The effect of iron deficiency anemia on experimental dental caries in mice

Archives of Oral Biology 105 (2019) 13–19

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The effect of iron deficiency anemia on experimental dental caries in mice Dania Bahdila, Kenneth Markowitz, Siddhi Pawar, Krupa Chavan, Daniel H. Fine, ⁎ Kabilan Velliyagounder

T

Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA

ARTICLE INFO

ABSTRACT

Keywords: Dental caries Iron deficiency anemia Mice Streptococcus mutans Iron

Objective: The objective of this study was to examine the relationship between iron deficiency and caries susceptibility in a mouse model. Materials and methods: Three-week-old C57BL/J6 mice were fed a cariogenic diet containing either standard iron (48 ppm Fe) or low iron (4 ppm Fe) levels. Concurrently, groups of mice with both diets were orally inoculated with Streptococcus mutans (1 × 108) cells on three consecutive days. At the end of the 5th week after infection, mice were sacrificed and jaws were collected for caries scoring, rating the number and severity of lesions using a modified Keyes method applicable to mice. Results: Blood analysis by the end of the 5th week revealed marked reduction in the hemoglobin and hematocrit levels of the mice fed the iron deficient diet (IDA and IDA-S. mutans). Anemic mice in both groups lacked the incisor enamel pigmentation observed in mice fed an iron deficient diet. Anemic infected mice had the highest caries severity scores reflecting extensive deep lesions (P < 0.05). S. mutans infected mice fed a standard iron diet had similar numbers of lesions and severity scores as un-infected IDA animals (p < 0.05). IDA did not alter S. mutans CFU counts in infected animals (P < 0.05). Conclusion: These results demonstrated that IDA mice are at a higher risk of developing deep dental caries compared to non-anemic mice; highlighting the protective role of iron against dental caries.

1. Introduction Dental caries is defined as destruction of the enamel and dentin (hard tissue surfaces) of the teeth, via the bacterial acidic byproducts, produced by dietary carbohydrates fermentation. This imbalance is attributed to the exposure to high sugar diet, poor oral hygiene, changes in the flow and contents of saliva, insufficient fluoride, increase load of cariogenic bacteria, defective immunological factors and genetics (Selwitz, Ismail, & Pitts, 2007). Dental caries is considered one of the principal public health concerns according to the World Health Organization (WHO). In the pediatric population, specifically preschool age children, early childhood caries (ECC) and severe early childhood caries (S-ECC) are relatively prevalent. ECC affects 4.5 million preschool children annually. High sucrose diet, which has poor nutritional value is one of the principles causes for ECC along with socio-economic level being another factor. Damage to the permanent teeth, severe pain and infections resulting in hospitalization are possible consequences of this condition (Casamassimo, Thikkurissy, Edelstein, & Maiorini, 2009). Children with ECC often suffer from malnutrition and weigh 20% less than their ideal weight (Tinanoff & O’Sullivan, 1997; Tinanoff &

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Reisine, 2009). ECC is known to be a complex, multifactorial disease, Streptococcus mutans is widely accepted as the main cariogenic bacteria for ECC along with a shift in the oral ecology favoring other cariogenic microorganisms (Takahashi & Nyvad, 2011). Several studies suggest that iron deficiency anemia (IDA) is the most common nutritional deficiency in the world, affecting up to 2 billion people (Killip, Bennett, & Chambers, 2007) with most of those afflicted residing in the non-industrializing countries. The World Health Organization defined IDA in children as: 1) Hb concentration lower than 11 g/dL or hematocrit lower than 33% for 6–59 month of age children or pregnant women, 2) Hemoglobin concentration lower than 11.5 g/dL or hematocrit lower than 34% for 5–11 year of age children, 3) hemoglobin concentration lower than 12 g/dL or hematocrit lower than 36% for 12–14 year of age children (WHO, 2001). The pathophysiology of iron deficiency anemia is related to different etiologies. IDA could be related to poor iron intake in cases of malnutrition or increased of the body iron demand during growth, pregnancy or lactation. This deficiency has been associated with impaired motor and cognitive development in children in addition to poor school performance (Zlotkin et al., 2004).

