journal of dentistry 42 (2014) 1502–1507

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In situ effect of a commercial CPP-ACP chewing gum on the human enamel initial erosion Catarina Ribeiro Barros de Alencar a, Ana Carolina Magalha˜es b,1, Maria Aparecida de Andrade Moreira Machado a, Thais Marchini de Oliveira a, Heitor Marques Hono´rio a, Daniela Rios a,* a Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of Sa˜o Paulo, Alameda Dr. Octa´vio Pinheiro Brisolla, 9-75, Bauru/SP – PO Box 73, 17012-101, Brazil b Department of Biological Sciences, Bauru School of Dentistry, University of Sa˜o Paulo, Alameda Dr. Octa´vio Pinheiro Brisolla, 9-75, Bauru/SP – PO Box 73, 17012-101, Brazil

article info

abstract

Article history:

Objective: This study evaluated the in situ rehardening effect of a commercial chewing gum

Received 2 June 2014

containing casein phosphopeptide – amorphous calcium phosphate (CPP-ACP) on initial

Received in revised form

erosion lesions.

13 August 2014

Methods: Seventy-two human enamel blocks, after selection (initial surface hardness – SHi)

Accepted 15 August 2014

and in vitro short-term acidic exposure (cola drink for 3 min – SHd) were randomly assigned to three groups. The factors under study were treatment (3 levels: GI chewing gum with CPPACP, GII chewing gum without CPP-ACP and GIII control group without gum) and intraoral

Keywords:

period (2 levels: 2 and 24 h). Twelve volunteers wore intraoral palatal devices for 24 h in 3

Tooth erosion

crossover phases. On each phase, after 2 h the surface hardness was assessed (SHf1) and the

Dental enamel

blocks were reinserted and the devices were used for additional 22 h (SHf2). In phases of GI

Chewing gum

and GII volunteers chewed the respective gum during 30 min, for 4 times with an interval of

Casein phosphopeptide-amorphous

4 h. Percentage of surface hardness recovery (%SHR) was calculated after 2 and 24 h. The

calcium phosphate

data were analysed by repeated measures ANOVA and Tukey’s test. Results: Chewing gum with CPP-ACP (2 h = 50.0% < 24 h = 95.9%) showed higher hardness recovery than chewing gum without CPP-ACP (2 h = 30.0% < 24 h = 71.1%) and control (2 h = 15.7% < 24 h = 40.9%) ( p < 0.05). Conclusions: The results suggest that saliva increased hardness of softened enamel after the use of conventional chewing gum (GII) and this effect was enhanced by the prolonged intraoral period (24 h) and by the use of CPP-ACP chewing gum (GI). Clinical significance: Since chewing gum is an alternative to enhance salivary defenses after erosive challenges, CPP-ACP chewing gum might be a supplementary strategy to potentiate the mineral precipitation of initial erosion lesions. # 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +55 14 32358218; fax: +55 14 32271486. E-mail addresses: [email protected] (C.R.B. de Alencar), [email protected] (A.C. Magalha˜es), [email protected] (M.A. de Andrade Moreira Machado), [email protected] (T.M. de Oliveira), [email protected] (H.M. Hono´rio), [email protected], [email protected] (D. Rios). 1 Tel.: +55 14 32358346; fax: +55 14 32271486. http://dx.doi.org/10.1016/j.jdent.2014.08.008 0300-5712/# 2014 Elsevier Ltd. All rights reserved.

journal of dentistry 42 (2014) 1502–1507

1.

