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Enamel surface loss after erosive and abrasive cycling with different periods of immersion in human saliva
Archives of Oral Biology 109 (2020) 104549
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Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio
Enamel surface loss after erosive and abrasive cycling with different periods of immersion in human saliva
T
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Raquel Marianna Lopesa, , Juliana Sant’ Anna da Silvaa, Samira Helena João-Souzab, Vinícius Maximianoa, Alana Cristina Machadoa, Taís Scaramuccia, Ana Cecília Correa Aranhaa a b
Department of Restorative Dentistry, School of Dentistry, University of São Paulo, Av. Prof. Lineu Prestes, 2227 Cidade Universitária, São Paulo, 05508-000, SP, Brazil Department of Restorative, Preventive and Pediatric Dentistry, University of Bern, Freiburgstrasse 7 CH-3010, Bern, Switzerland
A R T I C LE I N FO
A B S T R A C T
Keywords: Erosion Abrasion Enamel Remineralisation Human saliva
Objective: This in vitro study aimed to evaluate different periods of exposure to clarified human saliva for the ability to protect enamel against erosive tooth wear. Methods: For this purpose, sixty specimens (4 × 4 × 1.5 mm) were prepared from third human molars. For all groups, the period before abrasion was performed by remineralisation with human saliva (except in G1). The specimens were randomly divided into six groups (n = 10) according to the different remineralisation times of exposure to clarified human saliva: no exposure to saliva (G1) and 30 min (G2), 60 min (G3), 90 min (G4), 120 min (G5), and 240 min (G6) of exposure to human saliva. A 5-day cycling was performed with 5 min of erosion (1% citric acid; pH 2.3), 4x/day. After the first and last erosive episodes, the abrasion challenge was performed with slurry of fluoride toothpaste (1450 ppm F−, as sodium monofluorophosphate) plus human saliva (1:3), with an electric toothbrush (15 s, with a total of 120 s of slurry immersion). Surface loss (SL) was determined using an optical profilometer (n = 10) and for qualitative analysis, environmental scanning electron microscopy (ESEM) was performed (n = 3). The SL data were statistically analysed by one-way analysis of variance (α = 0.05). Results: No significant differences were detected among the groups for SL (p > 0.05), and ESEM showed similar aspects of eroded enamel. Conclusions: The period of in vitro exposure to clarified human saliva was not able to protect against enamel erosion.
1. Introduction Dental erosion is described as the loss of dental hard tissue due to the frequent action of non-bacterial acids on the surface of the tooth (Ganss, 2006). The presence of these acids leads to softening of the enamel surface due to demineralisation and, therefore, the eroded surface becomes more susceptible to abrasion removal. When this softening process is associated with mechanical forces, it results in erosive tooth wear (Lussi & Carvalho, 2014; Shellis, Ganss, Ren, Zero, & Lussi, 2011) The interaction between erosion and oral hygiene practices may be the main factor leading to the clinical manifestation of erosive tooth wear; therefore, it appears reasonable that the teeth should not be brushed immediately after an erosive challenge (Lussi, Jaeggi, & Zero, 2004). Hence, it is advisable that patients with dental erosion should
use a soft brush and gently brush the teeth with medium or low abrasive toothpastes (Magalhaes, Wiegand, & Buzalaf, 2014) The increased prevalence of this condition may be explained by changes in lifestyle and nutritional habits and has intensified the search for preventive measures (Hellwig, Lussi, & Goetz, 2013; Buzalaf, Magalhães, & Rios, 2018; Jaeggi & Lussi, 1999; Lussi & Carvalho, 2014). Low pH and buffer capacity of foods and drinks are the major risk factors (Buzalaf et al., 2018), and the rising consumption of soft drinks appears to be an increasingly important factor involved in the aetiology of erosive tooth wear (Lussi et al., 2004). The first biological factor for erosion prevention is saliva (Voronets, Jaeggi, Buergin, & Lussi, 2008). It forms the acquired pellicle on the dental surface, has buffering capacity, can dilute, clear, and neutralise the acids; and can provide calcium and phosphate to reduce the demineralisation rate and enhance remineralisation (Buzalaf, Hannas, &
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Corresponding author. E-mail addresses: [email protected] (R.M. Lopes), [email protected] (J.S.A. da Silva), [email protected] (S.H. João-Souza), [email protected] (V. Maximiano), [email protected] (A.C. Machado), [email protected] (T. Scaramucci), [email protected] (A.C.C. Aranha). https://doi.org/10.1016/j.archoralbio.2019.104549 Received 10 June 2019; Received in revised form 3 September 2019; Accepted 4 September 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. The 5-day model of erosion and abrasion cycling.
Chartpak, Leeds, MA, USA) were positioned on two areas of the polished surface of each specimen, leaving a window of 4 mm × 1 mm exposed for subsequent testing. The specimens were randomly divided into six experimental groups (n = 10), according to the period of saliva exposure.