Corresponding author at: Department of Oral Biology, Rutgers School of Dental Medicine, 110 Bergen St, C830, Newark, NJ, 07103, USA. E-mail address: [email protected] (K. Velliyagounder).

https://doi.org/10.1016/j.archoralbio.2019.05.002 Received 22 February 2019; Received in revised form 30 April 2019; Accepted 2 May 2019 0003-9969/ © 2019 Published by Elsevier Ltd.

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Studies on human populations have observed an association between iron deficiency and ECC (Clarke et al., 2006; Tang, Huang, & Huang, 2013). The role of dietary iron in caries susceptibility has been examined in animal models. Supplementing the diet with iron has been observed to reduce caries (Eshghi, Kowsari-Isfahan, Rezaiefar, Razavi, & Zeighami, 2012; Miguel, Bowen, & Pearson, 1997; Rosalen, Pearson, & Bowen, 1996). It has also been reported that rats fed cariogenic diets that also included low Fe levels (16 ppm) developed more carries than rats fed a conventional diet (Sintes & Miller, 1983). Since the diets tested differed in several respects this study did not compared the effect of iron deficiency alone when compared to a standard diet. Based on these previous studies, we hypothesized that iron deficiency would increase caries susceptibility in a mouse model. We examined the impact of iron deficiency leading to anemia, on caries development in S. mutans infected and uninfected animals. This study will advance our knowledge about the biological association between dental caries and IDA and the influence of nutrition on caries risk.

Table 1 Compositions of cariogenic diets.

2. Material and methods 2.1. Animals Twenty-four, 3-week old C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, Maine, USA) and maintained in the transgenic animal facility of the Rutgers New Jersey Medical School, Newark, New Jersey. Mice were housed on a 12 h light/dark cycle with the room temperature maintained at 25 ± 1 °C. The protocol was approved by the institutional animal care and use committee (IACUC; ID: PROTO201702519) of Rutgers Biomedical Health Sciences, Newark, New Jersey. 2.2. Diet Two customized diets were purchased from Harlan-Teklad (Madison, WI, USA). To limit background iron in both, cellulose was omitted, and reagent grade, pretested calcium phosphate was used in the mineral mix. For the standard iron diet, 48 ppm Fe was added as FeSO4 (Cat# TD.170952) and the iron deficient diet contained approximately 2–6 ppm Fe (Cat# TD.110669). Both were designed to be cariogenic containing a sucrose level of 56%. The nutrition was the same for both diets aside from the iron concentration. The diets were irradiated, color-coded and were in pelleted form (Table 1).

Formula

Standard diet Fe: 48 ppm (g/Kg)

Iron deficient diet Fe: 4 ppm (g/kg)

Casein low Cu&Fe DL-Methionine Sucrose Corn Starch Corn Oil Ferrous Sulfate, heptahydrate Choline Bitartrate Ethoxyquin, antioxidant Thiamin (81%) Riboflavin Pyridoxine HCl Niacin Calcium Pantothenate Folic Acid Biotin Vitamin B12 (0.1% in mannitol) Vitamin A Palmitate (500,000 IU/g) Vitamin E, DL-alpha tocopheryl acetate (500 IU/g) Vitamin D3,cholecalciferol (400,000 IU/g in sucrose) Vitamin K, MSB complex Sucrose, fine ground Calcium Phosphate, dibasic Sodium Chloride Potassium Citrate, monohydrate Potassium Sulfate Magnesium Oxide Manganous Carbonate Zinc Carbonate Cupric Carbonate Potassium Iodate Sodium Selenite, pentahydrate Chromium Potassium Sulfate, dodecahydrate Sucrose, fine ground Blue Food color Red food color

200.0 3.0 544.851 150.0 50.0 0.239 2.8 0.01 0.6 0.6 0.7 3.0 1.6 0.2 0.02 1.0 0.8 10.0

200.0 3.0 545.09 150.0 50.0 – 2.8 0.01 0.6 0.6 0.7 3.0 1.6 0.2 0.02 1.0 0.8 10.0

0.25

0.25

0.15 1.08 500.0 74.0 220.0 52.0 24.0 3.5 1.6 0.3 0.01 0.01 0.55

0.15 1.08 500.0 74.0 220.0 52.0 24.0 3.5 1.6 0.3 0.01 0.01 0.55

124.03 0.1

124.03

Protein Carbohydrate Fat Kcal/g 4.0

0.1

% by weight

% by weight

17.7 66.6 5.2

17.7 66.6 5.2

The bold text signifies that ferrous sulfate is the only constitute difference between the two diet.

