In vitro  cariostatic effects of various iron supplements on the initiation of dental caries
Thakib A. Al-Shalan

Department of Preventive Dental Sciences, College of Dentistry, King Saud University, P.O. Box 60169, Riyadh 11545, Saudi Arabia

Previous animal and laboratory studies have shown the possible cariostatic effect of iron. The purpose of this study was to in vitro study the effect of different concentrations of four iron supplements on the initiation of dental caries.

Materials and methods: Four products of iron supplements were used namely fre-in-sol, ferotonic, feromin and ferose. Two hundred extracted teeth were distributed randomly into 10 groups. Eight groups were from the four iron products in two concentrations (100% and 50%) in addition to the positive and negative control groups. Mutans streptococci bacteria (6715) grown in Todd Hewitt Broth were used. Assessment of decalcification and cavitation was done daily for 60 days.

Results: It showed that different iron - supplement products play cariostatic effect in the initia­tion of the dental caries. With the exception of 100% and 50% ferose, both 100% and 50% con­centrations of all supplements have cariostatic effect. The mean dates for decalcification varied with lowest for the positive control (12.2 days) and the highest was for 50% feromin. Cavitation was seen in the positive control and 100% ferose groups with mean on the first day of cavitation of 57 days.

Conclusion: It was concluded that iron may have cariostatic effect on the in vitro development of dental caries in human teeth.

Dental caries is a very common disease affecting humans. It is caused by Mutans streptococci (MS) bacteria which was pres­ent in the oral cavity and have been identified as the principal bacteria causing dental caries in human (Hamada and Slade, 1980). Dental caries ranks among the most significant human disease. In Saudi Arabia, the prevalence of dental caries in chil­dren has been reported in different studies (Al-Shammery et al., 1990; Al-Tamimi and Peterson, 1998; Wyne, 2008)A recent study Wyne (2008) reported the prevalence of dental caries in Saudi children was 74% because caries can continue throughout life and preventive measures must be part of every­one's lifestyle.

Preventive methods include fluoride, fissure sealants, die­tary advice and patient education, as well as recall visits (Duckworth and Bebington, 1993). It is known that fluoride is effective in reducing dental caries (Bawden et al., 1978; Bjarnason and Finnbogason, 1991). Another possible caries preventive method is by using minerals (Lonnerdal et al., 1983).

It has been found that iron decreases the caries develop­ment in desalivated rats (Rosalen et al., 1996; Miguel et al., 1997). Results of previous experiments suggest that iron (Fe) added to a cariogenic diet can reduce the incidence of dental caries in animals and humans (Opperman and Rolla, 1980; Davey and Embery, 1992; Larsson et al., 1992). Devulapalle and Mooser (2001) have shown that iron ions are strong inhib­itors of the glucosetransferase enzyme (GTF). Gierat-Kuchar-zewska and Karasinski (2006) and Clarke et al. (2006) have shown that iron plays an important role in the development of dental caries. Furthermore, Berlutti et al. (2004) concluded that iron could have a key role in the protection of the oral cavity from Streptococcus mutans pathogenicity.

Our previous study investigated the effect of one iron sup­plement (Fre-in-sol) on the initiation of dental caries (Al-Sha-lan and Al-Askar, 2006). Results have shown that iron plays a cariostatic role in the development of dental caries. Knowing the fact that pediatricians prescribe different iron supplements for children suffering from anemia, it might be interesting to study the effect of other iron supplements products on the ini­tiation of dental caries. Therefore, the aim of this study was to in vitro study the effect of different concentrations of four iron supplements on the initiation of dental caries.

2.1. Teeth preparation

Two hundred extracted caries and restoration-free human per­manent premolar teeth were used. These teeth were collected, cleaned and stored using thymol at room temperature until used. Teeth were prepared as described previously (Al-Shalan and Al-Askar, 2006). In brief, teeth were mounted using a cold cure acrylic resin covering the tooth to cemento-enamel-junc-tion. Selected area on the buccal surface of the teeth was cov­ered with a drop of colored nail polish. Then the whole coronal part was covered with transparent nail polish. After the var­nish dries, the colored parts were removed to leave one ex­posed enamel surface of approximately 0.5 cm². Teeth were randomly distributed to ten groups (Table 1).

2.2. Iron sources

Four products of iron supplements were used. These products were: (1) Fre-in-sol (Bristol Myers Squibb Company, New Jer­sey, USA), (2) Ferotonic (Ram Pharmaceutical, Amman, Jor­dan), (3) Feromin (Riyadh Pharma, Riyadh, KSA) and (4) Ferose (Spimaco Al Qassim Pharmaceutical Plant, Saudi Ara­bia). As supplied, two concentrations (100% and 50%) were used per group. The 100% concentration means that the con­tent of the bottle was used directly without dilution. Whereas, 50% concentration was prepared by adding 50% of the product to 50% distilled water (dH₂o) in sterilized containers. A to­tal of eight experimental groups were made.

