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Influence of Dental Plaque on Human Enamel In Situ/Ex Vivo Study
pyrig ORIGINALoARTICLE N ot C for Pu bli cat ion Erosion: te ss e n c e
Heitor Marques Honórioa/Daniela Riosb/Carlos Ferreira Santosc/ Marília Afonso Rabelo Buzalaf c/Maria Aparecida de Andrade Moreira Machadob
Purpose: The objective of the present in situ study was to evaluate the influence of dental plaque on human enamel erosion. Materials and Methods: Thirteen volunteers wore acrylic palatal devices with four enamel specimens that were prepared from freshly extracted impacted human third permanent molars (4 · 4 mm), randomly selected and distributed into two vertical rows, corresponding to the following groups: GI, erosion of dental plaque-free samples, and GII, erosion of dental plaque-covered samples. For the formation of dental plaque, the specimens were placed 1 mm below the level of the appliance and covered with a plastic mesh to allow the accumulation of dental plaque. The palatal device was continuously worn by the volunteers for 14 consecutive days and then immersed in a soft drink (Coca-Cola, 150 ml) for 5 min, three times a day. Half of the surfaces of specimens were coated with nail varnish for profilometry tests. The study variables included the depth of enamel surface wear (profilometer, vertical ranges in lm) and the percentage of superficial microhardness change (%SMHC). Data were analysed using the t test (P < 0.05). Results: The %SMHC and depth of enamel surface wear were significantly higher for GI (-87.82% ± 3.66 and 4.70 lm ± 1.65) than for GII (-13.79% ± 4.22 and 0.14 lm ± 0.03). Conclusions: It was concluded that the dental plaque formed in situ was able to protect the enamel surface against erosion by a cola soft drink, thus reducing the depth of enamel surface wear and the %SMHC. Key words: dental erosion, dental plaque, dental wear, enamel, soft drinks Oral Health Prev Dent 2010; 8: 179–184.
ental erosion is the loss of surface tooth structure due to the action of acids of gastric or dietary origin, without the influence of microorganisms
D a
Department of Clinic and Surgery (Pediatric Dentistry), Alfenas Federal University, Gabriel Monteiro da Silva Street, 714, 37130000 Alfenas-MG, Brazil.
b
Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, USP—University of São Paulo, Al. Octávio Pinheiro Brisolla, 9-75, 17012-901 Bauru-SP, Brazil.
c
Department of Biological Sciences, Bauru School of Dentistry, USP—University of São Paulo, Al. Octávio Pinheiro Brisolla, 9-75, 17012-901 Bauru-SP, Brazil.
Correspondence: Heitor Marques Honório, Department of Pediatric Dentistry, Alfenas Federal University, Gabriel Monteiro da Silva Street, 714, 37130-000 Alfenas-MG, Brazil. Tel: +55 35 32991424. Email:
[email protected]
Vol 8, No 2, 2010
Submitted for publication: 05.05.08; accepted for publication: 06.03.09.
