Abstract
Objectives
Aim of this investigation was to study the temperature-dependent in vitro enamel erosion of five acidic drinks and citric acid under controlled conditions in an artificial mouth.
Methods
The erosive potential of Orange juice, Coca-Cola Zero, Sprite Zero, two fruit teas and citric acid (control) was investigated on bovine enamel specimens at temperatures between 5 °C and 55 °C. The pH values and total calcium content of all test drinks were determined. Specimens were immersed into an artificial mouth to imitate physiological oral conditions for 60 h. Cyclic de- and re-mineralization was performed, imitating the intake of six drinks in six h followed by a six-hour remineralization phase, where only artificial saliva ran over the specimens. Total erosive enamel loss was determined by contact profilometry. Differences in substance loss at different temperatures were tested for statistical significance (p-values ≤ 0.05) by means of ANOVA.
Results
Rising liquid temperature did not result in a considerable change of pH. Highest substance loss was observed for citric acid (33.6 ± 6 μm to 38.7 ± 6 μm), while only little erosion was induced by fruit tea (0.8 ± 1 μm to 5.9 ± 1 μm). Rising liquid temperature did not result in significantly increased substance loss for citric acid, orange juice and Coca-Cola Zero. Sprite Zero and both fruit teas, however, caused significantly (p < 0.001) more enamel loss at elevated temperature.
Conclusions
Not all investigated drinks showed a temperature-induced change in erosivity.
Clinical significance
For some erosive beverages it can be recommended to keep the consummation temperature as low as possible to decrease the risk of erosive tooth substance loss.
1
Introduction
Dental erosion is defined as a loss of tooth substance by chemical processes without involvement of bacteria [ ]. Erosive tooth wear is on the rise, affecting between 4% and up to 100% of adults examined, depending on the population studied and the examination standard used [ ]. Furthermore, prevalence of erosion increases, especially among younger age groups [ ]. Parallel to this development, a globally increasing consumption of soft drinks has been observed [ ]. While high soft drink consumption is associated with overweight, obesity and diabetes [ ], it is also affecting dental health due to an increased risk for dental caries [ ] and dental erosion [ , , ]. Furthermore, erosion that is caused by beverages has not only been linked to the consumption of acidic carbonated drinks, but also to the intake of fruit juices, acidic sports drinks, wines, cider and acidic herbal teas [ ].
Apart from frequency and timing of intake, the erosive effect of acidic beverages is also influenced by factors including type and concentration of acid, pH value, amount of titratable acid and buffer capacity [ , ]. Since most chemical reactions are affected by temperature, it was hypothesized that the consumption of hot erosives results in increased enamel loss. Several studies confirmed this assumption by finding increasing erosive action of liquids with rising temperature [ ]. The influence of the temperature of erosive drinks in interaction with the remineralizing action of saliva, however, has rarely been investigated. Saliva is one of the most relevant factors for the prevention and development of dental erosion. It develops its protective properties by (1) diluting and clearing of acid, (2) neutralizing and buffering of acid, (3) by formation of the acquired pellicle through adsorption of salivary proteins and glycoproteins, which protect the enamel surface from demineralization and (4) by providing calcium, phosphate and fluoride necessary for maintaining a supersaturated state close to the tooth surface and for rehardening of eroded tooth substance [ , ]. The relevance of saliva and its flow rate on the initiation and magnitude of erosion was shown experimentally, as well in clinical studies [ , ]. Patients with reduced salivary flow rate or reduced buffering capacity are particularly susceptible to erosive tooth loss [ ].
Individual drinking habits determine the contact time between acidic agens and tooth substance, the liquid flow rate and of course the localization of erosive action [ ]. Therefore, erosion may not only be prevented by reducing the consumption of acidic foods and beverages but also through alteration of the drinking habits [ , ]. Beverage temperature would be one of the factors that can be modified.
Consequently, the aim of the present investigation was to study the temperature-dependent in vitro bovine enamel erosion of five differently tempered beverages in interaction with remineralizing action of artificial saliva under controlled conditions in an artificial mouth.
2
Materials and methods
2.1
Experimental procedure
Fig. 1 schematically illustrates the experimental procedure. The temperature-dependent erosive potential of five beverages and citric acid (control) was investigated at temperatures between 5 °C and 55 °C. This set-up resulted in 16 test groups with twelve independent enamel specimens each. Contact profilometry was used to determine the erosive substance loss. After initial profilometry, specimens were immersed in the artificial mouth for 60 h, corresponding to 5 days with a respective duration of 12 h. During these 12 h, cyclic de- and re-mineralization was performed to imitate the intake of six drinks in six h followed by a six-hour remineralization phase, during which only artificial saliva ran over the samples.