2.3. Artificial caries experiment

Ten 24-well ELISA plates (2.0 ml volume/well) were used. Each plate was assigned to one group. The mounted teeth were placed in a ELISA well containing immersed solution media of 1.00 ml of THB containing MS bacteria strain (6715), 100 llof 10% sucrose and 100 il iron in different concentrations/prod­ucts. For the positive control group, no iron was used. No bac­teria were used for the negative control group. For both control groups, 100 ildH₂o was used. The plates were incu­bated in an anaerobic chamber at 37 °C.

2.4. Caries progress evaluation

The progress of dental caries initiation and progression was evaluated by visual examination (decalcification) and tactile examination (cavitations). The decalcification and caries pro­gression were recorded daily for 60 days. The evaluations were done independently by two examiners and the lower value was taken.

2.5. Statistical analysis

Data was entered and analyzed using descriptive statistics to show the general behavior of the data. Chi-square test was used to determine the relationship between the groups. If the chi-square was not valid due to the small number of outcome, Fisher exact test was used. The significant level was fixed at 0.05 probabilities.

3. Results

3.1. Visual examination

Fig. 1 shows that all teeth in group one and ten (positive con­trol and 50% ferose, respectively) developed decalcification. Other groups (except the negative control) showed decalcifica-tion in some teeth. Table 2 summarizes the descriptive data of the visual examination. The means of the first day of decalcification was 12.2 days for group one and 17.5 days for group 10. Additionally, group three (100% fre-in-sol) and group eight (50% feromin) showed only two teeth decalcified. In these two groups, the means of the initial day of decalcification was 29 days for group three (100% fre-in-sol) and 50 days for group eight (50% feromin). Furthermore, group four (50% fre-in-sol), five (100% ferotonic), six (50% ferotonic) and seven (100% feromin) showed only three teeth decalcified. The means of the first day of decalcification was 36 days for group three and 45 days for the other three groups. In group nine (100% ferose), 16 teeth showed decalcification and the means of initial day of decalcification was 35.12 days.

The number (%) of sound teeth group at the end of the study using visual examination is shown in Table 3. All teeth in positive control group and 50% ferose group developed decalcification. In group nine (100% ferose), 4 teeth (out of 20) did not show decalcification. Group four (50% fre-in-sol), five (100% ferotonic), six (50% ferotonic) and seven (100% feromin) showed 85% of teeth were sound (N = 17). Additionally, Table 3 shows that group three (100% fre-in-sol) and group eight (50% feromin) showed 90% of the teeth were sound (N = 18). At the end of the experiment, 72 teeth were decalcified and 128 teeth were sound.

Multiple comparisons between groups (number of teeth decalcified) diagnosed by visual examination using chi-square test is shown in Table 4. Statistical analysis between different groups showed significant differences (P < 0.05) between the control and other groups except group ten. Significant differ­ence (P = 0.0001) was found between group one (positive con­trol) and all other groups except group nine which showed marginal significant difference (P = 0.053). Other significant difference was found between group nine and ten (100% ferose,  50%  ferose,  respectively) and all other groups (P = 0.000).

3.2. Tactile examination

Table 5 summarizes the descriptive data of the tactile examina­tion used for the diagnosis of cavitations. It shows that only five teeth developed cavitation. Four teeth were from group one (positive control) and one tooth from group nine (100% ferose). The means on the first day of cavitations was 57 days. Furthermore, data shows that 80% ofgroup one (positive con­trol), 95% from group nine (100% ferose) and all other groups were sound. Statistical analysis between different groups is shown in Table 6. Significant difference between group one (positive control) and all other groups (P = 0.024) except group nine (100% ferose) which showed no significant differ­ence (P = 0.091).

The objective of this study was to investigate the effect of dif­ferent iron - supplement products on the initiation of dental caries using an in vitro model. An in vitro caries model was used for ease of control of different factors (e.g. saliva and host factors) that may play a role in the initiation and progression of dental caries. An in vitro model was used previously (Al- Shalan and Al-Askar, 2006; Lammers, 1990; Kotsanos, 1991; Featherstone, 1996; Hall, 1997). In addition, extracted teeth were collected from young patients where the teeth were ex­tracted for orthodontics reason. The results from the in vitro models might be regarded as guidance for future in vivo stud­ies. Such studies are important before human trials are conducted.

The iron products used in this study are used widely for the treatment of anemia (Oski, 1993). Additional products are available and future studies might investigate their effect on the initiation of dental caries. In this study, 100% of the prod­ucts were supplied and the 50% dilution was used. It is possi­ble that the iron concentration in each product is different. Therefore, a better control will be to standardize the iron con­centration per product which may be investigated in future studies.