(Imfeld, 1996; Meurman and Ten Cate, 1996; Zero, 1996; Lussi et al, 2004; Lussi, 2006). This demineralisation dissolves the surface enamel prisms without formation of a subsurface lesion, thus being different from dental caries (Lussi et al, 1993; Meurman and Ten Cate, 1996; Amaechi and Higham, 2001). As this demineralising action is local and superficial, evidence in the literature shows the importance of physical barriers to protect the tooth structure against the acidic action of erosive agents (Skjørland et al, 1995; Amaechi et al, 1999; Hannig and Balz, 1999, 2001; Nekrashevych and Stösser, 2003; Hannig et al, 2004; Hara et al, 2006). The acquired pellicle is a physical barrier that protects the tooth against erosive attacks; it is composed of a protein layer that is formed on the tooth surface (Hara et al, 2006), acting as a diffusion 179
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The present in situ study was approved by the Research and Ethics Committee of the Bauru School
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barrier or permeability membrane. This selective barrier prevents the direct contact between acids and the tooth surface, thus reducing the dissolution of hydroxyapatite. Protection of the tooth surface by the acquired pellicle has been well established in the literature and has been demonstrated by several in vitro and in situ studies (Skjørland et al, 1995; Amaechi et al, 1999; Hannig and Balz, 1999, 2001; Nekrashevych and Stösser, 2003; Hannig et al, 2004; Hara et al, 2006). Clinically, dental erosion is more frequently evident on the palatal surfaces of maxillary teeth and less on the other surfaces (Järvinen et al, 1992; Amaechi et al, 1999). Johansson et al (2002) showed that dental plaque accumulation on the palatal surfaces of maxillary anterior teeth was lower in high-erosion subjects when compared with low-erosion groups. Some authors have mentioned that the dental plaque might also act as a mechanical barrier providing additional protection to the dental enamel (Lussi et al, 2004; Cheung et al, 2005; Hara et al, 2006). Dental plaque could also have a buffering capacity that is mainly provided by macromolecules of bacterial cell walls and dental plaque matrix and to a lesser extent by extracellular buffers (Shellis and Dibdin, 1988). However, few studies have investigated the association of erosion with the presence of dental plaque (Cheung et al, 2005). This may be connected with the definition of erosion itself, in which there is loss of tooth mineral without the influence of microorganisms (Ten Cate and Imfeld, 1996; Zero, 1996; Lussi, 2006). Owing to this reason, it was reported in most of the studies that the dental plaque was removed when an erosion situation was simulated (Hara et al, 2003; Rios et al, 2006). However, in daily clinical conditions, the dental plaque may be present on the dental enamel during the acidic action of an erosive beverage, and its influence on dental erosion is not totally clarified. Thus, the present in situ study aimed to evaluate the influence of dental plaque formed from the erosive attack of a cola soft drink on the percentage of superficial microhardness change (%SMHC) and wear of human enamel when subjected to the erosive action of this acidic beverage.
pyrig No Co t fo r PNo. 94/ of Dentistry, University of São Paulo (Proc. ub one 2005). The present study was performed in lica phase of 14 consecutive days. The factor under eval-tion te on dental uation was the influence of dental plaque ss e n c e
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erosion. Thirteen volunteers wore acrylic palatal appliances, each containing four dental enamel specimens. The groups under study were: GI, erosive challenge only, and GII, erosive challenge in the presence of dental plaque. The enamel specimens of GI were subjected to erosion and the erosive agent was a cola soft drink. On GII, the cola soft drink was used as an erosive agent and also as a substrate for dental plaque formation. The study variables investigated were the depth of enamel surface wear (lm) and the %SMHC (Fig 1).
Preparation of enamel specimens Enamel specimens (4 · 4 mm) were prepared from freshly extracted impacted human third permanent molars (Figs 1A–D) that were stored and sterilised in a 2% formaldehyde solution, pH 7.0, for 30 days at room temperature. Enamel surfaces of the specimens were ground flat with water-cooled carborundum discs (320, 600 and 1200 grades of Al2O3 papers; Buehler Ltd, Lake Bluff, IL, USA), and polished with felt paper wet by diamond spray (1 lm; Buehler Ltd), resulting in the removal of about 100 lm depth of the enamel, which was controlled with a micrometer (Figs 1E and F). A surface Knoop microhardness test was performed (five indentations in different regions of the specimens, 25 g, 5 s, HMV2000; Shimadzu Corporation, Tokyo, Japan) to select 52 human enamel specimens (Fig 1G). The specimens with a mean superficial microhardness of around 356 ± 20 Knoop hardness number were randomly divided into two groups. To maintain reference surfaces for the determination of lesion depth, two layers of nail varnish were applied on half of the surface of each specimen (Fig 1H).
Palatal device preparation Custom-made acrylic palatal devices were made with four cavities (5 · 5 · 4 mm), two in each side of the appliance (left and right). One enamel specimen was randomly assigned to each of the four sites and fixed with wax. The position of each group in the device was randomly determined for each volunteer; however, there was a difference in the preparation of cavities of GII compared with GI. For GII, a 4-mm deep space was created in the acrylic palatal appliance, Oral Health & Preventive Dentistry
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Fig 1 Flow chart illustrating the experimental design.
leaving a 1.5-mm space for dental plaque accumulation. A plastic mesh was fixed in the acrylic surface above the enamel specimens to protect from mechanical disturbance (Fig 1I). In GI, the specimens were fixed at the same level as the appliance without the plastic mesh.