Introduction

Erosive substance loss is a dynamic process involving acid demineralization and saliva remineralization. The initial stage of erosion lesion is characterised by enamel softening, which occurs as a result of partial demineralization of tooth surface. At this stage of the process, the remineralization of the lesion is still possible,1,2 since the remaining tissue may act as a scaffold for replacement of minerals lost.3 However, the softened enamel is highly susceptible to removal by abrasive forces and in case of wear of the demineralised enamel the lesion is not liable to recovery.1,2,4 Saliva is recognised as an important biological factor in minimizing enamel wear in erosive/abrasive attack.5 It is considered that calcium, phosphate and fluoride contained in saliva can assume the repairing effect of initial enamel lesions.6 In addition, the stimulation of salivary flow rate can enhance this salivary remineralizing effect by an increase in bicarbonate buffer and salivary mineral content, which can facilitate mineral redeposition onto the enamel surface, decreasing enamel loss.7 Accordingly, previous investigation has showed that saliva stimulation by the use of sugar-free chewing gum promoted a reduction of human and bovine enamel loss when enamel was subjected to erosive/abrasive challenge.8 A commercial chewing gum (Trident Total1, Kraft Foods/ Cadbury Adams, Bauru, SP, Brazil) containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP, patented as Recaldent1) is available on the market. CPP-ACP is a milk derived protein that has proposed to inhibit demineralization and enhance remineralization of enamel carious lesion.9–11 The main function of casein phosphopeptides (CPP) is to modulate the bioavailability of calcium phosphate levels, preserving them in an amorphous or soluble form termed as amorphous calcium phosphate (ACP)12 which maintains ionic phosphate and calcium in supersaturation.13 The ability of the CPP-ACP added to sugar-free chewing gum to remineralise enamel sub-surface lesions has been demonstrated on a clinical trial14 and several randomised and controlled in situ studies.9–11,15,16 The effect of CPP-ACP on erosive lesions has traditionally been evaluated through its incorporation into pastes or creams and the results are controversial. Some studies showed CPP-ACP preventive ability against erosion17–20, while

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others found no effect.21–23 There is only one study testing the effect of chewing gum containing CPP-ACP that showed improvement on the mineral precipitation of eroded bovine enamel.24 However this study used bovine enamel and 24 h of remineralization, which does not simulate the clinical interval of salivary exposure between erosive attacks. Therefore this in situ study was designed to evaluate the CPP-ACP chewing gum effect on initial erosive lesions of human enamel, on two different periods of salivary exposure (2 h and 24 h). It was hypothesised that CPP-ACP chewing gum would improve the remineralization ability of saliva when compared to the stimulated flow rate by a conventional chewing gum.

2.

Materials and methods

2.1.

Experimental design

The study was conducted with a single-blind and randomised protocol of three-way crossover phases of 24 h with an interval of one week between them. The factors under evaluation were treatment at three levels (GI – Sugar free chewing gum with CPP-ACP, GII – Conventional sugar free chewing gum, without CPP-ACP and GIII – negative control group, without chewing gum) and intraoral remineralization period at two levels (2 h and 24 h). The main components of the chewing gum evaluated are described in Table 1. Human enamel blocks with artificial initial erosion lesion (n = 72) were randomly divided into the volunteers (n = 12) that wore palatal appliances during 24 nonconsecutive hours in each one of the 3 phases. On each phase, after 2 h the devices were removed for surface hardness evaluation (SHf1), thereafter they were reinserted and used for additional 22 h, when hardness was reassessed (SHf2). In phases of GI and GII volunteers chewed the respective gum during 30 min, for 4 times with an interval of 4 h. Thus, hardness evaluation was performed on the same block after 2 h (1  30 min chewing gum) and 24 h (4  30 min chewing gum) of intraoral maintenance. Percentage of surface hardness recovery (%SHR) was calculated after 2 and 24 h.

2.2.

Enamel samples preparation

Enamel blocks (4  4  3 mm) were obtained from unerupted human third molars recently extracted, kept in 0.1% thymol

Table 1 – Main components of the chewing gum evaluated, according to manufacturers information. Trident total1 (CPP-ACP)a

Trident fresh1 (without CPP-ACP)a

Gum base Sweeteners: Sorbitol, manitol, maltitol, aspartame, acesulfame-k and sucralose Flavouring agents: Humectant: Glycerin, triacetin Emulsifier: Soylecithin Dyes: Titanium dioxide, brilliant blue fcf

Gum base Sweeteners: Sorbitol, manitol, maltitol, xylitol, aspartame, acesulfame-k and sucralose Flavouring agents: Humectant: Glycerin Emulsifier: Soylecithin Dyes: Titanium dioxide, patent blue V, anthocyanins, brilliant blue fcf

Thickener: Gum Arabic Glazing agent: Carnauba wax Casein phosphopeptide-amorphous calcium phosphate (Recaldent1) a

Manufactured by Trident, Cadbury Adams Indu´stria e Come´rcio, Bauru, Sa˜o Paulo, Brazil.

