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Original Paper Caries Res 2006;40:43–46 DOI: 10.1159/000088905

Received: March 28, 2002 Accepted after revision: February 16, 2005

The Effect of Adding Calcium Lactate to Xylitol Chewing Gum on Remineralization of Enamel Lesions R. Sudaa T. Suzukia R. Takiguchib K. Egawab T. Sanob K. Hasegawaa a

Department of Periodontology and b First Department of Oral Anatomy, Showa University Dental School, Tokyo, Japan

Key Words Calcium lactate ! Chewing gum ! Enamel lesion ! Remineralization ! Xylitol

Abstract The purpose of the study was to determine whether adding calcium lactate to chewing gum containing xylitol enhances remineralization of enamel surfaces using an early caries lesion model. Enamel slabs were cut from human extracted sound teeth and artificial subsurface lesions created within each. Half the enamel slabs were used as controls and stored in a humidifier while half were mounted into oral appliances worn by 10 volunteers (22–27 years old, 2 males and 8 females) in a threeleg trial, during which they wore the appliance without chewing gum, chewed gum containing xylitol + calcium lactate or chewed gum containing only xylitol 4 times a day for 2 weeks. Calcium concentrations in the enamel surfaces of control and test slabs were measured by Xray spectrometry and degrees of remineralization were calculated. The mean degree of remineralization was greater after chewing xylitol-Ca gum (0.46 8 0.10) than after no gum (0.16 8 0.14) or after chewing xylitol gum (0.33 8 0.10) (p ! 0.01). In conclusion, chewing gum containing xylitol + calcium lactate could enhance remineralization of enamel surface compared to chewing gum containing only xylitol or no gum chewing. Copyright © 2006 S. Karger AG, Basel

© 2006 S. Karger AG, Basel 0008–6568/06/0401–0043$23.50/0 Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com

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Xylitol, a five-carbon natural sugar alcohol, is widely used as a noncariogenic sweetener, which is not fermentable by most oral bacteria [Trahan, 1995]. Several studies show that xylitol also reduces demineralization of the subsurface enamel and increases its hardness in vitro and in vivo [Scheinin et al., 1993; Smits and Arends, 1988]. It has been reported that xylitol migrates with calcium ions in an electric field [Angyal and Millis, 1979; Kieboom et al., 1979]. Calcium lactate is generally used as a calcium supplement and food additive. The purpose of this study was to determine whether the effect of remineralization of enamel surface by xylitol can be enhanced when calcium lactate is added to gum containing xylitol.

Materials and Methods Enamel Lesions Premolars extracted for orthodontic reasons or impacted third molars with sound enamel surfaces were used to make enamel slabs. After extraction, the teeth were fixed in 18% v/v formalin acetate solution. Blocks of the outer enamel surface, approximately 8 ! 4 mm (2 mm deep) were cut using a water-cooled diamond blade saw. Each slab was covered with acid-resistant nail varnish except for two mesiodistal windows (1 ! 6 mm) separated from each other by about 1 mm. The slabs were immersed in 40 ml of demineralization buffer consisting of 20 g/l Carbopol 907 (carboxypolymethylene), 500 mg/l hydroxyapatite (Bio-Gel HTP) and 0.1 M lactic acid, pH 4.8, for 4 days at 37 ° C. The solution was changed after 2 days. After demineralization, each enamel slab was sec-

Reiko Suda Department of Periodontology, Showa University Dental School 2-1-1 Kitasenzoku, Ohta-ku, Tokyo 145-1958 (Japan) Tel. +81 3 3787 1151, ext. 352, Fax +81 3 3787 9290 E-Mail suda@senzoku.showa-u.ac.jp


