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| 2010-22 |
| 22-1 |
ISSN (Print) 1013-9052
EISSN 1658-3558
P.O. Box 52500,
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Kingdom of Saudi Arabia
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In vitro effect of different concentrations of iron on the initiation of dental caries: Pilot study
Thakib A. Al-Shalan,*BDS, MS, PhD, Amal Al-Askar,** BDS
Previous animal studies have shown that iron reduced the incidence of dental caries. Objective: The purpose of this in vitro study was to examine the effect of different concentrations of iron-supplement on the initiation of dental caries on human teeth.
Dental caries remains the most infectious disease affecting humans. It is caused by bacteria that are harbored in dental plaque. Mutans streptococci (MS) have been identified as the principal bacteria causing dental caries in humans.1 MS bacteria, under specific conditions, can ferment sugars and other carbohydrates from foods and drinks to produce lactic acid and other short chain organic acids.2 If the concentration of the acid depresses the pH adjacent to the tooth surface below 5.5, then the enamel may dissolve. Glucosyltransferase (GTF) enzymes from MS play a pivotal role in dental caries and are considered the most significant virulence factor.1 Different methods of caries prevention are in the dental literature.3 Previous studies had investigated the effect of minerals on the inhibition of dental caries.4-8 The results of previous experiments suggested that iron (Fe) added to a cariogenic diet could reduce the incidence of dental caries in animals.9.10 In addition, Rosalen et al.11 and Miguel et al.12 have found that iron decreases the caries development in de-salivated rats. Moreover, Larsson et al.13 found that diet supplemented with iron salts, either in food or in drinking water may have cariostatic effect attributable primarily to local action on the teeth. Other studies have also shown that Fe ions have a strong inhibitory effect on the GTF enzyme. This may explain the inhibitor effect of iron.14 No previous studies have investigated the effect of different concentrations of iron on the initiation of dental caries on human teeth using controlled environment such as artificial caries study. Therefore, the purpose of this in vitro study was to determine the effect of different concentrations of iron on the initiation of dental caries on human teeth.
Bacteria Strain MS bacterial strain (6715) was used in this study. The stock organism was stored in skim milk at -80o C until use. Cultures of these MS strain were initiated from frozen stocks by incubating five loops of the bacteria stock in 500 ml Todd-Hewitt broth (DIFCO, Detroit, MI) grown anaerobically in brewer jar filled with 80% N2, 10% H2, and 10% Co2 at 37OC. Teeth Preparation Sixty extracted caries-free and restoration-free human premolars were used. These teeth were collected, cleaned and stored using thymol at room temperature until used. Teeth were mounted using cold cure acrylic resin, which covered the tooth up to the cemento-enamel-junction. Selected area on the buccal surface of the teeth were covered with a drop of nail polish (colored) then the whole coronal part was covered with transparent nail polish (two layers). After the varnish dried, the colored part was removed to leave one exposed enamel surface of approximately 0.5 cm2. The teeth were randomly divided into five groups (Table 1). Artificial Caries Experiment The experimental design is shown in Figure 1. Three 24-well ELISA plates (2ml volume/well) were used. The mounted teeth were immersed in solution media containing MS bacteria strain (1x107 cells), 200ul 10% sucrose and 200 ul Fe in different concentrations (Table1). The teeth in solution were incubated in anaerobic chamber at 37oC for 24 hours. Fresh sucrose, iron and bacteria were added daily for four weeks. Caries Progress Evaluation The progress of dental caries was randomly evaluated by daily visual examination for decalcification and by tactile examination for cavitation using the explorer. The dates and degree of enamel decalcification and caries progression were recorded daily. Statistical Analysis Data were entered using FOXPRO data base program. Differences in caries initiation and progression between different groups were analyzed using SPSS program. Analysis of variance test (ANOVA) and Tukey post hoc tests were used to compare the differences between the groups.
Table 2 summarizes the number of sound, decalcified, and carious teeth in all the groups. All the teeth in the positive control (Group 1) developed cavitations. All experimental groups were free from cavitations. For the decalcification, Group 3 (50% Fe) showed the least number of teeth (one tooth) with decalcification followed by Group 2 (100% Fe) which had 3 teeth with decalcification.