Intraoral phase Throughout the experimental phase, the volunteers brushed their teeth with a fluoride-containing dentifrice (1100 ppm F as NaF, pH 6.8; Crest, USA). The palatal device was worn for one phase of 14 consecutive days (Fig 1J). One day before the experimental phase, the device was worn and the specimens were not subjected to erosive challenge, to allow the formation of a salivary pellicle (Hara et al, 2003). During the following 14 days, erosive challenges were offered extraorally three times a day (7 am, noon and 6 pm; Fig 1K). In each erosive challenge, the device was immersed into a cup containing 150 ml of a freshly opened bottle of a cola soft drink (Coca-Cola, Companhia Fluminense de Refrigerantes, Porto Vol 8, No 2, 2010
Real, Rio de Janeiro, Brazil) for 5 min (Fig 1K). The treatment performed in GI and GII was exactly the same. The only difference between GI and GII was the space that was created in the acrylic palatal appliances. Specimens from GI were fixed at the same level as the appliance, whereas for those from GII a 1.5-mm space was left for dental plaque accumulation. Taking this aspect into consideration, the cola soft drink was used just as an erosive agent in GI, whereas this acidic beverage was used as the erosive agent and also as the substrate for dental plaque formation in GII. The volunteers were instructed to wear the appliances continuously, even during the night, but to remove them during mealtime (three times a day), during the aforementioned procedures or when drinking water. Oral hygiene practice was followed exactly after mealtime when the appliance was removed from the mouth.
Mean mass of dental plaque formed After the study period, the dental plaque was collected with plastic spatulas and weighed in 181
Wear (lm)*
a
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-87.82 ± 3.66 -13.79 ± 4.22b
4.70 ± 1.65 0.14 ± 0.03d
*Means followed by distinct letters are significantly different (P < 0.05).
microcentrifuge tubes on a precision analytical scale for calculating the mean mass of dental plaque accumulated on the specimens of GII.
Microhardness analysis By the end of the 14th day, the volunteers stopped wearing the palatal devices. The specimens were removed from the device, and the nail varnish over the reference surfaces was carefully cleansed with acetone-soaked cotton wool. Surface microhardness of the enamel specimens was measured again using a microhardness tester (HMV-2000) with a Knoop diamond under a 25-g load for 5 s. Ten indentations were made on each specimen, five on the previously protected enamel surface (control), which was unaffected by the experimental period (SMH), and five on the experimental areas (SMH1). The %SMHC was calculated (%SMHC = [SMH1-SMH]/SMH] · 100) (Fig 1L).
Wear analysis The enamel wear was determined with respect to the reference surfaces, by profilometry (Hommel Tester T 1000, Hommelwerke, VS, Schwenningen, Germany). Five readings were taken on each specimen. These profilometric traces were taken from the reference surface, crossing the exposed surface. The average wear depth of an experimental unit was computed using the 10 readings: two specimens · five readings (Figs 1M and N).
Statistical analysis The assumptions of equality of variances and normal distribution of errors were checked for the tested variables. Since the assumptions were satisfied, the t test (P < 0.05) was performed. The analyses 182
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% SMHC ± SD*
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Table 1 Results of t test (P < 0.05) with dependent variables % SMHC and wear profile (lm) for the study groups.
pyrig No Co t fo r P version were performed using the SAS System u 6.11 software (SAS Institute, Cary, NC, USA).blic ati on te ss e n c e
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The results showed a mean dental plaque accumulation of 30.95 ± 10.49 mg on the specimens of GII. No dental plaque was observed on the specimens of GI, as these were at the same level as the appliance. Before the calculation of wear and final surface microhardness of specimens, the aspect was visually analysed. The specimens of GI exhibited a loss of surface shine and polishing and an apparent increase in roughness. After dental plaque removal, the specimens of GII had the same aspect exhibited at baseline, with full maintenance of the surface shine. Finally, analysis of the data using the t test reported higher %SMHC and wear for GI (-87.82% and 4.70 lm) than for GII (-13.79% and 0.14 lm) (Table 1).