tioned through the midline and divided into two 4 ! 4 mm (2-mmdeep) slabs and the cut surface of each slab was covered with nail varnish. One half of each slab was retained as the demineralization control and stored in a labeled plastic tube with MilliQ water. The other half of the slab was inset into the center of an intraoral palatal appliance with wax. Chewing Gums Chewing gums tested in the present study were Trident XyliCal® (Warner-Lambert K.K.), which contained 2.5 g xylitol and 94 mg calcium lactate per 16 g (xylitol-Ca gum) and gum containing only 2.5 g xylitol (xylitol gum). Both gums had the same taste. Volunteers were unaware of the type of gum used for the experiment. Subjects Ten volunteers (2 males and 8 females, aged 22–27, mean age 24.1 8 1.5) were recruited and gave their informed consent. All volunteers were dental hygienists or clinical study residents working in the Dental Hospital of Showa University. All of them met the following requirements: (1) No untreated caries. (2) Nonstimulated salivary flow rate 60.2 ml/min. After swallowing saliva in the mouth, the volunteers kept saliva in their mouth for 2 min without swallowing. Whole saliva was transferred to plastic tubes and the salivary flow rate was calculated. (3) Stimulated salivary flow rate 61.0 ml/min. Each volunteer chewed 2 pieces of either gum base, xylitol gum, or xylitol-Ca gum for 5 min. Whole saliva was transferred to plastic tubes without swallowing and salivary flow rate was calculated. After chewing each gum, the volunteer rinsed their mouth with water and took a 5-min rest before chewing the next gum. The order of gum chewing was randomized in each volunteer. Caries activity was tested by means of caries activity test kit (Orion Diagnostics, Espoo, Finland). This kit consisted of Dentobuff® Strip, Dentcult®-SM and Dentocult®-LB. Following the manufacturer’s instructions, the results of each test were scored for each subject (1–3 for Dentobuff Strip, 0–4 for Dentcult-SM and 0–3 for Dentocult-LB). Caries activity for each volunteer was then calculated as the mean score of 3 tests. Thus, a score of 3.3 indicated the highest caries activity and a score of 0.3 indicated the lowest. Intraoral Palatal Appliance An intraoral palatal appliance was made in acrylic resin for the upper jaw of each volunteer. Location of the clasps was designed so as not to inhibit natural chewing. A 5 ! 5 mm (3-mm-deep) hole was made in the center of the palatal plate and one enamel slab of the paired slabs (TE) was mounted with wax. Trial Each volunteer chewed 2 pieces of each chewing gum for 20 min wearing the intraoral appliance. The appliance was removed after another 20 min of wearing, and then, stored in the humidifier. This procedure was repeated 4 times a day (2 times in the morning and 2 times in the afternoon). Volunteers were instructed to brush their teeth after eating and to start chewing the gum at least 1 h later. They were also instructed to take a 90-min or longer break between the first and the second as well as between the third and the fourth chewing sessions. Volunteers chewed either xylitol-Ca gum or xylitol gum for 2 weeks. During the control phase, the volunteers wore the appliance

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Caries Res 2006;40:43–46

as explained previously, but did not chew gum. After completion of each phase, a 1-week rest was provided. The experiment was conducted until all volunteers went through all three phases of the experiment (i.e. two phases for chewing two varieties of gum and one no-gum phase). Every time one phase was completed, the appliances were collected and the enamel slabs removed to determine the extent of remineralization. During the 1-week rest, the appliance was kept at room temperature in a humidifier. On the 7th day, a new enamel slab was attached to the appliance. All the volunteers kept a diary covering the times they chewed the gum, the number of pieces chewed and the details of their meals. They were instructed to use fluoride toothpaste (PC Clinica®, Lion Co., Tokyo, Japan). Analysis of Remineralization on Enamel Slab During analysis each enamel slab was identified by a random number so that examiners did not know the treatment which they had received. Each pair of test slabs and control slabs was dehydrated by a graded alcohol series and absolute acetone, then embedded in polyester resin (Rigolac 70F and Rigolac 2004, Nissin EM, Japan). The embedded slabs were sawn through the midline with a diamond disk. The cut surface of each slab was ground with alumina grinding papers and coated with carbon in a HUS-5GB evaporator (Hitachi, Tokyo, Japan). Subsequently, the slabs were subjected to analysis in a scanning electron microscope (S-2500CX; Hitachi, Tokyo, Japan, 15 kV, 0.1 nA) fitted with an energy dispersive X-ray spectrometer with automatic background subtraction (Quantum Kevex delta 4, Kevex Instruments, USA). X-rays characteristic of calcium and phosphorus were collected from ten randomly selected 7.5 ! 10 !m areas (acquisition time 200 s per analyzed area) on both nondemineralized enamel (previously covered with nail varnish) and demineralized enamel on control and test slabs. Calcium and phosphorus concentrations (wt%) were calculated from signal count ratios by comparison with tracing of a fluorapatite standard (Hitachi, Tokyo, Japan). Mean concentrations of Ca and P were calculated for each area. Statistical Analysis The degree of demineralization for each control and test specimen ("dc and "dt) was obtained by dividing the mean calcium concentration of the demineralized area by the mean calcium concentration of the nondemineralized area. The degree of remineralization was expressed as ("dc – "dt)/"dc. Differences between the gums with respect to the degree of remineralization were analyzed by one-way ANOVA and Fisher’s PLSD pairwise test. The correlation between caries activity and the remineralization rate during the control phase (NC) was analyzed by Spearman’s correlation coefficient.

Results

Scanning electron microscope observations (not shown) indicated that the demineralized regions showed subsurface loss of mineral, with a relatively intact surface layer. The mean calcium concentration of nondemineralized enamel was 36.3–36.8 wt% and the mean phosphorus Suda/Suzuki/Takiguchi/Egawa/Sano/ Hasegawa