Kato, 2012). In vitro and in situ studies have shown that a period of exposure to saliva after an erosive episode and before brushing would reduce surface loss due to enamel remineralisation (Attin, Buchalla, & Putz, 2001; Alencar et al., 2016; Attin, Knöfel, Buchalla, & Tütüncü, 2001; Hellwig et al., 2013; Jaeggi & Lussi, 1999). However, this issue remains controversial, as the remineralisation of softened enamel by postponing brushing may not be sufficient to protect against enamel loss due to the histopathology of enamel erosion (Lussi, Schlueter, Rakhmatullina, & Ganss, 2011). Indeed, a similar degree of enamel re-hardening was shown with 2 h and 12 h of exposure to saliva (Alencar et al., 2016), and another study questioned the recommendation to postpone toothbrushing after erosive challenges (Lussi, Lussi, Carvalho, & Cvikl, 2014). The possible protection of eroded enamel by saliva should be further investigated. Investigation is also required for the period of saliva exposure needed after erosive challenges to protect against enamel loss during subsequent abrasion episodes. Thus, the aim of this study was to evaluate enamel surface loss after 5-day cycling with different periods of immersion in human saliva with 30 min to 4 h between the erosive and abrasive challenges. The null hypothesis was that there is no difference in the results of the diverse periods of saliva exposure before the brushing abrasion in surface loss.
2.2. Sample size calculation Sample size calculation for ANOVA models was performed with the program Sigma Plot 13.0 (Systat Software Inc., San Jose, CA, USA). Based on a previous study (Buedel, Lippert, Zero, Eckert, & Hara, 2018), it was considered a difference between means of 2.51 μm, a standard deviation of 0.99, a number of groups of 6, α = 0.05, and a power of 0.8, we have calculated a sample size of 6 specimens per group. Considering this result and other similar investigations of the literature, we have used 10 specimens per group in this study. 2.3. Experimental design The study protocol was approved by the Ethics Committee on Research with Humans of the University of São Paulo (process #2.441.489). This study tested one experimental factor, remineralisation time, at six levels – (G1) without salivary exposure before toothbrushing and (G2) 30 min, (G3) 60 min, (G4) 90 min, (G5) 120 min, and (G6) 240 min of exposure to human saliva with an erosion-abrasion cycling model, using human enamel specimens (n = 10). The response variable was enamel surface loss, measured by optical profilometry. The specimens from each group were analysed by environmental scanning electron microscopy (ESEM) for qualitative analysis (n = 3).
2. Materials and methods 2.1. Specimen preparation Sixty enamel slabs (4 mm × 4 mm × 1.5 mm) were cut from the crowns of human third molars using a microtome (Isomet 1000 Buehler Ltd, Lake Buff, IL, USA). The teeth were stored in 0.1% thymol solution at 4 °C, until the beginning of the experimental procedures. The slabs were embedded into acrylic resin (Varidur, Buehler Ltd), and the specimens were ground flat and polished (Ecomet 250c grinder-polisher; Buehler Ltd– Padrão FEPA), using aluminium oxide papers (grits #600, #1200, and #4000, Buehler Ltd.), under water cooling conditions. Between each paper treatment and at the end of the polishing procedures, the specimens were sonicated in distilled water for 3 min. Adhesive unplasticised polyvinyl chloride tapes (Graphic Tape;
2.4. Erosive/abrasive challenges A 5-day modified erosion-abrasion model was used (Scaramucci, Borges, Lippert, Frank, & Hara, 2013). The erosive challenge was performed with 1% citric acid solution (pH 2.6). The specimens were immersed into this solution (5 ml/specimen) for 5 min, four times a day, without agitation and at room temperature. After erosion and before saliva exposure, the specimens were rinsed with distilled water and gently dried with a soft absorbent paper (Fig. 1). 2
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2.8. Environmental scanning electron microscopy evaluation
The abrasive challenge was represented by toothbrushing, performed twice a day, 15 s per specimen, in a total of 120 s of slurry immersion, with electric brushes, (Oral B Professional Care 5000 Oral B Schwalbacham Taunus, Germany), equipped with a pressure alert feature that signalled when the pressure reached the value of 2 N, with a red light in the body of the brush. The head of the brush was positioned parallel to the surface of the specimens using a custom-made holder for toothbrushing. Slurries of standard toothpaste with 1450 ppm F− (Colgate Máxima Proteção, Colgate-Palmolive, Osasco, SP, Brazil) were prepared with human saliva (1:3 w/w) and used for brushing. A single operator performed the toothbrushing procedures. A different toothbrush was used for each group. The specimens were stored overnight in humid environment at 4 °C.
Three specimens from each group (total of 18 specimens) were analysed by ESEM (Hitachi Analytical Microscope TM3000 Table Top, Hitachi, Tokyo, Japan) to qualitatively verify the enamel surface after cycling. Representative micrographs were obtained at magnification ×2000, using the Anally observation conditions of the software TM300 (TM3000, Hitachi), with 15 kV, at the centre of each specimen. No sample preparation was required. 2.9. Statistical analysis Data were analysed for normal distribution and homoscedasticity with the Shapiro–Wilk and Brown–Forsythe tests, respectively. The surface loss data showed a normal distribution and were analysed with one-way analysis of variance. The significance level was set at 5%. The software Sigma Plot 13.0 (Systat Software Inc., San Jose, CA, USA) was used for the calculations.
2.5. Remineralisation All remineralisation periods were performed by immersing the specimens in clarified human saliva (5 ml/specimen). After the first and fourth erosive challenges, the pre-abrasion remineralisation period was determined as a function of the experimental groups, as follows: no salivary exposure time (G1), 30 min (G2), 60 min (G3), 90 min (G4), 120 min (G5), and 240 min (G6). After abrasion, remineralisation was performed for 30 min until the next erosion. The second and third erosive challenges were followed by a fixed period of 1 h of remineralisation as the original cycling model used (Scaramucci et al., 2013). These two additional erosive challenges (second and third) remained in agreement with the original model to simulate a daily erosive consumption condition and to test if the different periods of remineralisation before the brushing treatment would be able to protect the enamel surface even after successive erosive challenges.