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journal of dentistry 42 (2014) 1502–1507

solution at pH 7.0. The blocks were cut using a ISOMET low speed saw cutting machine (Buehler Ltd., Lake Bluff, IL, USA) with two diamond disks (Extec Corp., Enfield, CT, USA), which were separated by a 4-mm thickness spacer. The blocks’ surfaces were ground flat with water-cooled silicon carbide discs (320, 600, and 1200 grade papers; Buehler, Lake Bluff, IL, USA) and polished with felt paper wet by diamond spray (1 mm; Buehler, Ltd., Lake Bluff, IL, USA). The blocks were cleaned using an ultrasonic device for 10 min and checked regarding the presence of white spots and cracks using a microscope (40). The baseline surface hardness (SHi) was determined using the average values of five indentations performed at distances of 100 mm from each other (Knoop diamond, 25 g, 10 s, Hardness tester from Buehler, US). Blocks with mean SHi of 344.2 (16.0 KHN) were sterilised by exposure to ethylene oxide gas.

2.3.

Initial erosion lesion

The enamel blocks were demineralised in vitro by immersion in cola drink (Coca Cola1 Ribeira˜o Preto, Brazil, pH 2.4, 0.32 ppm F) for 3 min, under agitation (Flatbed oscillator, 60 rpm) at 25 8C (17.6 ml per block). Next, the surface hardness was measured again (SHd = mean of 5 indentations taken 50 mm below the initial) and 72 enamel blocks were selected (mean of 221.3  14.3 KHN) for randomization among 12 volunteers and three groups of treatments. The mean surface hardness of each group was GI/221.1  13.6 KHN, GII/ 220.4  15.9 KHN, and GIII/222.5  13.8 KHN.

2.4.

toothpaste (Total 12, 1.100 ppm F, Colgate, Brazil) 3 times a day. The volunteers were warned to not use any other fluoride product. The volunteers wore the acrylic palatal appliance containing 2 eroded human enamel samples during 24 nonconsecutive hours in each phase. In the intraoral phase of Groups I and II, immediately after the insertion of the appliance, volunteers were requested to use a unit of the chewing gum for 30 min. The volunteers were trained to chew the gum between the posterior teeth, ensuring that the gum did not get in contact with the appliance and blocks. After 2 h (30 min of chewing gum + 90 min intraoral maintenance) the blocks were removed from the appliances and the surface hardness was measured (SHf1 – mean of five indentations with 50 mm of distance in relation to indentations made after demineralization). The same blocks were reinserted and the devices used for additional 22 h (+3 cycles of 30 min chewing gum with an interval of 4 h and 10 h for overnight – Groups I and II). Thereafter, the blocks were removed from the appliances for surface hardness reassessment (SHf2 – mean of five indentations with 100 mm of distance in relation to indentations made after demineralization). To confirm the absence of removal of the softened enamel by the tongue abrasion, the indentations length of SHd (surface hardness after initial erosion lesion) were re-checked after the in situ phase. Considering the values of the indentation length (L), the depth of indentation impression was calculated according to the equation: D = L/2.tana, where a = 3.75 deg.25,26 The mean depth (SD) of indentations of the studied groups before and after the in situ phase were respectively: 1.27 (0.05) mm and 1.26 (0.05) mm.

Volunteers and in situ phase 2.5.

