under the concentration of 0.1% [37-39] once varnish
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prolongs the contact method of the fluoride with enamel surface [40].
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The CPP-ACP complexes marketed as Recaldent™ are added to the dentifrices (MI Paste) for topical application on tooth enamel surfaces in order to promote enamel remineralization and also to prevent tooth erosion [9, 10] through the stabilization of Ca and PO4. In this way, the calcium phosphate nano-complexes formation on the tooth surface will act as a source of minerals for the remineralization process [8-10].
There is an affinity between CPP-ACP and acquired film proteins. The process of remineralization of early enamel lesions by CPP-ACP includes protein-protein interactions. These interactions with specific proteins/peptides may facilitate the release of calcium and phosphate ions, thus the remineralization of underlying dental enamel will occur [41]. Sn and F-containing solutions show anti-erosive properties, which Sn presents the main role [18]. When F-containing solutions (1500ppm) with different Sn concentrations
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(2100, 144 or 400ppm) are applied on the enamel surface, Sn precipitates and under acid challenge, there is the incorporation of Sn into the enamel [19].
When combining AmF/NaF/SnCl2 (Elmex® Protection Email Toothpaste), a significant
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reduction in enamel loss due to erosion is found, which may be due to the precipitation of stannous salts acting as a protective layer against acid attack [20]. Also, a previous
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study [42] with the Elmex® Protection Email Toothpaste showed a significant lower
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surface loss, however, it was not significantly better than a regular fluoridated dentifrice.
The addition of Ca minerals to fluoride dentifrices may increase the level of Ca both in
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plaque and saliva, which may reduce the chance of caries lesion formation and contribute to reduce the rate of enamel demineralization and promote its
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remineralization [43, 21].
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Calcium silicate forms hydroxyapatite (HAP) which can be deposited on the eroded enamel promoting a repair potential. Also, calcium silicate may protect the sound enamel from acid attacks [44]. NR-5 ™ technology has been developed based on the combination of calcium silicate, sodium phosphate and fluoride salts and proposes at increasing the process of natural mineralization of human saliva by providing additional Ca and PO4. Thus,
Regenerate™ is a novel dentifrice, which contains calcium silicate, sodium phosphate salts and sodium monofluorophosphate [22], and it is able to deposit calcium silicate effectively on the surface of the enamel and from the first toothbrushing, there is an increase of the mineral phase of HAP which keeps going for an equivalent period of 4 weeks of toothbrushing, at least [22]. Our study tested the preventive effect of the aforementioned materials containing fluoride (Duraphat, Colgate-Palmolive), CPP-ACP (MI Paste® (GC America), Sn-F (E Elmex® Erosion Protection Toothpaste (GABA
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International AG, Therwil, Switzerland) and calcium silicate (Regenerate Enamel Science™ (Unilever) and all the tested materials showed a preventive effect on the
eroded enamel when compared to non-treated groups, so, the hipotesis tested could be
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accepted.
This study showed that the enamel surface exposed to the acid challenge may result in
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the erosion process which changes the enamel structure and such changes were detected
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by the laser speckle. Our results showed that erosion surface decreased its mean intensity to 70% of its sound counterpart, which is associated with loss in microhardness of the enamel leading to damage and changes in its structure. All the four
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treatments used in this work were unable to completely proctect the enamel from the erosion process; nevertheless, on average, the eroded enamel decreased by 43% and the
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speckle intensity was 84% of its sound intensity, indicating less eroded area in
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comparison to its untreated region (p < 0.05 for all groups).
5. CONCLUSION Within the limitations of this in vitro study, it could be concluded that all the preventive materials tested were effective in the partial prevention of enamel erosion. Also, it was
possible to evaluate the preventive power of dental materials for professional and daily use regarding to enamel erosion through the laser speckle imaging.
Conflicts of interest The authors declare that there was no financial or commercial conflicts of interest in any of the products employed in this investigation.
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Acknowledgements The authors would like to thank FAPESP for the support of this work under the grant
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2015/25180-7.
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FIGURE CAPTIONS
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Figure 1. Schematic design of the sample preparation showing the aspect of the final
sample with the three different areas: 1. Control (sound area), 2. Area without previous
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treatment before erosive challenge.
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preventive treatment before erosive challenge, 3. Area with previous preventive
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Figure 2. Image of the experimental setup used in this work.
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Figure 3. Sample images after preventive treatment and erosive challenge compared to the sound tissue. (a) the image is under white illumination and shows the erosion + preventive treatment, the sound tissue and the eroded tissue; (b) the image is under
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RAW laser speckle; (c) the image processed showing different amounts of red points
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according to the treated areas (False color image with medium intensity).
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Figure 4. Speckle changes means on the enamel for all groups. Error bars respresent standard error of the means.