3. Results There were no differences in enamel surface loss among the groups (p > 0.05). Fig. 2 illustrate the results of enamel surface loss in μm (mean and standard deviation). The results of the ESEM evaluation showed that all groups shared similar enamel surface characteristics (Fig. 3). A prism-like pattern of the demineralised enamel could be observed, with the presence of some deposits on the enamel surface. 4. Discussion According to our results, none of the periods of salivary exposure (up to 240 min) prior to toothbrush abrasion was able to decrease enamel loss when compared to brushing immediately after an erosive challenge. This finding is in line with the outcomes of a previous study, which showed that exposure of eroded enamel to saliva for up to 4 h after an erosive attack could not increase surface microhardness or reduce enamel erosive wear (Lussi et al., 2014). In view of this result, the authors recommended that postponing toothbrushing of enamel after an erosive attack should be reconsidered. In the present investigation, all groups tested presented loss of enamel surface, which could be attributed to the abrasion force or the aggressiveness of the erosive challenges. A study evaluating the susceptibility of demineralised enamel to abrasion showed that electric toothbrushes could lead to significantly greater surface loss compared to manual brushing (Wiegand, Begic, & Attin, 2005). As concerning the aggressiveness of the erosive challenge, in the present study was used 1% citric with a natural pH of 2.6 and an immersion time of 5 min. This could have led to large-scale demineralisation, rendering the surface unable to be remineralised. An in vitro study showed increased abrasion resistance of eroded enamel with up
2.6. Human saliva collection The collection of saliva was performed after obtaining local Ethics Committee approval, and according to a previous study (João-Souza et al., 2018). Stimulated saliva was collected from volunteers aged between 20 and 35 years, who had no active caries or salivary dysfunction and were not using any medication that could influence saliva production. The collection was performed in the morning, and the volunteers were instructed not to drink or eat for 1 h prior to the collection. To stimulate salivary secretion, volunteers chewed a piece (5 cm × 5 cm) of parafilm for 10 min, and the saliva produced was collected in an iced tube. To obtain clarified saliva, after collection, the saliva of all volunteers was pooled and centrifuged at 4,000 rpm (20 min; -4 °C; 3,226 g-force). The supernatants were separated from the pellet and stored at −80 °C. One day prior to each experimental procedure, the amount of saliva required was thawed at 4 °C. The saliva was placed at room temperature for 2 h before the beginning of the cycle. 2.7. Enamel surface loss evaluation Surface loss was measured by an optical profilometer (Proscan2100, Scantron, Taunton, UK). An initial measurement was performed to select the specimens with curvature < 0.3 μm (Turssi, Hara, Amaral, França, & Basting, 2014). After the 5-day cycling, the tapes were removed and an area 2 mm long (X) × 1 mm wide (Y) was scanned at the centre of the specimens. The length covered the treated area and the two reference surfaces. The step size was set at 0.01 mm and the number of steps at 200 in the X-axis and at 0.1 mm and 10, respectively, in the Y-axis. The depth of the treated area was calculated based on the subtraction of the average height of the test area from the average height of the two reference surfaces, using the Proscan Application software v. 2.0.17. For this analysis, a 3-point height tool was used, and a single blind operator performed the measurements.
Fig. 2. Enamel surface loss in μm (mean and standard deviation). 3
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Fig. 3. Representative environmental scanning electron microscopy images with magnification 2000 x after cycling. G1: no exposure to saliva; G2: 30 min; G3: 60 min, G4: 90 min, G5: 120 min, and G6: 240 min.
showed that tooth brushing with fluoride toothpaste, associated or not to fluoride mouthrinse, right after an erosive challenge appeared to have more promising protective effect than did 2 h of exposure to saliva in situ and toothbrush abrasion with fluoride-free toothpaste (Ganss, Schlueter, Friedrich, & Klimek, 2007). It seems that the application of fluoride appeared to provide greater enamel protection than did the waiting period before brushing. The proteins present in the saliva act on the formation of the acquired salivary pellicle, which also plays a role on the protection against erosion. However, the protective effect of the pellicle may vary depending on different conditions. Indeed, Amaechi, Higham, Edgar, and Milosevic (1999)) observed differences in the thickness of acquired salivary pellicle within dental arches and among people, and that the protective effect of the pellicle against erosion was proportional to its thickness. We could expect that longer periods of exposure to human saliva would result in thicker salivary pellicle and, consequently, less enamel surface loss. However, considering the limitations of an in vitro study and the model used in the present study, it can be hypothesized that the protective effect of the saliva against dental erosion was limited also due to the removal of the salivary pellicle by the frequent erosive challenges. The micrographs showed a prism-like pattern, typical of eroded enamel. Unexpectedly, scratches from toothbrush abrasion could not be observed. This may be explained by the high frequency of erosive challenges performed during the cycles. The fact that the same surface characteristics were observed in all groups supports the non-significant results of surface loss. A limitation of the present study was that we used an in vitro erosion-abrasion model, which does not allow for direct comparisons with clinical situations. However, it allowed for the standardisation of all parameters, such as the toothbrush challenges and the exact periods of exposure to saliva. Besides, to more closely simulate the clinical scenario, an erosion-remineralisation-abrasion cycle using human saliva was performed. Delaying brushing after an acid challenge does not appear to be effective in a situation of frequent acid exposure. Controlling the frequency of the acid challenges and the pH of the beverages may be more effective methods to prevent the progression of erosive wear than the waiting period for brushing. Corroborating this finding, one
