Dental caries is a widespread infectious disease caused by environmental and genetic factors. Thus far, studies have identified several environmental factors influencing dental caries; however, little remains known about the underlying genetic factors. Recent studies using mice have reported the major genes responsible for dental caries to be located on mouse chromosome 2. Using congenic mice, this study aimed to clarify if the chromosomal region on mouse chromosome 2 influenced dental caries.
Materials and methods
We examined the dental caries scores obtained from caries induction, salivary secretion volume, and enamel hardness in the strains C57BL/6Slc, C3H/HeSlc, B6-Chr.2 C3H , and three types of congenic mouse strains that we generated.
We successfully generated three types of congenic mouse strains. The caries scores of congenic mice, which had the C3H/HeSlc-derived interval between D2Mit126 (84 Mega base pair; Mbp) and D2Mit226 (163 Mbp), were significantly lower than that of any other mouse strain studied herein ( p < 0.05). Moreover, the salivary secretion volume of the congenic mice described above tended to be more than that of any other congenic strain. However, enamel hardness was not significantly different among the strains.
Several caries-resistant genes could be located between D2Mit126 and D2Mit226. Salivary secretion volume was one of the most important factors related to dental caries, and the genes influencing the rate of salivary secretion might be located in the same region.
Dental caries is a common chronic infectious disease globally. Susceptibility to dental caries is influenced by various factors, such as host factors, oral flora, and the environment. Environmental and bacterial factors have been studied as the causes of dental caries, but not much is known about host genetic factors.
Studies on twins have indicated that dental caries has a significant genetic contribution, of about 40–60% . In other studies on twins raised in different environments, significant genetic variance (45–67%) for dental caries was proved by monozygotic twins and was supported by the findings in dizygotic twins . The results of these studies reinforced the fact that dental caries is determined by a genetic component as well .
Animal models have also demonstrated genetic contribution to dental caries susceptibility: Kurihara et al. identified two inbred strains of mice—C57BL/6NJcl, which is highly susceptible to dental caries, and C3H/HeNJcl, which is highly resistant to dental caries. Quantitative trait locus (QTL) analysis of these strains showed that chromosome 2 had a high likelihood of being related to dental caries susceptibility . To prove the effect of this QTL located on chromosome 2, Nomi et al. produced a consomic mouse strain, termed B6-Chr.2 C3H , in which a C57BL/6Slc-derived interval of chromosome 2 was replaced with that from the caries-resistant strain C3H/HeSlc. B6-Chr.2 C3H mice had a six times lower dental caries susceptibility and higher salivary secretion volume compared to C57BL/6Slc . They suggested that this QTL (the C3H/HeSlc interval) on chromosome 2 played a role in susceptibility to dental caries and salivary secretion.
In this study, to elucidate the genetic component of dental caries susceptibility on chromosome 2, we induced dental caries using several types of congenic mice containing different C3H/HeSlc-derived intervals on chromosome 2 in a C57BL/6Slc genetic background. Considering the results of the caries score obtained from caries induction, the volume of stimulated saliva secretion, and enamel hardness, we also aimed to narrow down the chromosomal region influencing dental caries susceptibility using congenic mice.
Materials and methods
Mice and breeding conditions
C57BL/6Slc and C3H/HeSlc mice were purchased from Sankyo Lab Service Co. (Tokyo, Japan). B6-Chr.2 C3H mice were established in our laboratory and maintained at Sankyo Lab Service Co. All mice were maintained at room temperature (25 °C ± 1 °C), a relative humidity of 55% ± 5%, and a 12 h light/dark cycle. All the animal use protocols of this study were reviewed and approved by the Nihon University Institutional Review Board (Chiba, Japan; AP14MD005-1, AP17MD010).
Polymerase chain reaction (PCR) conditions
PCR was performed by the usual methods using Ex Taq ® (TaKaRa, Shiga, Japan). The amplification conditions were as follows: DNA denaturation at 94 °C for 3 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; and final extension at 72 °C for 10 min. After the PCR products were electrophoresed in 4% agarose gels, the gels were stained with ethidium bromide and examined under ultraviolet light.