Ethical approval for the study was granted by the local Institutional Ethics Committee (protocol no 169/2011). This study was conducted in full accordance with the Declaration of Helsinki. Twelve healthy adult subjects (ten female and two males) with an average age of 27.2 years (range 23–38 years) who respected the inclusion criteria (residing in the same fluoridated area (0.70 mg F/l), physiological stimulated salivary flow rate (>1 ml/min), adequate oral health – with no caries or erosion lesions) without violating the exclusion criteria (systemic illness, pregnancy or breastfeeding, under orthodontic intervention, use of fluoride compounds in the last two months) took part in this study. Sample size calculation was based on a previous in situ study.24 A sample size of 10 volunteers was estimated based on a a-error of 5%, b-error of 20%, 8.7 as estimated standard deviation and 15.0 as minimum detectable difference in means. Considering possible losses inherent to in situ studies, 12 volunteers were selected. The intraoral palatal devices were made of the upper arch for each volunteer with acrylic resin on the plaster model. Each intraoral device had two vertical rows, one on the right and the other on the left side, with one cavity (6  6  3 mm) in each side, for enamel blocks fixation. The samples were fixed with wax and were carefully adapted to the level of the resin surface of the appliance, in order to avoid dental plaque accumulation. Seven days prior to and during the experiment period, the volunteers brushed their teeth with standardised fluoride

Percentage of surface hardness recovery (SHR)

Mean values obtained from initial hardness (shi), after demineralization (shd) and remineralization periods (SHf1 and SHf2) were used to calculate the percentage of surface hardness recovery (% SHR = ([shf shd]/[shi shd])  100).

2.6.

Statistical analysis

Statistical analysis was performed with SigmaPlot version 12.3 (2011 Systat Software, Germany). The assumption of equality of variances were satisfied and Shapiro–Wilk test checked the normal distribution ( p > 0.05). Then after, Repeated Measures ANOVA followed by Tukey’s test were applied, considering treatment as independent variable and both intraoral remineralization periods the dependent variables (repeated measures: 2 h and 24 h). The significant limit was set at 5%.

3.

Results

All twelve volunteers completed the in situ protocol and no side effects were reported. There was a statistical significant difference for factors under study (treatment and period) and no interaction between (Table 2). The effect of both chewing gums contributed to higher surface hardness recovery compared to the control group (no chewing gum), whereas the highest recovery was found when the CPP-ACP containing chewing gum was used. For all studied

journal of dentistry 42 (2014) 1502–1507

Table 2 – Means and standard deviation values of percentage of surface hardness recovery (%SHR) for the experimental groups for different in situ remineralization periods (n = 12). Experimental groups GI (CPP-ACP) GII (without CPP-ACP) GIII (no chewing gum)

2 h %SHR (SD)

24 h %SHR (SD)

50.04 (6.54) a 30.30 (3.43) c 15.70 (3.90) e

95.86 (3.54) b 71.07 (8.04) d 40.93 (5.14) f

Groups whose means are followed by distinct letters differ significantly. (Repeated measures ANOVA/Tukey’s test, p < 0.0001).

groups, 24 h of remineralization provided greater hardness recovery compared to 2 h.

4.

Discussion

In an attempt to prevent erosive wear of dental enamel, due to difficulties on removing the etiological factors of dental erosion, different treatments have been proposed and studied.27–33 The common feature of all these treatment proposals is the search for an effective mechanism of action, able to increase remineralization of softened enamel layer and to promote enamel resistance against the effect of erosive acids, mainly by the formation of a mechanical barrier (reducing demineralization). CPP-ACP nanocomplexes were developed to deliver high concentrations of bioavailable calcium and phosphate ions intraorally, inhibiting demineralization and promoting remineralization of white spot carious lesions.34 It is accepted that the mechanism by which CPP-ACP reduces enamel dental caries might be different from dental erosion, due to the different structural characteristics of the lesions. In the white spot carious lesions, the surface layer covering the subsurface lesion is a relatively intact mineral-rich and porous layer. These pores are at least partly filled with organic materials, which provide the passage of the external solutions or agents to maintain contact with the deeper tissues.35 On the other hand, on erosive lesions, the process of remineralization seems to involve deposition of mineral into the porous zone, rather than crystal regrowth.17,36 Therefore, it can be conceivable that antierosion materials directly react with the eroded tooth surface, promoting ‘surface-only’ remineralization.22 Considering these aspects, the present study evaluated the use of a chewing gum with CPP-ACP as an attempt to understand the remineralizing effect of CPP-ACP on initial erosion lesions. In early stages of erosion, calcium dissolution typically results in enamel surface softening,37,38 which can be measured through the substrate resistance to the pressure exerted by a Knoop and Vickers indenter. However, the first indenter is more sensitive to changes in the superficial layer of the erosive lesion39 and for this reason was utilised. An in vitro protocol to produce initial erosive lesions was determined through a pilot study in which the surface hardness of the blocks was evaluated at every minute of exposure in cola drink. The data showed that up to 3 min the surface hardness decreased and above this time, there was a slight increase in the hardness value, suggesting that the softened surface layer had been partially lost and the