2.3. Bacterial strain and growth conditions A streptomycin resistant clinical strain of S. mutans-VSK1 (serotype c) was grown at 37 °C in brain heart infusion (BHI) broth supplemented with 200 μg/ml of streptomycin (BD & company, Sparks, MD, USA) for 12 h, and 1 × 108 cells suspended in phosphate-buffered saline (PBS) were used for oral inoculation. Prior to inoculation, all animals were screened for indigenous streptomycin-resistant bacteria as previously described (Velusamy, Markowitz, Fine, & Velliyagounder, 2016). In all experimental groups, the results were negative. In addition, as previously described, the mice were screened for endogenous S. mutans using PCR methods (Velusamy et al., 2016). As had been observed in other studies using the mouse model, the animals did not have detectable S. mutans.

in each experimental group and littermates were randomly distributed in stainless steel cages. The animals were then divided into the following experimental groups: 1) mice maintained on a standard-iron (48 ppm)/cariogenic diet (Control), group 2) mice infected with S. mutans maintained on a standard-iron (48 ppm)/cariogenic diet (S. mutans), group 3) mice maintained on an iron-deficient (4 ppm)/cariogenic diet (IDA) and group 4) mice infected with S. mutans maintained on an iron-deficient (4 ppm)/cariogenic diet (IDA-S. mutans). 2.5. IDA and caries induction The objective of this study was to determine whether IDA mice infected with S. mutans alone could increase the caries susceptibility when compared to uninfected IDA mice. In the post weaning mice (21 days old), we inoculated S. mutans or PBS and began the cariogenic diet on the same day. Mice were transferred to new cages where they were infected with S. mutans for three consecutive days by inoculating approximately 1 × 108 cells (100 μl) onto the teeth using a micropipette. Simultaneously, control, S. mutans infected, IDA and IDA-S. mutans infected groups were fed either the standard iron diet or iron deficient

2.4. Experimental design and sample size calculation Based on our previous dental caries studies using the mouse model, power was calculated by using the G*Power software (Faul, Erdfelder, Lang, & Buchner, 2007). We set the effect size of a severity score difference of 10.0 between groups, a power of 0.9 and a significance level of 0.05 for an experiment with 4 experimental groups, which calculated our sample size to be 24 mice (6 mice per group). Six animals were used 14

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Fig. 1. Schematic diagram showing experimental sequence of different cariogenic diets, S. mutans inoculation, plaque collection blood drawn, euthanization and caries assessment.

diets, (both containing 56% sucrose) and given 10% (w/v) sucrosecontaining sterile drinking water. To monitor the body weight of the all the experimental groups we measured them weekly throughout the 5 weeks of experiment and prior to euthanization (Fig. 1).

2.9. Statistical analysis All data were expressed as means ± standard deviation. Differences between the group means were assessed for statistical significance using Student’s t-test or ANOVA with post hoc Tukey’s HSD test between the groups. Non-parametric analysis of caries scoring was done using the Kruskal-Wallis and post hoc tests. P-values of less than 0.05 were considered statistically significant.

2.6. Detection of S. mutans colonization To confirm S. mutans colonization, plaque samples were collected at 10th, 20th and 35th days following inoculation using a micro brush (Easyinsmile, Passaic, NJ, USA) from the tooth surface. Plaque sampling was accomplished by gently placing the tip of the brush into the animal’s mouth and rotating the brush over the upper and lower teeth. These plaque samples were collected from both inoculated and uninoculated mice. The plaque samples were then diluted to BHI medium, serially diluted and then plated onto mitis salivary agar (MSA) containing streptomycin (200 μg/ml) and incubated at 37 °C in an anaerobic chamber. After 24 h of incubation, colony-forming units (CFUs) were enumerated.