After completion of the 60 h of experiment, final profilometry was performed. Total erosive enamel loss was determined by calculating the difference between baseline and final profiles.
2.2
Enamel specimen preparation
Enamel specimens were obtained from intact bovine incisors. After separation of the crown from the root, a water-cooled diamond core drill was used to prepare enamel samples with a diameter of 3 mm out of the labial tooth surfaces. Thereafter, specimens were embedded in acrylic resin blocks (Paladur, Heraeus Kulzer, Hanau, Germany). Enamel surfaces were ground with water-cooled silicon carbide paper discs (# 1000, # 2000, # 4000; Struers, Birmensdorf, Switzerland). A notch was placed in all samples to enable exact repositioning for the profilometric measurements.
2.3
Artificial mouth
All samples were submitted to alternating episodes of de- and remineralization in a so-called artificial mouth, which was described in detail previously [ ]. This special custom-made set-up was constructed to imitate physiological oral conditions. Hereby, the twelve specimens per group were centrally placed in a superfusion chamber at a constant temperature of 37 °C. The system further consisted of two electric pumps (IPC, Ismatec; Cole-Parmer GmbH, Wertheim, Germany), which supplied the specimens with artificial saliva (pump 1) or the tempered test drink (pump 2) through individual tubes via 12-channel T-tube connector. Test liquids were stored at the required temperature in an isolated tank, which was controlled by a thermostat (Julabo F25, Julabo GmbH, Seelbach, Germany). Furthermore, test liquids were permanently pumped through the system to ensure the stability of the desired temperature level throughout the experiment. Liquid flow rate (280 mm/min), as well as the valve assembly controlling the intermittent alternation of erosive test liquids and artificial saliva were controlled by custom designed software (PPK laboratory, University of Zurich, Switzerland). Pump 1 was programmed to constantly supply the specimens with artificial saliva. Only during the demineralization phase the valve assembly was programmed to switch from pump 1 to pump 2, which now provided the specimens with tempered test liquid. Each of the six simulated drinks was taken in 10 “sips”, with one “sip” lasting for 10 s, followed by 50 s of clearance with artificial saliva. This set-up should represent the consummation of one drink in 10 min. For the following 50 min, only artificial saliva was circulated over the samples for remineralization. This sequence was repeated for 6 times (representing the consummation of six drinks in six h), followed by six h of remineralization with saliva (see Fig. 1 ).
2.4
Artificial saliva
Artificial saliva was prepared freshly every day. The composition of saliva was prepared as follows [mmol/l]: NaCl: 9.92; CaCl 2 : 1.53; NH 4 Cl: 2.99; KCl: 17.0; NaSCN: 1.97; KH 2 PO 4 : 2.42; CO(NH 2 ) 2 : 3.33; Na 2 HPO 4 : 2.40 [ ]. The pH value was 6.4. Artificial saliva was supplied to the enamel samples via individual tubes, which were fed by pump 1 from the storage tank.
2.5
Test liquids
Following beverages were investigated at different temperatures: Orange Juice (Eckes-Granini Suisse S.A., Henniez, Switzerland), Coca-Cola Zero (Coca-Cola HBC Switzerland AG, Brüttisellen, Switzerland) and Sprite Zero (Coca-Cola HBC Switzerland AG, Brüttisellen, Switzerland) were tested at the temperatures of 5 °C and 20 °C, whereas fruit tea 1 (“Fruit tea”, Twinings of London™, Wander AG, Neuenegg, Switzerland; Ingredients: hibiscus, natural cranberry, pomegranate and strawberry flavour, 29% other natural flavors, apple pieces, rosehip and sweet wood) and fruit tea 2 (“Fruit tea Strawberry, Raspberry and Rhubarb”, Lipton ® , Unilever Deutschland GmbH, Hamburg, Germany; Ingredients: hibiscus, apple, flavour, rosehip, sweet wood, 2% strawberry, 2% raspberry, 2% rhubarb and maltodextrin) were tested at 20 °C, 37 °C and 55 °C. Citric acid served as control, and was investigated at all temperatures (5 °C, 20 °C, 37 °C and 55 °C).