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measurement of the hardness was being conducted in an underlying layer. Furthermore, the difficulty of viewing the baseline indentations after 3 min of immersion confirmed the enamel loss, determining the optimum time for the development of initial erosion lesions. In a recent in situ study it was adopted the exposure protocol of 2 min in Sprite Light1 (Coca Cola Company, Atlanta, GA), as representative of an episode of rapid consumption of erosive beverage.23 The results showed that mechanical and gustatory salivary stimulation with chewing gum, increased hardness of the eroded layer, which could be a signal of enamel remineralization. The salivary stimulation might have increased saliva production, especially of the parotid gland, which resulted in higher amount of calcium and phosphate ions available for precipitation onto enamel.7,40 Another hypothesis for the hardness recovery is the remove of the softened enamel by the tongue abrasion, reaching a harder surface when compared to the eroded one. Gregg et al.41 and Vieira et al.42 showed that the tongue exert an abrasive effect on enamel softened by erosion, thereby partly removing the softened layer. However it is important to point out that those studies used protocols to simulate a very severe abrasive challenge, which is not the case of the present study. On our study, to diminish the possibility of enamel wear by tongue friction, the volunteers were advised to not touch the blocks with the tongue. In addition, the mean depth of all indentations impression done on the initial erosion lesion was similar to the mean depth of these indentations measured after the in situ phase of 24 h, showing that the surface was almost the same. Thus, this hypothesis was rejected. The first intraoral remineralization period was designed to reflect a more realistic condition, where after an erosive challenge the dental tissue would be exposed to the remineralizing effect of saliva for 2 h, until the occurrence of a new acid attack. At this interval the volunteers chewed only one tablet containing CPP-ACP and this treatment resulted on 50% of hardness recovery, which was higher than the recovery of the conventional chewing gum treatment (30%). These findings are in accordance with several studies that have used other vehicles to CPP-ACP delivery in order to promote an increase in enamel surface hardness of erosive lesions.17–19,43 In a previous in situ study, using bovine enamel, even after the volunteers chewed a new gum 3 times for 30 min each, during 24 h, the hardness recovery was about 29%. The main difference between the studies was the type of enamel since the human enamel used in the present study might be more prone to mineral precipitation. However this hypothesis must be confirmed on future researches. Although not capable of simulating clinical conditions, the intraoral period of 24 h was evaluated as a strategy for assessing the maximum rehardening potential of CPP-ACP contained in chewing gum. The results showed that after 4 times of 30 min use of CPP-ACP gum, in the absence of new erosive challenges and/or mechanical abrasion, there was almost entirely enamel hardness recovery, with baseline microhardness being achieved. The rehardening ability of the CPP-ACP gum treatment was 25% and 58% higher than the effect of conventional gum (without CPP-ACP) and saliva, respectively. The results of the present study suggest that CPP-ACP chewing gum can benefice patients at risk of dental erosion by

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rehardening initial erosion lesions. However, before extrapolating this therapy into clinical situation the inhibitor effect of CPP-ACP chewing gum against enamel demineralization and also its ability to prevent enamel loss when there is erosive challenge should be further tested.

5.

Conclusions

Saliva rehardening effect was increased by the use of conventional chewing gum and by prolonged period of remineralization. In addition, the presence of CPP-ACP in the chewing gum promoted the highest rehardening ability. Future studies are required to evaluate the effect of this preventive strategy on the inhibition of erosive enamel loss.

Acknowledgements The authors would like to gratefully acknowledge all the volunteers who participated in this study and FAPESP for the financial support (Proc. no. 2011/16326-7).

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In situ effect of a commercial CPP-ACP chewing gum on the human enamel initial erosion.

This study evaluated the in situ rehardening effect of a commercial chewing gum containing casein phosphopeptide - amorphous calcium phosphate (CPP-AC...
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