2868
Long-term Hyperglycemia Naturally Induces Dental
Caries but Not Periodontal Disease in Type 1 and
Type 2 Diabetic Rodents
Yutaka Nakahara, Kiyokazu Ozaki, and Tetsuro Matsuura
Diabetes 2017;66:2868–2874 | https://doi.org/10.2337/db17-0291
Periodontal disease (PD) in patients with diabetes is de-
scribed as the sixth complication of diabetes. We have
previously shown that diabetes increases dental caries,
and carious inflammation might have a strong effect on
the adjacent periodontal tissue in diabetic rodent models.
However, the possibility that hyperglycemia may induce
PD in diabetic animals could not be completely elimi-
nated. The goal of this study was to confirm the presence
COMPLICATIONS
of PD in diabetic animal models by preventing carious
inflammation with fluoride administration. F344 rats in-
jected with alloxan (type 1 diabetic model) and db/db mice
(type 2 diabetic model) were given either tap water alone
or tap water containing fluoride. A cariostatic effect of fluo-
ride was evident in the diabetic animals. Meanwhile, fluoride
treatment drastically attenuated periodontal inflammation in
addition to preventing dental caries. Furthermore, with fluo-
ride treatment, periodontitis was notably nonexistent in the
periodontal tissue surrounding the normal molars, whereas
the caries-forming process was clearly observed in the
teeth that were enveloped with persistent periodontitis, sug-
gesting that enhanced periodontal inflammation might have
been derived from the dental caries in the diabetic rodents
rather than from the PD. In conclusion, long-term hypergly-
cemia naturally induces dental caries but not PD in type 1
and type 2 diabetic rodents.
Periodontal disease (PD) in patients with diabetes is de-
scribed as the sixth most common complication of diabetes
(1). Several recent studies have reported that the severity of
PD in patients with type 1 and type 2 diabetes is higher than
that in individuals without diabetes (2). Diabetic rodent
Laboratory of Pathology, Faculty of Pharmaceutical Sciences, Setsunan Univer-
sity, Hirakata, Osaka, Japan
Corresponding author: Tetsuro Matsuura, matsuura@pharm.setsunan.ac.jp.
Received 8 March 2017 and accepted 27 July 2017.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0291/-/DC1.
Y.N. and K.O. are joint first authors and contributed equally to this study.
Diabetes Volume 66, November 2017
models subjected to experimental manipulations such as
ligature placement have been used as diabetic PD models
(3–8). Furthermore, several studies have reported that only
long-term hyperglycemia can lead to naturally occurring PD
without experimental manipulation in diabetic rats (9,10).
Our previous studies revealed that both PD and dental
caries, which have completely different etiologies, appar-
ently occur in these diabetic models (11–13), and the onset
and progressive phases of dental caries may be strongly
involved in the development of periodontal inflammation.
However, we were not able to exclude the possibility that
hyperglycemia might induce PD-derived inflammation in
diabetic animals. If hyperglycemia induces PD in diabetic
animals, then PD-derived inflammation would indepen-
dently emerge in periodontal tissues by preventing car-
ious inflammation. On the basis of this hypothesis, the
current study was designed to assess the utility of al-
loxan-induced diabetic rats and diabetic db/db mice as
potential PD models by protecting dental caries with fluo-
ride treatment, which is considered effective against dental
caries not only in humans but also in experimental animals
(14,15).
RESEARCH DESIGN AND METHODS
Animals and Housing Conditions
Six-week-old female F344 rats were obtained from Japan
SLC, Inc. (Hamamatsu, Japan). Eight-week-old male db/db
and db/+ mice were purchased from Charles River Labora-
tories (Yokohama, Japan). The animals were housed and
handled under the same conditions as reported in previous
studies (12,13,16,17). All animal experiments were approved
Y.N. is currently affiliated with Drug Safety Research Laboratories, Astellas
Pharma Inc., Tsukuba, Ibaraki, Japan.
© 2017 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and the
work is not altered. More information is available at http://www.diabetesjournals
.org/content/license.
diabetes.diabetesjournals.org
by the Committee for Animal Experiments of Setsunan
University.
Experimental Design of the Rat Study (Type 1 Diabetic
Model)
Thirty rats (7 weeks of age) were intravenously administered
a single dose (35 mg/kg body weight) of alloxan according
to the previous studies (13,17) and were divided into three
groups. Each group of 10 diabetic rats was given either tap
water alone (the DM F0 group) or tap water containing
10 and 50 ppm sodium fluoride (the DM F10 and DM
F50 groups, respectively). Each group of 10 nondiabetic
rats (without alloxan dosing) was also given either tap wa-
ter alone (the non-DM F0 group) or tap water containing
50 ppm sodium fluoride (the non-DM F50 group). The
surviving 43 rats were autopsied at 20 weeks of age for
morphological examination.