3. Results 3.1. Effect of iron deficient diet on animal weight, Hb and Hct Mice in all experimental groups gained weight through the duration of the experiment. There was no significant weight difference between the groups at any time point. We compared the groups maintained on a standard iron diet with the groups fed ID diet for 5 weeks. The Hb and Hct levels of the control and S. mutans infected mice fed a standard iron diet were within the normal range and did not differ significantly from each other (Hb, 14.70 ± 0.57; Hct, 50.45 ± 2.05). On the other hand, the Hb (3.75 ± 0.49; P < 0.01) and Hct (4.05 ± 0.21; P < 0.01) levels of IDA and IDA-S. mutans mice were significantly reduced when compared to the standard iron diet groups respectively. The IDA groups also had significantly reduced Hct levels when compared to the control or S. mutans infected group. The Hb and Hct levels between the IDA and IDA-S. mutans did not significantly differ from each other. These findings confirmed that IDA was successfully induced in our study (Table 2).

2.7. Blood collection and euthanization At the end of the study (35 days after infection), blood was collected by cardiac puncture under anesthesia and the complete blood count (CBC) was determined using an automated H1 Technicon system (Antech Diagnostics, New Hyde Park, NY, USA). Mice were then euthanized by carbon dioxide asphyxiation; death was confirmed by cervical dislocation. 2.8. Caries scoring

3.2. Effect of iron deficient diet on S. mutans colonization

The heads of sacrificed mice were collected and defleshed manually. The mandibles and maxillae were removed, mechanically cleaned and photographed. Determination of caries scores was done by blinded scoring of the jaws and performed by a single calibrated examiner using an Olympus (SZ61) dissecting microscope (Olympus, Center Valley, PA, USA). Caries lesions were rated using a modified Keyes method applicable to mice (Keyes, 1958). Each molar was given a score reflecting the number of carious surfaces and depth of lesion invasion including superficial/slight invasion (E = 1), moderate invasion (Dm = 2), extensive invasion (Ds = 3). Caries were classified as moderate when it extended one to two third of the way through the tooth. A total caries score was determined for each animal. The decay was classified as extensive when the lesion extended more than two-thirds the distance from the surface to the center of the tooth. The consistency of the evaluator in rating caries was assessed in an exercise where the caries score of jaws was rated on separate occasions. The intra examiner agreement was over 90%.

In order to induce dental caries, mice were inoculated with S. mutans (1 × 108) cells for three consecutive days. To confirm S. mutans colonization, plaque samples were collected at three time points, serially diluted and plated on streptomycin-MSA plates, and the CFUs were counted. The mean CFUs count (Log 10 CFUs/ml) at the end point of Table 2 Mean values of hemoglobin (Hb) and hematocrit (Hct) for each group. Blood parameters

Uninfected

S. mutans

IDA

IDA-S. mutans

Hb (g/dl) Hct (%)

14.70 ± 0.57 50.45 ± 2.05

14.85 ± 1.20 49.50 ± 4.95

3.75 ± 0.49* 12.55 ± 0.64#

4.05 ± 0.21* 13.50 ± 0.71#

* p < 0.01 when compared to the IDA and IDA-S. mutans groups. # p < 0.01 when compared to the control and S. mutans groups. 15

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Fig. 2. Effect of iron deficient diet on caries development in mice. Values are the mean caries scores or count ± standard deviations (SD) for experimental groups of six mice consisting of 1) Control group, 2) S. mutans infection group 3) The IDA group and 4) The IDA-S. mutans group. Asterisks indicate statistically significant differences between the groups as calculated by Student’s t-test or ANOVA with post hoc Tukey’s HSD test between the groups. Determination of caries count and scores was performed by blinded scoring of the jaws as described in the methods section. a. Mean number of carious surfaces per mouse for each group. b. Counts of lesions classifies as enamel lesion (E) moderate dentin invasion (Dm) or deep dentinal invasion (Ds). c. Mean severity score for mice in each of the four groups.

experimental groups (Control and IDA 0.00 ± 0.00 and S. mutans 0.50 ± 0.55 respectively; all P < 0.01). The mice in the IDA-S. mutans group also had significantly greater number of multi-surface lesions affecting the occlusal proximal and facial or lingual surfaces, than the S. mutans or IDA groups (both P < 0.05).