to 1 h remineralisation time (Attin, Buchalla, Gollner, & Hellwig, 2000). However, they used artificial saliva, which has a greater remineralisation potential, and the erosive challenge was weaker (1 min in soft drink against 5 min in 1% citric acid), resulting in less enamel softening and thus, less enamel available to be removed by the subsequent toothbrush abrasion. A more recent in vitro study also found less enamel surface loss with exposure to artificial saliva between erosive and abrasive challenges (Buedel et al., 2018). They showed that toothpaste abrasiveness affects the remineralisation of the eroded enamel. For enamel, for the highly abrasive toothpaste, it was observed that 30 min of exposure to saliva was able to significantly reduce surface loss, without additional protection for 60 and 120 min of exposure; for medium and low abrasiveness, after 120 min, remineralisation resulting in significantly less surface loss was observed. The authors observed that the results were not linear, suggesting that the benefit of remineralisation time before brushing in order to protect the enamel is limited. It is noteworthy that both studies cited above performed fewer episodes of erosion and brushing abrasion compared to the present study. Conversely, in the present study, more episodes of erosive challenges were performed per cycle. Although there was 1 h of saliva exposure between the challenges, the higher frequency of acid challenges may have affected our results, increasing the enamel softened layer and the surface loss for all groups. Moreover, these in vitro studies used artificial saliva. Despite artificial saliva being a useful substitute for human saliva in in vitro studies (Hara, González-Cabezas, Creeth, & Zero, 2008), it is supersaturated with respect to tooth minerals (Attin et al., 2000). This fact allows for greater mineral deposition on the softened enamel with surface re-hardening and reduced surface loss (Eisenburger, Addy, Hughes, & Shellis, 2001). In contrast, in our study we used clarified human saliva, which is not as supersaturated as the artificial saliva in relation to the enamel surface. Moreover, it contains proteins that will also influence on the process of des-/remineralization. In situ studies have shown that 1 h of exposure to saliva before performing toothbrush abrasion had a protective effect compared to no exposure to saliva (Attin, Buchalla et al., 2001; Attin, Knöfel et al., 2001; Jaeggi & Lussi, 1999). Moreover, 2 h of exposure to saliva in situ was shown to re-harden the softened enamel (Alencar et al., 2016; Mendonça et al., 2017). However, another study 4
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epidemiological study performed in seven European countries showed a prevalence of tooth wear of 29.4%, but did not find evidence that waiting after breakfast for toothbrushing affects the degree of tooth wear (Bartlett et al., 2013). In view of the above, it can be concluded that the period of exposure to human saliva after an erosive challenge and before toothbrush abrasion does not prevent enamel erosive wear in vitro.
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Authors’ contributions Raquel Marianna Lopes: Conception and design of the study, acquisition of data and manuscript drafting Juliana Sant`Anna da Silva: preparation of specimens, collected human saliva from donors, worked on the days of cycling and manuscript review Samira Helena João- Souza: collected human saliva from donors, worked on the days of cycling and manuscript drafting Vinicius Maximiano: worked on the days of cycling and manuscript review Alana Machado: worked on the days of cycling and manuscript review Tais Scaramucci: Data analysis and manuscript review Ana Cecília Corrêa Aranha: Conception and design of the study and manuscript review Funding This work was supported by the São Paulo Research Foundation (FAPESP) [grant numbers FAPESP 2015:15629-7 to R.M.L. and FAPESP 2017:14085-9 to J.S.A.S.]. Declaration of Competing Interest None. References Alencar, C. R., Mendonça, F. L., Guerrini, L. B., Jordão, M. C., Oliveira, G. C., Honório, H. M., et al. (2016). Effect of different salivary exposure times on the rehardening of acid-softened enamel. Brazilian Oral Research, 30, e104. https://doi.org/10.1590/ 1807-3107BOR-2016.vol30.0104. Amaechi, B. T., Higham, S. M., Edgar, W. M., & Milosevic, A. (1999). Thickness of acquired salivary pellicle as a determinant of the sites of dental erosion. Journal of Dental Research, 78(December 12), 1821–1828 PubMed PMID: 10598912. Attin, T., Buchalla, W., & Putz, B. (2001). In vitro evaluation of different remineralization periods in improving the resistance of previously eroded bovine dentine against tooth-brushing abrasion. Archives of Oral Biology, 46, 871–874. https://doi.org/10. 1016/S0003-9969(01)00039-5. Attin, T., Buchalla, W., Gollner, & Hellwig, E. (2000). Use of variable remineralization periods to improve the abrasion resistance of previously eroded enamel. Caries Research, 34, 48–52. https://doi.org/10.1159/000016569. Attin, T., Knöfel, S., Buchalla, W., & Tütüncü, R. (2001). In situ evaluation of different remineralization periods to decrease brushing abrasion of demineralized enamel. Caries Research, 35, 216–222. https://doi.org/10.1159/000047459. Bartlett, D. W., Lussi, A., West, N. X., Bouchard, P., Sanz, M., & Bourgeois, D. (2013). Prevalence of tooth wear on buccal and lingual surfaces and possible risk factors in young european adults. Journal of Dentistry, 41, 1007–1013. https://doi.org/10.
