Introduction
The caries-preventive mechanism of topical fluoride is based on formation of CaF2 or “CaF2-like material” on the surface of hard dental tissues. CaF2 interferes with de- and re- mineralization processes during acid attacks by elevating fluoride levels through slow dissolution (1–3). The amount of CaF2 formed is known to depend on several factors, such as fluoride concentration, the time of exposure, the pH of the solution, phosphate and salivary calcium concentration (4–9). The availability of calcium ions is an important factor for calcium fluoride formation (7). Larsen and Richards showed that presence of saliva, even in small amounts, is important for the success of topical fluoride treatment, presumably because of its calcium content. (6) However, little is known about the effects of smoking on the composition of saliva and, in particular, on its inorganic constituents. Recent studies showed that tobacco smoking has an important impact on the chemical composition of natural saliva (10). The aim of this preliminary study was to assess the difference in fluoride uptake by enamel from a smoker’s saliva and a non-smoker’s saliva, assuming the difference in salivary calcium concentration.
Materials and methods
Enamel Slabs
Impacted human third molars were provided by oral surgeons (Department of Oral Surgery, School of Dental Medicine, University of Zagreb, Croatia) and stored in a humid environment until use. The fluoride content in drinking water was under 0.1 ppm F/L. The teeth were brushed with non-fluoride pumice powder and four enamel slabs were cut (2 from buccal and 2 from lingual surfaces) from 14 teeth using a dental diamond disk. Only the slabs without any visible defects were selected after examination under a magnifying glass (10x magnification). The slabs from each tooth were randomly assigned to 4 groups, so that each group consisted of 14 slabs (total of 56 slabs). Two groups were immersed in the fresh unstimulated whole saliva (smoker or non–smoker) before treatment with the toothpaste slurry. One group of slabs was treated only with the toothpaste slurry and one group without treatment served as the control (Table 1). Before treatment, the slabs were rinsed with distilled water and dried at room temperature. All surfaces except the enamel surface were covered with dental wax and orthodontic ligature wire was attached to each slab for easier handling. The enamel surfaces were measured using a caliper, transferred to millimeter paper, and expressed in square millimeters. The slabs were stored in a humid environment.
| Saliva1 | Toothpaste slurry2 | |
|---|---|---|
| Group A | Smoker | + |
| Group B | Non-smoker | + |
| Group C | no treatment | + |
| Group D | no treatment | - |
1 Slabs were immersed in one of the saliva samples (Smoker, Non-smoker) for 5 minutes and gently dried. 2 +: Slabs were immersed in toothpaste slurry for 3 minutes with gentle agitation, rinsed with distilled water for 30 seconds and gently dried; -: slabs were not immersed in toothpaste slurry
Saliva Collection
Saliva donors for this study were patients from a private dental clinic, who volunteered for the study. For the purpose of this preliminary study, only two saliva samples were used. One of them was from a smoker, who smoked 30 cigarettes a day for the past 5 years, and the other was from a non-smoker. A brief medical history, including medications and smoking habits, was obtained using a questionnaire filled out by volunteers before screening. The volunteers received written information about the aims and the design of the study, and signed a written informed consent. Both subjects were healthy males, aged 27 and 28, with good oral hygiene and absence of active caries and periodontal disease. They were asked not to eat or drink, and to refrain from brushing the teeth and smoking for 1 hour before clinical examination and saliva collection.
Unstimulated saliva was collected at 9 a.m. Before sampling, each subject was briefed about the procedure and instructed to wash his mouth with plain water. Unstimulated whole saliva samples were collected over a 15 minute period into sterile plastic cups following the guidelines published previously (11). pH of the saliva was determined immediately after collection. The analysis was made using a pH electrode type 91 02 BN (Orion Res Inc., USA) connected to a potentiometer Orion EA 940 (Orion Res Inc., USA). The electrode was calibrated using standard solutions at pH of 4.0 and 7.0. The collected samples were stored at -20ş C until further processing.