Generation of congenic mouse strains
The congenic strains were produced as described previously . DNA was extracted from the tails of mice using the DNeasy Blood and Tissue kits (Qiagen, Hilden, Germany) following the instruction manual, and the genotypes were confirmed by PCR using Massachusetts Institute of Technology primers, D2Mit237 (40 Mega base pair; Mbp), D2Mit90 (65 Mbp), D2Mit126 (84 Mbp), D2Mit100 (106 Mbp), D2Mit107 (133 Mbp), D2Mit226 (163 Mbp), D2Mit200 (179 Mbp) . Based on the genotyping, we selected the mice and crossed them to the mice of the subsequent generations. Finally, the male and female mice that were checked for homozygous genotypes on chromosome 2 were intercrossed to establish congenic mouse strains.
Bacterial strains and culture conditions
Streptococcus mutans JC-2 (serotype c ), which is resistant to streptomycin (200 μg/mL), was cultured in brain-heart infusion (BHI; Difco, Detroit, MI, USA) under an atmosphere of 95% N 2 and 5% CO 2 at 37 °C for 18 h. The bacterial cells were collected by centrifugation and suspended in 5 mL of BHI broth .
Dental caries induction
The mice were weaned at 21 days and fed on Diet#2000 (CLEA Japan Inc., Tokyo, Japan). Each mouse was infected for 7 days with S. mutans by inoculating a bacterial solution of 10 9 colony-forming units into the oral cavity. Bacterial colonization on the dental surface was confirmed on the 28th day by placing a sterilized swab into the mouth and culturing the oral swab material on Mitis Salivarius agar plates containing streptomycin (200 μg/mL). At 49 days of age, the mice were euthanized under CO 2 . Their mandible bones were removed, and the soft tissue was dissolved at 42 °C for 24 h in 2% KOH .
Calculation of the caries score
Both sides of the molars on the mandible were examined by microscopy and micro-computed tomography (micro-CT). The micro-CT images were taken at 90 kV and 200 μA for 3 min. The depth and extent of dental caries were examined using three parts of the images (buccal, central, and lingual). The caries score was evaluated according to the modified Keyes method applicable to mice . The maximum scores were as follows: 4 points at the first and second fissures in the mandibular first molar (M1), 2 points at the first fissure in the mandibular second molar (M2), and 1 point at the third fissure in M1 and first fissure in mandibular third molar (M3). The caries scores were calculated by adding the scores of M1, M2, and M3 on the left and right mandibules.
Measurement of the volume of stimulated saliva secretion
The volume of stimulated saliva secretion was measured as reported earlier . Mice of all strains studied herein (C57BL/6Slc, C3H/HeSlc, B6-Chr.2 C3H , and 3 congenic mouse strains) were intraperitoneally injected with pilocarpine (0.05 μg/100 g body weight) at 49 days of age. After injection, saliva was collected from the oral cavity of each mouse for 30 min using a pipette. The total saliva was weighed and statistically tested for significant differences by ANOVA.
Measurement of enamel hardness
Enamel hardness was measured as previously reported . The mandibles were extracted from the mice of each strain, and the soft tissue was manually removed. Mandibules were mounted on slide glass with composite resin. The enamel hardness of the lingual side of the first molar on the left mandible was measured with a Dynamic Ultra Micro Hardness Tester (Shimadzu, Kyoto, Japan). Enamel hardness was measured at three sites on the left first molar in one mouse of each strain, and the average was calculated.
Generation of congenic mouse strains
We generated three types of congenic mouse strains: one with a C3H/HeSlc-derived interval between D2Mit226 and D2Mit200 in a C57BL/6Slc genetic background (congenic 1); the second, with a C3H/HeSlc-derived interval between D2Mit126 and D2Mit226 (congenic 2); and the third, with a C3H/HeSlc-derived interval between D2Mit126 and D2Mit100 (congenic 3) ( Fig. 1 ).
Calculation of the caries score in each strain
The mandibles of each mouse strain were observed by microscopy and micro-CT ( Fig. 2 ). The means of the caries scores were 3.6 ± 4.9 in B6-Chr.2 C3H , 23.4 ± 1.1 in congenic 1, 14.8 ± 1.8 in congenic 2, and 27.2 ± 4.3 in congenic 3. They differed significantly between B6-Chr.2 C3H and the congenic strains, and between each congenic mouse strain ( Fig. 3 ). As control groups, C57BL/6Slc and C3H/HeSlc were also infected with S. mutans under the same conditions. The means of the caries scores were 47.6 ± 7.9 in C57BL/6Slc and 0.2 ± 0.4 in C3H/HeSlc. These results corresponded with those of earlier studies , and the difference was significant at p < 0.05, as determined by Tukey’s test.