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Enamel surface loss after erosive and abrasive cycling with different periods of immersion in human saliva
T
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Raquel Marianna Lopesa, , Juliana Sant’ Anna da Silvaa, Samira Helena João-Souzab, Vinícius Maximianoa, Alana Cristina Machadoa, Taís Scaramuccia, Ana Cecília Correa Aranhaa a b
Department of Restorative Dentistry, School of Dentistry, University of São Paulo, Av. Prof. Lineu Prestes, 2227 Cidade Universitária, São Paulo, 05508-000, SP, Brazil Department of Restorative, Preventive and Pediatric Dentistry, University of Bern, Freiburgstrasse 7 CH-3010, Bern, Switzerland
A R T I C LE I N FO
A B S T R A C T
Keywords: Erosion Abrasion Enamel Remineralisation Human saliva
Objective: This in vitro study aimed to evaluate different periods of exposure to clarified human saliva for the ability to protect enamel against erosive tooth wear. Methods: For this purpose, sixty specimens (4 × 4 × 1.5 mm) were prepared from third human molars. For all groups, the period before abrasion was performed by remineralisation with human saliva (except in G1). The specimens were randomly divided into six groups (n = 10) according to the different remineralisation times of exposure to clarified human saliva: no exposure to saliva (G1) and 30 min (G2), 60 min (G3), 90 min (G4), 120 min (G5), and 240 min (G6) of exposure to human saliva. A 5-day cycling was performed with 5 min of erosion (1% citric acid; pH 2.3), 4x/day. After the first and last erosive episodes, the abrasion challenge was performed with slurry of fluoride toothpaste (1450 ppm F−, as sodium monofluorophosphate) plus human saliva (1:3), with an electric toothbrush (15 s, with a total of 120 s of slurry immersion). Surface loss (SL) was determined using an optical profilometer (n = 10) and for qualitative analysis, environmental scanning electron microscopy (ESEM) was performed (n = 3). The SL data were statistically analysed by one-way analysis of variance (α = 0.05). Results: No significant differences were detected among the groups for SL (p > 0.05), and ESEM showed similar aspects of eroded enamel. Conclusions: The period of in vitro exposure to clarified human saliva was not able to protect against enamel erosion.
1. Introduction Dental erosion is described as the loss of dental hard tissue due to the frequent action of non-bacterial acids on the surface of the tooth (Ganss, 2006). The presence of these acids leads to softening of the enamel surface due to demineralisation and, therefore, the eroded surface becomes more susceptible to abrasion removal. When this softening process is associated with mechanical forces, it results in erosive tooth wear (Lussi & Carvalho, 2014; Shellis, Ganss, Ren, Zero, & Lussi, 2011) The interaction between erosion and oral hygiene practices may be the main factor leading to the clinical manifestation of erosive tooth wear; therefore, it appears reasonable that the teeth should not be brushed immediately after an erosive challenge (Lussi, Jaeggi, & Zero, 2004). Hence, it is advisable that patients with dental erosion should
use a soft brush and gently brush the teeth with medium or low abrasive toothpastes (Magalhaes, Wiegand, & Buzalaf, 2014) The increased prevalence of this condition may be explained by changes in lifestyle and nutritional habits and has intensified the search for preventive measures (Hellwig, Lussi, & Goetz, 2013; Buzalaf, Magalhães, & Rios, 2018; Jaeggi & Lussi, 1999; Lussi & Carvalho, 2014). Low pH and buffer capacity of foods and drinks are the major risk factors (Buzalaf et al., 2018), and the rising consumption of soft drinks appears to be an increasingly important factor involved in the aetiology of erosive tooth wear (Lussi et al., 2004). The first biological factor for erosion prevention is saliva (Voronets, Jaeggi, Buergin, & Lussi, 2008). It forms the acquired pellicle on the dental surface, has buffering capacity, can dilute, clear, and neutralise the acids; and can provide calcium and phosphate to reduce the demineralisation rate and enhance remineralisation (Buzalaf, Hannas, &
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Corresponding author. E-mail addresses: [email protected] (R.M. Lopes), [email protected] (J.S.A. da Silva), [email protected] (S.H. João-Souza), [email protected] (V. Maximiano), [email protected] (A.C. Machado), [email protected] (T. Scaramucci), [email protected] (A.C.C. Aranha). https://doi.org/10.1016/j.archoralbio.2019.104549 Received 10 June 2019; Received in revised form 3 September 2019; Accepted 4 September 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. The 5-day model of erosion and abrasion cycling.
Chartpak, Leeds, MA, USA) were positioned on two areas of the polished surface of each specimen, leaving a window of 4 mm × 1 mm exposed for subsequent testing. The specimens were randomly divided into six experimental groups (n = 10), according to the period of saliva exposure.