Fruit teas were prepared according to the package instructions. One tea bag was used per 200 ml of boiling hot water. The brewing time was set to five min. In order to avoid air bubbles disturbing the tube system, the originally carbonated drinks Coca-Cola Zero and Sprite Zero were decarbonated through shaking and use of a magnetic stirrer (IKA, Staufen im Breisgau, Germany) for a few minutes until the degassing process was finished. Further, the orange juice was centrifuged with 3000 rpm for three min (Hermle, Gosheim, Germany) to remove larger suspended particles that could have blocked the system.
The pH values of all liquids were determined at the tested temperatures (pH-meter 827; Methrom, Herisau, Switzerland). In addition, total calcium content was evaluated (atomic absorption spectroscopy, contrAA ® 300, Analytik Jena AG, Jena, Germany) as described elsewhere [ ]. Furthermore, all beverages were titrated (686 Titroprocessor and 665 Dosimat, Metrohm Schweiz AG, Herisau, Switzerland) at room temperature with 0.1 M NaOH solution to pH 5.5 for determination of the titrable acid.
2.6
Profilometry
Erosive substance loss was investigated by contact profilometry (Perthometer S2; Mahr, Göttingen, Germany). The stylus tip of the profilometer had a diameter of 2 μm, and moved with a speed of 0.13 mm/s. Five profiles with a set distance of 250 μm between each profile were recorded before (baseline profiles) and after the experiments (final profiles). A custom-made jig ensured the reproducible positioning of the samples. After completion of the experiments, the exact superimposition of the baseline and respective final profiles was performed with custom designed software (4D Client, Custom designed software; University of Zurich, Switzerland) to calculate the average loss of substance per profile [ ].
2.7
Statistical analysis
For the descriptive statistics, the means and standard deviations of the substance loss for all liquids and tested temperatures were calculated. Differences in substance loss at different temperatures within each beverage were tested statistically by means of ANOVA, as assumptions of homogeneity and normality of errors were not critically violated. Differences with a p-value of ≤0.05 were considered statistically significant. Pairwise posthoc comparisons within each beverage group at different temperatures were calculated using Tukey’s Honest Significance Difference Test. The statistical software R was used for the entire analysis [ ].
2
Materials and methods
2.1
Experimental procedure
Fig. 1 schematically illustrates the experimental procedure. The temperature-dependent erosive potential of five beverages and citric acid (control) was investigated at temperatures between 5 °C and 55 °C. This set-up resulted in 16 test groups with twelve independent enamel specimens each. Contact profilometry was used to determine the erosive substance loss. After initial profilometry, specimens were immersed in the artificial mouth for 60 h, corresponding to 5 days with a respective duration of 12 h. During these 12 h, cyclic de- and re-mineralization was performed to imitate the intake of six drinks in six h followed by a six-hour remineralization phase, during which only artificial saliva ran over the samples.
After completion of the 60 h of experiment, final profilometry was performed. Total erosive enamel loss was determined by calculating the difference between baseline and final profiles.
2.2
Enamel specimen preparation
Enamel specimens were obtained from intact bovine incisors. After separation of the crown from the root, a water-cooled diamond core drill was used to prepare enamel samples with a diameter of 3 mm out of the labial tooth surfaces. Thereafter, specimens were embedded in acrylic resin blocks (Paladur, Heraeus Kulzer, Hanau, Germany). Enamel surfaces were ground with water-cooled silicon carbide paper discs (# 1000, # 2000, # 4000; Struers, Birmensdorf, Switzerland). A notch was placed in all samples to enable exact repositioning for the profilometric measurements.
2.3
Artificial mouth
All samples were submitted to alternating episodes of de- and remineralization in a so-called artificial mouth, which was described in detail previously [ ]. This special custom-made set-up was constructed to imitate physiological oral conditions. Hereby, the twelve specimens per group were centrally placed in a superfusion chamber at a constant temperature of 37 °C. The system further consisted of two electric pumps (IPC, Ismatec; Cole-Parmer GmbH, Wertheim, Germany), which supplied the specimens with artificial saliva (pump 1) or the tempered test drink (pump 2) through individual tubes via 12-channel T-tube connector. Test liquids were stored at the required temperature in an isolated tank, which was controlled by a thermostat (Julabo F25, Julabo GmbH, Seelbach, Germany). Furthermore, test liquids were permanently pumped through the system to ensure the stability of the desired temperature level throughout the experiment. Liquid flow rate (280 mm/min), as well as the valve assembly controlling the intermittent alternation of erosive test liquids and artificial saliva were controlled by custom designed software (PPK laboratory, University of Zurich, Switzerland). Pump 1 was programmed to constantly supply the specimens with artificial saliva. Only during the demineralization phase the valve assembly was programmed to switch from pump 1 to pump 2, which now provided the specimens with tempered test liquid. Each of the six simulated drinks was taken in 10 “sips”, with one “sip” lasting for 10 s, followed by 50 s of clearance with artificial saliva. This set-up should represent the consummation of one drink in 10 min. For the following 50 min, only artificial saliva was circulated over the samples for remineralization. This sequence was repeated for 6 times (representing the consummation of six drinks in six h), followed by six h of remineralization with saliva (see Fig. 1 ).