Calcium Determination
Agilent 7500cx (Agilent Technologies, Waldbronn, Germany) inductively coupled plasma mass spectrometer (ICP-MS) with a collision cell was used for the measurements of saliva calcium concentration. Saliva samples (1 ml) were digested with nitric acid (2 ml of 65% HNO3 and 1 ml of H2O), using a high pressure microwave digestion system (UltraCLAVE, Milestone, Italy). After cooling down, samples were diluted with 1% (v/v) HNO3 to a total volume of 15 ml and Ca was analyzed by ICP-MS. All standard solutions were prepared from a single 1 g/l PlasmaCAL standard (SCP Science, Canada). Seronorm® Trace Elements Serum Control Level I and Level II (Sero AS, Billingstad, Norway) were used to control the accuracy of measurements. Samples of reconstituted serum reference material were prepared using the same procedure as saliva samples.
Human Subjects
The Ethics Committee of the School of Dental Medicine University of Zagreb, Croatia, approved the study protocol.
Fluoride treatment
The toothpaste used in this experiment was Elmex®, GABA International AG, Münchenstein, Switzerland (Silica based toothpaste, 1400 ppm F-, Amine fluoride (Olaflur), pH=4.6). The toothpaste/deionized water slurry (1:3 w/w) was made for use in treatment. Toothpaste slurries were prepared fresh, 15 min before each treatment. The procedure was repeated after a 6-hour overnight period for each of the groups.
Fluoride Analysis
The amount of KOH-soluble fluoride was determined by the method of Caslavska et al. (12). Each slab was exposed to 2 ml of 1 M KOH for 24 h with gentle agitation at room temperature. The solutions were neutralized with 2 ml of HNO3 and buffered with 0.5 ml of TISAB III® (Total Ionic strength Adjusting Buffer, Orion Research Inc., Cambridge, Mass., USA), adjusting the final pH of the sample solution to approximately 6.0. Fluoride was analyzed using a fluoride electrode (Orion 96-09, Boston, Mass., USA). The amount of KOH-soluble fluoride was calculated as described by Dijkman and Arends (13).
Statistical analysis
The assumption of approximate normal distribution of the data was verified by Kolmogorov–Smirnov test with Lilliefors correction, as well as by the Shapiro-Wilk test. The Friedman test procedure was used to test the overall level of significance for the data. The non-parametric Wilcoxon matched-pairs test, following a significant Friedman result, was used to determine the significance of changes in the enamel uptake of KOH-soluble fluoride concentration between the groups. P < 0.05 was considered statistically significant. All statistical analyses were performed using the Statistica software package (version 7.1, StatSoft, Inc.).
Results
Calcium concentration in the saliva of the smoker was higher (52.68 mg/L) than in that of the non-smoker (23.95 mg/L). The fluoride concentration of saliva samples was 0.05±0.02 mg/L. pHs of the smoker's and nonsmoker's saliva samples were 6.55 and 6.48, respectively.
The results of the Kolmogorov–Smirnov test and the Shapiro-Wilk test were non-significant for groups A and B, indicating that the assumption of the normal distribution of the data was not violated. The results of the Kolmogorov–Smirnov test and the Shapiro-Wilk test were significant for groups C and D, indicating that the assumption of the normal distribution of the data was violated. Since the data did not have a normal distribution, a non-parametric test was used.
The overall level of significance for the data was previously analyzed using the Friedman procedure (n=14, d.f.=3, χ2 =34.71, P<0.015), and it yielded a significant result.
The amount of KOH-soluble fluoride on the enamel in the group treated with the smoker’s saliva and toothpaste was significantly higher than those of control group, toothpaste only group and non-smoker’s saliva and toothpaste group (P<0.015). The amount of KOH-soluble fluoride in the group treated with the non-smoker’s saliva and toothpaste was significantly higher than those of control group (P<0.015) but was not significantly different when compared to toothpaste only group (P>0.015). Summary data for the enamel uptake of KOH-soluble fluoride concentrations are presented in Table 2.