Kato, 2012). In vitro and in situ studies have shown that a period of exposure to saliva after an erosive episode and before brushing would reduce surface loss due to enamel remineralisation (Attin, Buchalla, & Putz, 2001; Alencar et al., 2016; Attin, Knöfel, Buchalla, & Tütüncü, 2001; Hellwig et al., 2013; Jaeggi & Lussi, 1999). However, this issue remains controversial, as the remineralisation of softened enamel by postponing brushing may not be sufficient to protect against enamel loss due to the histopathology of enamel erosion (Lussi, Schlueter, Rakhmatullina, & Ganss, 2011). Indeed, a similar degree of enamel re-hardening was shown with 2 h and 12 h of exposure to saliva (Alencar et al., 2016), and another study questioned the recommendation to postpone toothbrushing after erosive challenges (Lussi, Lussi, Carvalho, & Cvikl, 2014). The possible protection of eroded enamel by saliva should be further investigated. Investigation is also required for the period of saliva exposure needed after erosive challenges to protect against enamel loss during subsequent abrasion episodes. Thus, the aim of this study was to evaluate enamel surface loss after 5-day cycling with different periods of immersion in human saliva with 30 min to 4 h between the erosive and abrasive challenges. The null hypothesis was that there is no difference in the results of the diverse periods of saliva exposure before the brushing abrasion in surface loss.
2.2. Sample size calculation Sample size calculation for ANOVA models was performed with the program Sigma Plot 13.0 (Systat Software Inc., San Jose, CA, USA). Based on a previous study (Buedel, Lippert, Zero, Eckert, & Hara, 2018), it was considered a difference between means of 2.51 μm, a standard deviation of 0.99, a number of groups of 6, α = 0.05, and a power of 0.8, we have calculated a sample size of 6 specimens per group. Considering this result and other similar investigations of the literature, we have used 10 specimens per group in this study. 2.3. Experimental design The study protocol was approved by the Ethics Committee on Research with Humans of the University of São Paulo (process #2.441.489). This study tested one experimental factor, remineralisation time, at six levels – (G1) without salivary exposure before toothbrushing and (G2) 30 min, (G3) 60 min, (G4) 90 min, (G5) 120 min, and (G6) 240 min of exposure to human saliva with an erosion-abrasion cycling model, using human enamel specimens (n = 10). The response variable was enamel surface loss, measured by optical profilometry. The specimens from each group were analysed by environmental scanning electron microscopy (ESEM) for qualitative analysis (n = 3).
2. Materials and methods 2.1. Specimen preparation Sixty enamel slabs (4 mm × 4 mm × 1.5 mm) were cut from the crowns of human third molars using a microtome (Isomet 1000 Buehler Ltd, Lake Buff, IL, USA). The teeth were stored in 0.1% thymol solution at 4 °C, until the beginning of the experimental procedures. The slabs were embedded into acrylic resin (Varidur, Buehler Ltd), and the specimens were ground flat and polished (Ecomet 250c grinder-polisher; Buehler Ltd– Padrão FEPA), using aluminium oxide papers (grits #600, #1200, and #4000, Buehler Ltd.), under water cooling conditions. Between each paper treatment and at the end of the polishing procedures, the specimens were sonicated in distilled water for 3 min. Adhesive unplasticised polyvinyl chloride tapes (Graphic Tape;
2.4. Erosive/abrasive challenges A 5-day modified erosion-abrasion model was used (Scaramucci, Borges, Lippert, Frank, & Hara, 2013). The erosive challenge was performed with 1% citric acid solution (pH 2.6). The specimens were immersed into this solution (5 ml/specimen) for 5 min, four times a day, without agitation and at room temperature. After erosion and before saliva exposure, the specimens were rinsed with distilled water and gently dried with a soft absorbent paper (Fig. 1). 2
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2.8. Environmental scanning electron microscopy evaluation
The abrasive challenge was represented by toothbrushing, performed twice a day, 15 s per specimen, in a total of 120 s of slurry immersion, with electric brushes, (Oral B Professional Care 5000 Oral B Schwalbacham Taunus, Germany), equipped with a pressure alert feature that signalled when the pressure reached the value of 2 N, with a red light in the body of the brush. The head of the brush was positioned parallel to the surface of the specimens using a custom-made holder for toothbrushing. Slurries of standard toothpaste with 1450 ppm F− (Colgate Máxima Proteção, Colgate-Palmolive, Osasco, SP, Brazil) were prepared with human saliva (1:3 w/w) and used for brushing. A single operator performed the toothbrushing procedures. A different toothbrush was used for each group. The specimens were stored overnight in humid environment at 4 °C.
Three specimens from each group (total of 18 specimens) were analysed by ESEM (Hitachi Analytical Microscope TM3000 Table Top, Hitachi, Tokyo, Japan) to qualitatively verify the enamel surface after cycling. Representative micrographs were obtained at magnification ×2000, using the Anally observation conditions of the software TM300 (TM3000, Hitachi), with 15 kV, at the centre of each specimen. No sample preparation was required. 2.9. Statistical analysis Data were analysed for normal distribution and homoscedasticity with the Shapiro–Wilk and Brown–Forsythe tests, respectively. The surface loss data showed a normal distribution and were analysed with one-way analysis of variance. The significance level was set at 5%. The software Sigma Plot 13.0 (Systat Software Inc., San Jose, CA, USA) was used for the calculations.
2.5. Remineralisation All remineralisation periods were performed by immersing the specimens in clarified human saliva (5 ml/specimen). After the first and fourth erosive challenges, the pre-abrasion remineralisation period was determined as a function of the experimental groups, as follows: no salivary exposure time (G1), 30 min (G2), 60 min (G3), 90 min (G4), 120 min (G5), and 240 min (G6). After abrasion, remineralisation was performed for 30 min until the next erosion. The second and third erosive challenges were followed by a fixed period of 1 h of remineralisation as the original cycling model used (Scaramucci et al., 2013). These two additional erosive challenges (second and third) remained in agreement with the original model to simulate a daily erosive consumption condition and to test if the different periods of remineralisation before the brushing treatment would be able to protect the enamel surface even after successive erosive challenges.