2.4
Artificial saliva
Artificial saliva was prepared freshly every day. The composition of saliva was prepared as follows [mmol/l]: NaCl: 9.92; CaCl 2 : 1.53; NH 4 Cl: 2.99; KCl: 17.0; NaSCN: 1.97; KH 2 PO 4 : 2.42; CO(NH 2 ) 2 : 3.33; Na 2 HPO 4 : 2.40 [ ]. The pH value was 6.4. Artificial saliva was supplied to the enamel samples via individual tubes, which were fed by pump 1 from the storage tank.
2.5
Test liquids
Following beverages were investigated at different temperatures: Orange Juice (Eckes-Granini Suisse S.A., Henniez, Switzerland), Coca-Cola Zero (Coca-Cola HBC Switzerland AG, Brüttisellen, Switzerland) and Sprite Zero (Coca-Cola HBC Switzerland AG, Brüttisellen, Switzerland) were tested at the temperatures of 5 °C and 20 °C, whereas fruit tea 1 (“Fruit tea”, Twinings of London™, Wander AG, Neuenegg, Switzerland; Ingredients: hibiscus, natural cranberry, pomegranate and strawberry flavour, 29% other natural flavors, apple pieces, rosehip and sweet wood) and fruit tea 2 (“Fruit tea Strawberry, Raspberry and Rhubarb”, Lipton ® , Unilever Deutschland GmbH, Hamburg, Germany; Ingredients: hibiscus, apple, flavour, rosehip, sweet wood, 2% strawberry, 2% raspberry, 2% rhubarb and maltodextrin) were tested at 20 °C, 37 °C and 55 °C. Citric acid served as control, and was investigated at all temperatures (5 °C, 20 °C, 37 °C and 55 °C).
Fruit teas were prepared according to the package instructions. One tea bag was used per 200 ml of boiling hot water. The brewing time was set to five min. In order to avoid air bubbles disturbing the tube system, the originally carbonated drinks Coca-Cola Zero and Sprite Zero were decarbonated through shaking and use of a magnetic stirrer (IKA, Staufen im Breisgau, Germany) for a few minutes until the degassing process was finished. Further, the orange juice was centrifuged with 3000 rpm for three min (Hermle, Gosheim, Germany) to remove larger suspended particles that could have blocked the system.
The pH values of all liquids were determined at the tested temperatures (pH-meter 827; Methrom, Herisau, Switzerland). In addition, total calcium content was evaluated (atomic absorption spectroscopy, contrAA ® 300, Analytik Jena AG, Jena, Germany) as described elsewhere [ ]. Furthermore, all beverages were titrated (686 Titroprocessor and 665 Dosimat, Metrohm Schweiz AG, Herisau, Switzerland) at room temperature with 0.1 M NaOH solution to pH 5.5 for determination of the titrable acid.
2.6
Profilometry
Erosive substance loss was investigated by contact profilometry (Perthometer S2; Mahr, Göttingen, Germany). The stylus tip of the profilometer had a diameter of 2 μm, and moved with a speed of 0.13 mm/s. Five profiles with a set distance of 250 μm between each profile were recorded before (baseline profiles) and after the experiments (final profiles). A custom-made jig ensured the reproducible positioning of the samples. After completion of the experiments, the exact superimposition of the baseline and respective final profiles was performed with custom designed software (4D Client, Custom designed software; University of Zurich, Switzerland) to calculate the average loss of substance per profile [ ].
2.7
Statistical analysis
For the descriptive statistics, the means and standard deviations of the substance loss for all liquids and tested temperatures were calculated. Differences in substance loss at different temperatures within each beverage were tested statistically by means of ANOVA, as assumptions of homogeneity and normality of errors were not critically violated. Differences with a p-value of ≤0.05 were considered statistically significant. Pairwise posthoc comparisons within each beverage group at different temperatures were calculated using Tukey’s Honest Significance Difference Test. The statistical software R was used for the entire analysis [ ].
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