The objective of this in vitro study was to examine the effect of different concentrations of iron on the initiation of dental caries in human teeth. We used the in vitro caries model for the ease of control on the factors affecting dental caries. In addition, result from this in vitro study may help in the design of an in vivo future study. Results from this laboratory study have demonstrated the cariostatic effect of iron (ferrous sulfate) at different concentrations 100%, 50%, 25% and 12.5%. The best effect of iron was with 50% concentration which showed 91.7% fewer lesions followed by 100% concentration which showed 75% fewer caries lesions. These percentages decreased to 41.7% and 25% fewer carious lesions in groups 4 and 5, respectively. Our results confirm the findings of previous studies that showed a cariostatic effect of ferrous sulfate.11,12 It was suggested by McClure 15 that anion might influence the cariostatic effect of iron. In addition, it has been well documented that reactive oxygen species cause damage to various biomolecules. When the metal ion is bound to an enzyme, it is involved in the oxidation-reductions. The locally generated oxygen species may react at specific sites with the enzyme impairing its activity.16 In this study, FRE-IN-SOL iron supplement, which contains ferrous sulfate, was used because it is the available iron supplement in King Abdul-Aziz University Hospital (KAUH) and King Khalid University Hospital (KKUH). We aimed to use the same iron supplements prescribed by pediatricians in KKUH and KAUH. As this study was a pilot one, the period that the study was carried out was 29 days. This time allowed the caries initiation and some cavitations. Future studies may need to extend the time over 29 days. In addition, other iron supplement products could be used in future studies to assess the effect of each supplement. In this pilot study, visual examination was used to diagnose decalcification and cavitation. Traditionally, dental caries can be diagnosed by visual examination, tactile examination (by explorer), radiographic examination and/or by a combination of the above methods.17 Previous studies have used electron microscope to diagnose the caries in similar in vitro studies. Due to the number of the teeth and the objectives of the study, sectioning the teeth at different intervals was not possible. In a recent study, laser-based diagnostic system (DIAGNOdent) was compared to visual examination for the diagnosis of caries.1819 Visual examination was used in this study because decalcification is easy to diagnose after drying the tooth. In the study, staining was observed in some teeth. A previous study had shown that daily inorganic iron, not organic, may cause tooth-staining.20 In another study, staining was observed with both ferrous and ferric iron.8 The presence of extrinsic staining on the enamel surface of teeth receiving ferrous sulfate indicates the precipitation of inorganic iron. Ferrous ions react with molecular oxygen by oxidation and yield superoxide radical ions. By dismutation reaction, the superoxide radical ions produce hydrogen peroxide. The ferrous ion reacts with hydrogen peroxide to produce hydroxyl radical ion via the feton reaction. The results showed that DIAGNOdent was helpful to diagnose actual cavitation, while visual examination was important to diagnose early caries signs (white spot lesion as decalcification). Although visual examination may be subjective, recent study showed the importance of using visual examination with other advanced techniques (DIAGNOdent) for the diagnosis of decalcification and dental caries.
The results of this in vitro study enable us to conclude that iron plays a cariostatic role in the development of dental caries in artificial dental caries experiments regardless of its concentration.
The authors would like to thank Dr. Nazeer Khan for his help in the statistical analysis. This research is registered in College of Dentistry Research Center number NF-1819.
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Table1. Description of the groups and the material used
* 200 ul dH2o was used instead of iron. Table 2. Summary of total number (%) of teeth with sound, decalcified, and cavitated teeth (N=12/group)
1.
Group 1 vs Groups 2, 3, 4, 5 P
< 0.001 Group 3 vs Group 4 P = 0.009 Group 3 vs Group 5 P = 0.001 2. All pairs were significant (P<0.001) * Total number is 12, 12 is placed here for comparisonTable 3. Analysis of variance for starting day of decalcification and starting day of cavitation
*Statistical significance for starting day of decalcification and cavitation
Group 1
vs Groups 2,3,4, and 5 (P<0.0001)
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