3. Results There were no differences in enamel surface loss among the groups (p > 0.05). Fig. 2 illustrate the results of enamel surface loss in μm (mean and standard deviation). The results of the ESEM evaluation showed that all groups shared similar enamel surface characteristics (Fig. 3). A prism-like pattern of the demineralised enamel could be observed, with the presence of some deposits on the enamel surface. 4. Discussion According to our results, none of the periods of salivary exposure (up to 240 min) prior to toothbrush abrasion was able to decrease enamel loss when compared to brushing immediately after an erosive challenge. This finding is in line with the outcomes of a previous study, which showed that exposure of eroded enamel to saliva for up to 4 h after an erosive attack could not increase surface microhardness or reduce enamel erosive wear (Lussi et al., 2014). In view of this result, the authors recommended that postponing toothbrushing of enamel after an erosive attack should be reconsidered. In the present investigation, all groups tested presented loss of enamel surface, which could be attributed to the abrasion force or the aggressiveness of the erosive challenges. A study evaluating the susceptibility of demineralised enamel to abrasion showed that electric toothbrushes could lead to significantly greater surface loss compared to manual brushing (Wiegand, Begic, & Attin, 2005). As concerning the aggressiveness of the erosive challenge, in the present study was used 1% citric with a natural pH of 2.6 and an immersion time of 5 min. This could have led to large-scale demineralisation, rendering the surface unable to be remineralised. An in vitro study showed increased abrasion resistance of eroded enamel with up
2.6. Human saliva collection The collection of saliva was performed after obtaining local Ethics Committee approval, and according to a previous study (João-Souza et al., 2018). Stimulated saliva was collected from volunteers aged between 20 and 35 years, who had no active caries or salivary dysfunction and were not using any medication that could influence saliva production. The collection was performed in the morning, and the volunteers were instructed not to drink or eat for 1 h prior to the collection. To stimulate salivary secretion, volunteers chewed a piece (5 cm × 5 cm) of parafilm for 10 min, and the saliva produced was collected in an iced tube. To obtain clarified saliva, after collection, the saliva of all volunteers was pooled and centrifuged at 4,000 rpm (20 min; -4 °C; 3,226 g-force). The supernatants were separated from the pellet and stored at −80 °C. One day prior to each experimental procedure, the amount of saliva required was thawed at 4 °C. The saliva was placed at room temperature for 2 h before the beginning of the cycle. 2.7. Enamel surface loss evaluation Surface loss was measured by an optical profilometer (Proscan2100, Scantron, Taunton, UK). An initial measurement was performed to select the specimens with curvature < 0.3 μm (Turssi, Hara, Amaral, França, & Basting, 2014). After the 5-day cycling, the tapes were removed and an area 2 mm long (X) × 1 mm wide (Y) was scanned at the centre of the specimens. The length covered the treated area and the two reference surfaces. The step size was set at 0.01 mm and the number of steps at 200 in the X-axis and at 0.1 mm and 10, respectively, in the Y-axis. The depth of the treated area was calculated based on the subtraction of the average height of the test area from the average height of the two reference surfaces, using the Proscan Application software v. 2.0.17. For this analysis, a 3-point height tool was used, and a single blind operator performed the measurements.
Fig. 2. Enamel surface loss in μm (mean and standard deviation). 3
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Fig. 3. Representative environmental scanning electron microscopy images with magnification 2000 x after cycling. G1: no exposure to saliva; G2: 30 min; G3: 60 min, G4: 90 min, G5: 120 min, and G6: 240 min.
showed that tooth brushing with fluoride toothpaste, associated or not to fluoride mouthrinse, right after an erosive challenge appeared to have more promising protective effect than did 2 h of exposure to saliva in situ and toothbrush abrasion with fluoride-free toothpaste (Ganss, Schlueter, Friedrich, & Klimek, 2007). It seems that the application of fluoride appeared to provide greater enamel protection than did the waiting period before brushing. The proteins present in the saliva act on the formation of the acquired salivary pellicle, which also plays a role on the protection against erosion. However, the protective effect of the pellicle may vary depending on different conditions. Indeed, Amaechi, Higham, Edgar, and Milosevic (1999)) observed differences in the thickness of acquired salivary pellicle within dental arches and among people, and that the protective effect of the pellicle against erosion was proportional to its thickness. We could expect that longer periods of exposure to human saliva would result in thicker salivary pellicle and, consequently, less enamel surface loss. However, considering the limitations of an in vitro study and the model used in the present study, it can be hypothesized that the protective effect of the saliva against dental erosion was limited also due to the removal of the salivary pellicle by the frequent erosive challenges. The micrographs showed a prism-like pattern, typical of eroded enamel. Unexpectedly, scratches from toothbrush abrasion could not be observed. This may be explained by the high frequency of erosive challenges performed during the cycles. The fact that the same surface characteristics were observed in all groups supports the non-significant results of surface loss. A limitation of the present study was that we used an in vitro erosion-abrasion model, which does not allow for direct comparisons with clinical situations. However, it allowed for the standardisation of all parameters, such as the toothbrush challenges and the exact periods of exposure to saliva. Besides, to more closely simulate the clinical scenario, an erosion-remineralisation-abrasion cycle using human saliva was performed. Delaying brushing after an acid challenge does not appear to be effective in a situation of frequent acid exposure. Controlling the frequency of the acid challenges and the pH of the beverages may be more effective methods to prevent the progression of erosive wear than the waiting period for brushing. Corroborating this finding, one