concentration was 17.5–17.7 wt%. There were no statistically significant differences between control and test specimens or between treatment phases. The mean calcium concentration of demineralized areas in control specimens was 29.8–31.1 wt% and the phosphorus concentration was 14.1–14.9 wt%, with no significant difference between treatment phases. In test specimens, the calcium concentration increased in all treatment phases, being significantly greater in the xylitol and xylitol-Ca phases than in the no-gum phase (table 1). The phosphorus concentrations also increased: from 14.1 to 14.5 wt% in the no-gum controls; from 14.9 to 15.6 wt% on the xylitol-Ca gum phase and from 14.6 to 16.0 wt% in the xylitol gum phase. The mean degree of remineralization was significantly higher in the xylitol-Ca gum phase than in the xylitol gum phase and the degree of remineralization was significantly higher in both phases than in the no-gum phase (table 1). It was observed that subjects who showed a low degree of remineralization in the no-gum phase tended to show a greater degree of remineralization in the xylitol-Ca gum phase. The subjects were divided into two groups according to the degree of remineralization in the no-gum phase: !0.2 (low) and 60.2 (high). The mean difference in the degree of remineralization between the xylitol-Ca gum and the no-gum phases was significantly greater in the low group (0.42 8 0.32) than in the high group (0.12 8 0.05) (p ! 0.05; Mann-Whitney U test). The degree of remineralization in the no-gum phase was correlated with the number of mutans streptococci (rS = –0.75; p ! 0.05) and with caries activity score (rS = –0.75; p ! 0.05). However, there was no significant correlation with salivary buffer capacity, number of lactobacilli or nonstimulated salivary flow rate. The mean nonstimulated saliva flow rate was 0.76 8 0.50 ml/min. Flow rate was raised significantly by chewing either gum base, xylitol or xylitol-Ca gum (1.96 8 1.67, 4.40 8 2.79 and 4.87 8 2.28 ml/min, respectively). Flow rates when chewing xylitol or xylitol-Ca gum were not significantly different but the flow rates for both gums were significantly higher than for gum base.

Discussion

The reason that test specimens showed remineralization during the no-gum phase is probably due to saliva being supersaturated with respect to calcium phosphates [Grøn, 1973; Lagerlöf et al., 1994]. The degree of reminEnamel Remineralization by Xylitol and Calcium Lactate

Table 1. Changes in the calcium concentrations of artificially de-

mineralized enamel according to treatments (means 8 SD) Treatment

No gum Xylitol gum Xylitol-Ca gum

Calcium concentration, wt% control

experimental

29.881.6a 31.181.3a 30.581.1a

30.782.2a 33.080.8b 33.381.1b

Degree of remineralization

0.1680.14a 0.3380.10b 0.4680.10c

Within columns, means with the same superscript letter are not significantly different (one-way ANOVA and Fisher’s PLSD test).

eralization doubled with xylitol gum chewing and tripled with xylitol-Ca gum chewing, as compared to control. The salivary flow rate, which might influence remineralization of subsurface and surface enamel, also increased significantly during gum chewing. However, the stimulated flow rate did not differ significantly between the xylitolcontaining gums. It can be speculated that the calcium added to the xylitol-Ca gum can enhance remineralization. Subjects who showed a low degree of remineralization during the no-gum phase NC tended to show a greater increase in the degree of remineralization during the xylitol-Ca gum phase. Since caries activity and the number of mutans streptococci were inversely correlated with the degree of remineralization during the no-gum phase, chewing xylitol-Ca gum might be effective in preventing the development of early caries lesions in subjects with high caries activity. It should be noted that the enamel slabs were mounted in the center of the palatal appliance, and the appliance was not worn all day, so plaque accumulation on the surface of the enamel slab was not detected during the experiment. As the presence of plaque will probably influence remineralization processes, remineralization in stagnation sites might not be as high as observed in this study. However, xylitol was also effective in reducing mineral loss from the surface of enamel slabs applied to the buccal surface of a lower denture [Smits et al., 1988]. In conclusion, chewing gum containing calcium lactate as well as xylitol enhanced remineralization of subsurface enamel lesions more than gum containing xylitol only. It is also suggested that the effect of remineralization is greater in subjects with high caries activity.

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References

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Angyal SJ, Millis JA: Complexes of carbohydrates with metal cations. Aust J Chem 1979; 32: 1993–2001. Grøn P: Remineralization of enamel lesions in vivo. Oral Sci Rev 1973;3:84–99. Kieboom APG, Buurmans HMA, van Leeuwen LK, van Benschop HJ: Stability constants of (hydroxy)carbozylate- and aldol-Ca (II) complexes. J R Neth Chem Soc 1979;98:393–395. Lagerlöf F, Oliveby A: Caries-protective factors in saliva. Adv Dent Res 1994;8:229–238. Scheinin A, Söderling E, Scheinin U, Glass RL, Kallio ML: Xylitol-induced changes of enamel microhardness paralleled by microradiographic observations. Acta Odontol Scand 1993;51: 241–246.

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Smits MT, Arends J: Influence of extraoral xylitol and sucrose dippings on enamel demineralization in vivo. Caries Res 1988;22:160–165. Söderling E, Isokangas P, Tenovuo J, Mustakallio S, Makinen KK: Long-term xylitol consumption and mutans streptococci in plaque and saliva. Caries Res 1991;25:153–157. Trahan L: Xylitol: A review of its action on mutans streptococci and dental plaque – its clinical significance. Int Dent J 1995;45:77–92.

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