In addition to the experimental groups, positive and nega­tive control groups were used in this study. The objective of using positive control was to ensure that the model we used is effective in producing decalcification and/or cavitations. The negative control group was used to rule out the possibility of contamination that might occur. The results showed that both control groups served their purposes.

The diagnosis of dental caries is fundamental to the practice of dentistry. Early stage of dental caries is difficult to diagnose (Dodds, 1993; Rosen et al., 1996). Caries is diagnosed by examination of teeth including visual inspection, radiograph and tactile examination by dental explorer (Dodds, 1993).

4.1. Visual examination

In this study, visual examination was used for the diagnosis of decalcification. Results showed that the teeth in the positive control group developed decalcification on all the teeth. In contrast, the teeth in the negative control group did not devel­op any decalcification. This showed the good controls used. If a contamination took place, negative control might have developed decalcification in some teeth. This may ensure to a certain limit, the elimination of contamination possibility in our study.

Results showed that teeth in groups containing fre-in-sol, ferotonic and feromin developed less decalcification as com­pared to ferose groups. This might be attributed to different iron product used. Fre-in-sol, ferotonic and feromin contain ferrous sulphate and ferose contain iron (III) - hydroxide poly-maltose complex. Torell (1988) reported that application of different iron product in term of content have different cario-static effect. For ferrous sulphate groups, it was found to have cariostatic effect on experimental hamster studies when ap­plied topically to the teeth as when added to the drinking water or the diet (Dodds, 1993; Emilson and Krasse, 1972). Rosalen et al. (1996) and Miguel et al. (1997) had demonstrated that the iron sources containing ferrous sulphate reduced the develop­ment of dental caries in intact and desalivated rat.

For the ferose group, ferose contain iron III - hydroxide polymaltose complex. There is no cariostatic effect compared to other ferrous sulphate groups. This might be attributed to the hydrolysis of maltose that produced two molecules of glu­cose. Another possible effect is the formation of complex be­tween hydroxide plymaltose and iron that may lead to have very few iron to react with the dental enamel (Torell, 1988).

4.2. Tactile examination

Dental explorer was used for diagnosis of cavitation (Tactile examination). This examination was used in different studies (Penning et al., 1992; El-Housseiny and Jamjoum, 2001; Lussi et al., 2001). This technique has been criticized due to the possi­bility of transferring microorganism from one side to another. In addition, a sharp explorer can actually produce irreversible trau­matic defect in the early demineralized area (Ekstrand et al., 1987; Penning et al., 1992). All teeth in group 1 and group 10, and 16 teeth from group 9 developed decalcification. The result showed that only five teeth developed cavitation. Four teeth were from group 1 (positive control) and one tooth from group 9 (100% ferose). Significant difference was found between group 1 (positive control) and all other groups except group 9 (100% ferose). The findings were confirmed by visual examination. As the experiment extended for 60 days, there is a need to extend the time as all teeth diagnosed as cavitations by tactile examina­tion occurred in the last three days.

4.3. Effect ofIron on dental caries

Our results confirmed those of the previous studies that showed a cariostatic effect of ferrous sulphate (Miguel et al., 1997; Devulapalle and Mooser, 2001). Iron is able to cover the enamel surface with protecting layers. Acid protective layer effect has been demonstrated in vitro.(Al-Shalan and Al-As-kar, 2006; Clarkson et al., 1984). It can be assumed that these protective layers will be firmly bound to the organic parts of the enamel as hydrous iron oxides and clay materials have great affinity to organic materials (Craig, 1979). This affinity has been utilized in the clinical work to increase the adhesion between composite resins and dentine by mordanting with fer­ric chloride (Jedrychowski et al., 1981).

Gels and crystal of hydrous iron oxides are capable of adsorbing various ions and of nucleating various crystalline substances. From a dental point of view, the adsorption of cal­cium and phosphate ions is of special interest as it indicates that iron may have a repairing function (Torell, 1988). This can be by nucleating the precipitation of salivary calcium and phosphate ions as apatite or other phosphates on the en­amel surface. Therefore, the iron may have the effect of replac­ing minerals which can have been dissolved during the acid phases of the caries process. In addition, owing to the above-mentioned affinity to organic materials iron ought to be able to mediate the fixation of remineralized particles to the organic parts of the enamel (Torell, 1988).

The concept of a possible role of iron in the remineraliza-tion of human enamel is supported by several studies. Bachra and van Harskamp (1970) showed that extremely low concen­trations of iron could initiate precipitation of calcium apatite from meta stable calcifying buffer systems. Moreover, reminer-alized caries lesion was reported to contain more iron than in­tact enamel surface layers (Torell, 1988).

Based on the results of this in vitro study, we conclude Differ­ent iron supplement products used in this study play cario-static effect in the development of the dental caries. With the exception of 100% ferose, both 100% and 50% concentrations of all remaining products have cariostatic effect.

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