to 1 h remineralisation time (Attin, Buchalla, Gollner, & Hellwig, 2000). However, they used artificial saliva, which has a greater remineralisation potential, and the erosive challenge was weaker (1 min in soft drink against 5 min in 1% citric acid), resulting in less enamel softening and thus, less enamel available to be removed by the subsequent toothbrush abrasion. A more recent in vitro study also found less enamel surface loss with exposure to artificial saliva between erosive and abrasive challenges (Buedel et al., 2018). They showed that toothpaste abrasiveness affects the remineralisation of the eroded enamel. For enamel, for the highly abrasive toothpaste, it was observed that 30 min of exposure to saliva was able to significantly reduce surface loss, without additional protection for 60 and 120 min of exposure; for medium and low abrasiveness, after 120 min, remineralisation resulting in significantly less surface loss was observed. The authors observed that the results were not linear, suggesting that the benefit of remineralisation time before brushing in order to protect the enamel is limited. It is noteworthy that both studies cited above performed fewer episodes of erosion and brushing abrasion compared to the present study. Conversely, in the present study, more episodes of erosive challenges were performed per cycle. Although there was 1 h of saliva exposure between the challenges, the higher frequency of acid challenges may have affected our results, increasing the enamel softened layer and the surface loss for all groups. Moreover, these in vitro studies used artificial saliva. Despite artificial saliva being a useful substitute for human saliva in in vitro studies (Hara, González-Cabezas, Creeth, & Zero, 2008), it is supersaturated with respect to tooth minerals (Attin et al., 2000). This fact allows for greater mineral deposition on the softened enamel with surface re-hardening and reduced surface loss (Eisenburger, Addy, Hughes, & Shellis, 2001). In contrast, in our study we used clarified human saliva, which is not as supersaturated as the artificial saliva in relation to the enamel surface. Moreover, it contains proteins that will also influence on the process of des-/remineralization. In situ studies have shown that 1 h of exposure to saliva before performing toothbrush abrasion had a protective effect compared to no exposure to saliva (Attin, Buchalla et al., 2001; Attin, Knöfel et al., 2001; Jaeggi & Lussi, 1999). Moreover, 2 h of exposure to saliva in situ was shown to re-harden the softened enamel (Alencar et al., 2016; Mendonça et al., 2017). However, another study 4
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epidemiological study performed in seven European countries showed a prevalence of tooth wear of 29.4%, but did not find evidence that waiting after breakfast for toothbrushing affects the degree of tooth wear (Bartlett et al., 2013). In view of the above, it can be concluded that the period of exposure to human saliva after an erosive challenge and before toothbrush abrasion does not prevent enamel erosive wear in vitro.
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Authors’ contributions Raquel Marianna Lopes: Conception and design of the study, acquisition of data and manuscript drafting Juliana Sant`Anna da Silva: preparation of specimens, collected human saliva from donors, worked on the days of cycling and manuscript review Samira Helena João- Souza: collected human saliva from donors, worked on the days of cycling and manuscript drafting Vinicius Maximiano: worked on the days of cycling and manuscript review Alana Machado: worked on the days of cycling and manuscript review Tais Scaramucci: Data analysis and manuscript review Ana Cecília Corrêa Aranha: Conception and design of the study and manuscript review Funding This work was supported by the São Paulo Research Foundation (FAPESP) [grant numbers FAPESP 2015:15629-7 to R.M.L. and FAPESP 2017:14085-9 to J.S.A.S.]. Declaration of Competing Interest None. References Alencar, C. R., Mendonça, F. L., Guerrini, L. B., Jordão, M. C., Oliveira, G. C., Honório, H. M., et al. (2016). Effect of different salivary exposure times on the rehardening of acid-softened enamel. Brazilian Oral Research, 30, e104. https://doi.org/10.1590/ 1807-3107BOR-2016.vol30.0104. Amaechi, B. T., Higham, S. M., Edgar, W. M., & Milosevic, A. (1999). Thickness of acquired salivary pellicle as a determinant of the sites of dental erosion. Journal of Dental Research, 78(December 12), 1821–1828 PubMed PMID: 10598912. Attin, T., Buchalla, W., & Putz, B. (2001). In vitro evaluation of different remineralization periods in improving the resistance of previously eroded bovine dentine against tooth-brushing abrasion. Archives of Oral Biology, 46, 871–874. https://doi.org/10. 1016/S0003-9969(01)00039-5. Attin, T., Buchalla, W., Gollner, & Hellwig, E. (2000). Use of variable remineralization periods to improve the abrasion resistance of previously eroded enamel. Caries Research, 34, 48–52. https://doi.org/10.1159/000016569. Attin, T., Knöfel, S., Buchalla, W., & Tütüncü, R. (2001). In situ evaluation of different remineralization periods to decrease brushing abrasion of demineralized enamel. Caries Research, 35, 216–222. https://doi.org/10.1159/000047459. Bartlett, D. W., Lussi, A., West, N. X., Bouchard, P., Sanz, M., & Bourgeois, D. (2013). Prevalence of tooth wear on buccal and lingual surfaces and possible risk factors in young european adults. Journal of Dentistry, 41, 1007–1013. https://doi.org/10.
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