the study for S. mutans and IDA-S. mutans were 5.61 ± 0.30 and 5.74 ± 0.47 respectively (P < 0.01). In contrast, microbial sampling confirmed that the mice in the uninfected groups remained S. mutans free. 3.3. Caries assessment, number of carious lesions

3.5. Higher caries scores were associated with IDA and S. mutans infection

The modified Keyes scoring system reflects the depth and severity of carious lesions in each mouse. Molars were given scores reflecting the number of carious surfaces and depth of lesion invasion including superficial/slight invasion (E = 1), moderate invasion (Dm = 2), and extensive invasion (Ds = 3). The mean number of carious lesions per animal was determined for each group. The control-uninfected mice’s teeth were generally intact with a mean of 0.5 ± 0.55 lesions per mouse. As shown in Fig. 2a, mice in the other three groups had significantly higher numbers of carious lesions when compared to the control group. The S. mutans, IDA and IDA-S. mutans groups had 7.67 ± 1.75 (P = 0.015), 6.67 ± 0.82 (P = 0.026) and 8.83 ± 1.48 (P = 0.013) lesions per mouse respectively. Mice in the IDA-S. mutans group had significantly greater number of lesions than mice in the IDA group (P < 0.01). The number of caries in the IDA-S. mutans group were not significantly different than the caries count in the S. mutans infection group.

As shown in Fig. 2c mice in the control group had low caries scores (0.5 ± 0.55). S. mutans infected animals feed a normal diet had a caries score of 14 ± 5.25 this was significantly higher than the score for the control group (P = 0.044). The uninfected mice feed an irondeficient diet (IDA group) had a caries score of 9.67 ± 2.50 this was significantly higher than the score for the control group (P < 0.01) but did not differ significantly from the score of the S. mutans group. IDA-S. mutans group possessed the highest caries score when compared to the other groups with a mean of 23.33 ± 7.45. This was significantly higher than the score of the IDA group (P < 0.01), the S. mutans (P = 0.0044) or the control group (P < 0.001). 3.6. Molars involvement To determine the distribution of carious lesions for each molar the mean caries score of 1 st, 2nd and 3rd molars of mice in each experimental group is shown in Table 3. The small number of lesions observed in control mice was observed only on first molars. Although caries was observed in all three molar types in S. mutans and IDA mice, the distribution of lesions was unequal with the 1st molars having significantly higher caries scores than 2nd (P < 0.05) or 3rd molars (P < 0.01. In the S. mutans group mice, 3rd molars had significantly lower caries scores than 1 st molars. In contrast, caries was more evenly distributed in S. mutans- IDA mice, with 3rd molars having caries scores that did not differ significantly from 1 st or 2nd molars. Photos of jaws obtained from S. mutans-IDA mice with extensively decayed 3rd molars can be seen in Fig. 4.

3.4. S. mutans infected mice with IDA present higher numbers of severe lesions Fig. 2b shows the mean ± SD numbers of lesions in each mouse classified as restricted to enamel, having moderate dentin invasion or having deep dentin invasion. The small number of lesions observed in the controls was all restricted to enamel. In contrast, the other groups had lesions that extended into dentin as well as large numbers of enamel lesion. The IDA-S. mutans group had the highest number of lesions extending into deep dentin. For this group, mean number of the deep carious lesions (4.00 ± 3.03) was significantly higher than the other 16