DISCUSSION Erosive agents are characterised by their high subsaturation of calcium, phosphate and fluoride compared with hydroxyapatite and fluorhydroxyapatite (Barbour et al, 2003; Lussi et al, 2004; Lussi, 2006). This leads to superficial loss of dental structure and adjacent superficial demineralisation (Lussi et al, 1993; Meurman and Ten Cate, 1996; Amaechi and Higham, 2001). Thus, the results observed for specimens of GI are in accordance with those in the literature under similar situations (Rios et al, 2006), as a high percentage of loss of surface microhardness was observed, with significant wear. This group was not protected by plastic mesh, and the tongue could have exerted an abrasive effect, as a palatal device was used. However, Gregg et al (2004) showed that the chemical action of acidic fluids on tooth surfaces clearly must be the dominant factor, with the action of the tongue exerting a secondary abrasive localised effect. Moreover, the volunteers were advised not to touch the tongue on the blocks. Only 13.79% loss of surface microhardness and nearly no wear (0.14 lm) were observed in the specimens of GII. The mean amount of dental plaque formed during the 14-day experimental period was calculated to be 30.95 mg. This layer of dental plaque may have acted as a physical barrier to minimise the erosive effects. The results found for GII are in accordance with those in the literature. If the Oral Health & Preventive Dentistry
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acquired pellicle, whose thickness ranges from 0.5 to 1 lm (Amaechi et al, 1999), may act as a physical barrier in vitro providing a protection ranging from 30% to 100% against demineralisation, what might be expected from a 1-mm thick, 30 mg barrier? According to the results of the present study, the protection provided by the dental plaque against the erosive action of the acidic beverage may have been high. Additionally, the protective effect of dental plaque may also be due to its buffering capacity (Shellis and Dibdin, 1988) and to its ability to store large quantities of calcium, phosphate and fluoride ions both from saliva and from dissolution products of tooth mineral (Cheung et al, 2005). A previous study also reported a protective effect of dental plaque, but a higher value of dental wear (60 lm) for the specimens with dental plaque was observed (Cheung et al, 2005). This is certainly due to the different methodology used. A major difference between the two studies was the localisation of the specimens (palatal versus buccal) and the amount of dental plaque formed. In the study carried out by Cheung et al (2005), dental plaque was exposed to normal mechanical wear, as the specimens were not recessed. Furthermore, in the present study, the total erosive exposure during the experimental phase was much lower (210 min versus 1500 min), the beverage used was different (cola soft drink versus dry white wine) and a longer daily remineralisation was allowed (approximately 21 h versus 8 h). On the other hand, in the study carried out by Cheung et al (2005), the length of the experimental period was lower (10 days versus 14 days), and the dental plaque was allowed to accumulate on the specimens before the erosive challenge (growth starting 3 days earlier versus dental plaque growth during the experimental phase). In the present study, the accumulation of dental plaque along with the erosive challenge is probably causing more erosion to take place, at least during the first days of the study period. The low %SMHC (13.79%) in GII might then be due to the occurrence of erosion during the first days of the study period (when the dental plaque was not yet formed) or to the occurrence of a slight demineralisation caused by the influence of microorganisms in the dental plaque in response to the cariogenic challenge posed by the carbohydrate-containing acidic beverage (Martin-Villa et al, 1981; Birkhed, 1984). The present data showed that the in situ dental plaque protected the enamel surface against erosion by a cola soft drink. These results are important to understand and testify the clinical results of Johansson et al (2002), who pointed out that the formation of
pyr Co etigal No Honório t fo rofPdental dental plaque interferes with the occurrence ub erosion. However, the present results have to be interlica preted with caution, as the data of the present study tion ess not do not indicate or suggest that patients tshould e nc e brush their teeth to preserve the protecting dental plaque layer against erosive challenges. Further investigations are necessary to properly address this point.
ACKNOWLEDGEMENTS The authors gratefully acknowledge all the volunteers who participated in the present study and also José Roberto Pereira Lauris, Associate Professor of the Bauru School of Dentistry, for the statistical analysis. The present study was supported by CAPES.
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