#Friedman's ANOVA test yielded a significant result (P<0.05) *the Wilcoxon matched-pairs test significant (P<0.015): awhen value is compared with the A group data, bwhen value is compared with the B group data, cwhen value is compared with the C group data, dwhen value is compared with the D group data.
Discussion
The results of this study provide some novel observations regarding the association between salivary calcium and fluoride uptake by enamel. Salivary calcium concentration in this study was two times higher in the smoker’s saliva sample compared to the nonsmoker’s saliva sample. Salivary calcium is usually investigated in relation to periodontal health of tobacco smokers. A large number of studies have shown that smoking has unquestionable influence on the development of periodontal disease and has been identified as one of the risk factors for periodontitis (review by Johnson and Hill (14)). There are conflicting results regarding the difference in salivary calcium concentration of smokers and nonsmokers in the literature. Sewón found higher salivary calcium in subjects who smoke more than ten cigarettes per day than in non-smokers (15). They found that smokers have lower mineral density of bones and suggested that higher salivary calcium concentration related to skeletal calcium disturbances. Khan et al. found a higher level of salivary calcium in tobacco users than in non-users under resting conditions and following stimulation with nicotine (16). They suggested that higher salivary calcium concentration in smokers could come from different tobacco products. In another study, significantly higher salivary calcium levels were observed in smokers than non-smokers, but more frequent tooth-brushing was associated with reduced salivary content of calcium and phosphorus (17). Kiss et al. conducted a study with female smokers and non-smokers with or without periodontitis and their findings showed that patients with periodontitis who smoke exhibit higher salivary calcium levels than those in non-smokers (18). A separate study found higher, but not statistically significant, salivary calcium in young, moderate smokers (19), while yet another one found reduced calcium concentrations in saliva of smokers before and after periodontal therapy (20). These data support a clear association between smoking and salivary calcium concentrations, although the relationship remains to be fully elucidated.
Our results showed that all treatment groups had statistically significant higher concentrations of KOH-soluble fluoride compared to the control group with no treatment. There are studies supporting the idea that saliva combined with exposure to low fluoride topical treatment does not enhance the formation of calcium fluoride (21, 22), which is not supported by our findings. One reason for those findings could be longer exposure time (60 min) to the fluoride solution, which may have allowed the release of calcium from enamel in those studies. On the other hand, there are several different studies (6, 8) which reported that saliva increased the concentration of calcium fluoride. It has been suggested that the presence of saliva may increase the amounts of calcium fluoride because of its calcium content and mucinous nature (6). Our study demonstrated that the group treated with toothpaste and smoker’s saliva had significantly higher enamel uptake of KOH-soluble fluoride compared to the group with the toothpaste and nonsmoker’s saliva treatment. On the other hand, there was no significant difference between the group with toothpaste and nonsmoker’s saliva treatment and the group with the toothpaste but no saliva treatment. This result suggests that the amount of enamel uptake of KOH-soluble fluoride is affected not only by the presence, but the concentration of calcium in saliva, as well. Since these are novel findings, further research is needed to elucidate where salivary calcium in smokers’ saliva comes from. Another question that remains to be answered is which concentration of salivary calcium makes a difference in the amount of fluoride uptake by enamel.
A positive correlation between high salivary calcium levels and the number of intact teeth was established by Sewon and Mäkelä (23). Prior to their study, Ashley (24) and Shaw (25) established an inverse relationship between caries and mineral content of plaque and saliva (23, 24). Sewon et al. also found that periodontitis-affected subjects have more intact teeth than subjects who are free of the disease (26). Therefore, while further investigation remains necessary in the field of smokers’ saliva mineral content, our results provide a possible mechanistic explanation of existence of more intact teeth in subjects with periodontitis.
In conclusion, this study demonstrates that higher levels of salivary calcium found in smokers enhance the amount of KOH-soluble fluoride uptake. This result encourages us to conduct a larger scale study with more sample donors.