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our study consumed a high sucrose diet and only the iron level varied. After weaning the mice, we infected the animals with S. mutans and started feeding them the ID-diet simultaneously. By the end of 5th week after inoculation, low blood Hb and Hct levels confirmed the anemic status of the IDA and IDA-S. mutans infected animals. These hematological results were consistent with the results of previous IDA experimental animal models (Grant, Wiesinger, Beard, & Cantorna, 2003; Hubbard et al., 2013; Krijt et al., 2012; Nagababu et al., 2008). It is well known that IDA in children is usually associated with low body weight due to maternal and child malnutrition (Black, Quigg, Hurley, & Pepper, 2011). On the other hand, among children in industrial countries, IDA was more commonly associated with being overweight and obese (Brotanek, Gosz, Weitzman, & Flores, 2007; Hutchinson, 2016; Nead, Halterman, Kaczorowski, Auinger, & Weitzman, 2004). In our study where the mice had unlimited access to a high sucrose diet, we observed that the weight gain in IDA mice did not differ significantly from the gains experienced by mice fed the diet containing normal iron levels. We screened the mice for indigenous S. mutans (non-streptomycinresistant) flora by collecting plaque samples from each mouse before S. mutans inoculation and confirming its absence. The microbial sampling method was intended to specifically examine tooth plaque. Since the high sucrose diet was started simultaneously with S. mutans infection, we are confident that the high sucrose diet did not create ecological conditions in the mice’s oral cavities that would hinder S. mutans colonization. Our data indicates that IDA-S. mutans mice had similar levels of S. mutans colonization and numbers of caries but significantly highest caries scores when compared to other group. These higher scores reflect deeper, more extensive lesions as seen in Fig. 3. The greatest number of carious lesions was located on the occlusal surface due to the topography of mice teeth. Proximal lesions were observed less frequently. Small proximal lesions were much less noticeable due to the technical limitation of direct visualizations under the microscope. As was the case in this study, all the animals in our previous study examining the role of lactoferrin and S. mutans on caries in mice were given a high sucrose diet, all uninfected mice had low caries score. Infected lactoferrin knockout animals had high caries scores and infected wild type had caries scores that were intermediate between the uninfected and knockout infected groups (Velusamy et al., 2016). These observations support the use of the mouse model in investigating states of high caries risk. In our study, the control group had close to zero caries and the experimental group had high scores. In order to statistically resolve this difference a limited sample size is needed. In contrast, large numbers of rats were used to statistically resolve relatively small differences in caries scores between animals in different

Table 3 Mean caries score of molars in each group. Molars

Uninfected

S. mutans

IDA

IDA-S. mutans

1st molar 2nd molar 3rd molar

0.50 ± 0.55 00 ± 00* 00 ± 00#

8.50 ± 4.51 4.17 ± 2.86 1.33 ± 1.03#,!

6.17 ± 1.33 1.50 ± 1.05* 2.00 ± 1.10#

10.67 ± 6.44 6.67 ± 3.50 6.00 ± 4.86

* P < 0.05 significant difference between 1st molars and 2nd molars. # P < 0.05 significant difference between 2nd molars to 3rd molars. ! P < 0.05 significant difference between 2nd molars and 3rd molars.

3.7. Effect of iron deficient diet and enamel pigmentation The incisors of the mice feed an iron deficient diet were chalky white. In contrast, the incisors of mice fed a diet containing normal iron levels had typical brown pigmentation on the labial surface. Photographed of incisors of control and IDA mice are shown in Fig. 4. The presence or absence of S. mutans infection did not alter the appearance of the incisors in this study. 4. Discussions Clinical studies suggest a link between iron deficiency and caries, particularly in children (Abdallah, Gehan Hamza, & Alsahafi, 2016; Jayakumar & Gurunathan, 2017; Sadeghi, Darakhshan, & Bagherian, 2012; Tang et al., 2013). Children with high dental caries experience have been reported as having significantly lower salivary iron levels, suggesting that salivary iron is involved in defense against caries (Buche et al., 2016). Other studies however, failed to show a relationship between most hematological parameters of IDA and caries (Nur, Tanriver, Altunsoy, Atabay, & Intepe, 2016). Hence there is a need for further research on this important question. Few experimental studies utilizing animal models have directly assessed the effect of iron deficient diets on caries risk. Supplementing iron in the diet reduces caries in caries-susceptible rodents (Larsson, Johansson, & Ericson, 1992; Miguel et al., 1997; Oppermann, Rolla, Johansen, & Assev, 1980). The effect of iron deficiency in the absence of other diet manipulations on caries development has not been examined (Sintes & Miller, 1983). In order to further examine the relationship between systemic iron levels and caries risk we examined whether iron deficient S. mutans infected mice would have more extensive caries than S. mutans infected mice fed a cariogenic diet with normal Fe. We also examined caries development in ID animals that were lacking S. mutans infection. In this study we examined the effect of iron deficiency without other dietary variables on caries development. All animals in

Fig. 3. Five weeks after S. mutans infection, the mice were sacrificed, heads defleshed and the jaws cleaned. Digital images of jaws from all the mouse groups were taken using a light microscope at 10× magnification. The pictures show the effect of iron deficiency anemia and/or S. mutans infection on caries development. 17

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Fig. 4. Five weeks after S. mutans infection, the mice were sacrificed, heads defleshed and the jaws cleaned. The digital images of incisors from all mouse groups were taken using a light microscope at 10× magnification. The pictures showing the effect of iron deficiency anemia on enamel pigmentation.

treatment groups in a study examining the effectiveness of various forms of fluoride (Briner & Francis, 1971). We also observed caries in IDA mice that were not infected and free of S. mutans; these IDA mice had significantly higher caries scores than control and scores that did not differ significantly from non-IDA S. mutans infected animals. Several studies have examined the impact of iron on oral microbial populations. It has been reported that Fe reduces bacterial biofilm growth and enamel demineralization in a dose dependent manor in vitro and in vivo (Dunning, Ma, & Marquis, 1998; Emilson & Krasse, 1972; Rosalen et al., 1996). It is also possible that IDA might hinder oral defenses rather than by enhancing the oral pathogens aggression. It has been observed that 49.3% of the adult patients with IDA had dry mouth (Wu et al., 2014). Correcting IDA in pediatric patients significantly improved their salivary pH levels and buffering capacity (Mahantesha et al., 2015). Ironbinding proteins play an important role in host defense. The iron binding protein lactoferrin has been shown to be important in both animal models (Velusamy et al., 2016) and in humans (Fine, 2015). Further investigation regarding the iron binding proteins’ role in IDA and dental infection is crucial to understanding this relationship. The observation of high caries scores in non-S. mutans infected IDA animals indicate that indigenous members of the flora are behaving in a cariogenic fashion. The development of caries in non-S. mutans infected mice may suggest that various indigenous acid producing bacteria such as S. mitis, S. sanguinis, and Lactobacillus increase in the IDA mice’s microflora (de Soet, Nyvad, & Kilian, 2000). The effects of IDA on the oral cavity’s microbial community need further investigation. The observation of loss of pigmentation in the incisors of IDA mice in this study demonstrates that the iron deficient diet used had an impact on the animal’s dental hard tissues. The effect of dietary iron on the incisor enamel pigmentation was observed in both S. mutans infected and uninfected animals. We also observed that iron deficiency increased the susceptibility of the late erupting third molars to caries, resulting in these teeth developing extensive lesions in the IDA-S. mutans group. Previous dental caries studies in mice reported that the relatively small third molars are usually free from dental decay due to their late eruption time on the 24th- 36th days of life, agreeing with our observation of low third molar caries scores in S. mutans infected mice fed the Fe-containing cariogenic diet (Catalan et al., 2011). The results

of our study provide support for the hypothesis that the incorporation of iron improves the acid-resistance of the dental hard tissue (Lacruz et al., 2012). The protective role of iron was suggested to be through the formation of an acid resistant precipitation on the enamel surface, acting as a replacement mineral for ions lost during demineralization (Torell, 1988). This finding shed light on the rapid and progressive effect of IDA on dental caries and raises the possibility that the tooth structure of IDA mice is more susceptible to the caries process. Future investigation of the effect of IDA on the teeth development, mineralization and maturation are required. There are limitations in drawing clinical conclusions from the current study. The mouse’s short life expectancy, flora, dental anatomy and other differences make extrapolating the results of experiments with mice to human health difficult. These results do support the plausibility of a relationship between IDA and ECC. 5. Conclusions Based on this experiment, we conclude that IDA markedly increases the caries susceptibility of mice. This tendency towards more destructive lesions was observed in IDA animals that were uninfected with S. mutans. Iron binding proteins analysis, the actions of iron deficiency on tooth development and oral microbial ecology during anemia can be investigated in future studies to explore the effect of this deficiency on dental caries in specific and oral health in general. Declaration of Competing Interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments The authors received financial support from Oral Biology Department and declare no other potential conflicts of interest with respect to the authorship and/or publication of this article. 18

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