Experimental Design of the Mouse Study
(Type 2 Diabetic Model)
From 10 weeks of age onward, each group of 10 db/db mice
was given either tap water alone (db/db F0 group) or tap
water containing 25 or 50 ppm sodium fluoride (the db/db
F25 and db/db F50 groups, respectively). Simultaneously,
each group of 10 db/+ mice was given either tap water alone
(db/+ F0 group) or tap water containing 25, 50, or 100 ppm
sodium fluoride (the db/+ F25, db/+ F50, and db/+ F100
groups, respectively). The surviving 56 mice were autopsied
at 40 weeks of age for morphological examination.
Glucosuria and Glycemia Monitoring
Glucose levels in fresh urine and tail vein blood samples
were measured according to the previous studies (12,13,17).
Macroscopic Examination of Dental Caries in Mice
Macroscopic examination of dental caries in mice was per-
formed using a stereoscope under the same conditions as
described in a previous study (12).
Soft X-ray Examination of Dental Caries and Alveolar
Bone Resorption in Rats
Soft X-ray examination of dental caries and alveolar bone
resorption (ABR) in rats was performed under the same con-
ditions as described in previous studies (13,17).
Histopathological Examination of Carious and
Periodontal Lesions in Rats and Mice
Histopathological examination was performed on the man-
dible and maxilla of each rat and mouse. Dental caries
including pulpitis (P), apical periodontitis (AP), periodontal in-
flammation including gingivitis (GV), marginal periodontitis
(MP), and ABR were evaluated and graded histopathologically
according to the previous study (17).
Statistical Analysis
The x2 test and Wilcoxon rank sum test were used accord-
ing to the previous studies (12,13,17). A P value ,0.05 was
considered statistically significant.
Nakahara, Ozaki, and Matsuura 2869
RESULTS
Severe Hyperglycemia and Glucosuria Continue
in Diabetic Rats and Mice
In all diabetic alloxan-treated rats and db/db mice, severe
hyperglycemia and glucosuria continued during the experi-
mental period. In all nondiabetic alloxan-nontreated rats and
db/+ mice, the blood and urine glucose levels were normal.
Fluoride Macroscopically Suppresses Dental Caries and
ABR in Alloxan-Induced Type 1 Diabetic Rats
Result of soft X-ray examination revealed that the develop-
ment of dental caries was suppressed by fluoride treatment
in a dose-dependent manner in diabetic rats, and the lesions
were scarcely observed in the DM F50 group. In comparison,
almost half of the molars were affected with dental caries in
the DM F0 group. Additionally, the mean caries scores and
caries incidence in the DM F10 and DM F50 groups were
significantly lower (P , 0.01) than those in the DM F0
group. No carious lesions were observed in any molars in
the nondiabetic rats (Fig. 1A and Supplementary Table 1).
High mean scores and high incidence of ABR were ob-
served in the DM F0 group using soft X-ray examination;
however, bone resorption was markedly suppressed by fluo-
ride treatment, which decreased dental caries. Lesions were
not detected in any maxillary molars in the DM F10 and DM
F50 groups or in the mandibular molars in the DM F50
group. In the non-DM F0 and non-DM F50 groups, ABR was
not observed in any molars (Fig. 1B and Supplementary
Table 2).
In addition, ABR was characterized on the basis of its
presence in the apical area adjacent to carious molars (Fig.
1C). There were no radiolucent changes in the alveolar bone
around the noncarious molars in the fluoride-treated dia-
betic rats (Fig. 1D).
Fluoride Histologically Suppresses Periodontal Lesions
and Dental Caries in Alloxan-Induced Type 1 Diabetic
Rats
Histopathologically, fluoride treatment significantly (P ,
0.01) suppressed the incidence of dental caries in the di-
abetic rats. Together with the prevention of dental caries,
the incidence of AP was also markedly decreased (P , 0.01)
in the DM F10 and DM F50 groups compared with the DM
F0 group. Furthermore, MP was not observed in any molars
of the DM F10 and DM F50 groups. The incidence of GV in
these two groups was significantly decreased (P , 0.01)
compared with that in the DM F0 group and was compa-
rable to the incidence in fluoride-treated and -untreated
nondiabetic rats (Fig. 2A).
In the fluoride-untreated diabetic rats, MP was always
accompanied by moderate (++) caries with P and AP as well
as moderate (++) GV. The inflammatory cells in the
periodontal tissue (i.e., MP) were exclusively adjacent to
suppurative AP (Fig. 2B). Furthermore, neither AP nor MP
was detected in any region around the noncarious molars
regardless of the presence or absence of diabetes (Fig. 2C
and Supplementary Table 3 and 4).
2870 Diabetes Induces Caries but Not PD in Rodents Diabetes Volume 66, November 2017

Figure 1—Fluoride macroscopically suppresses dental caries and ABR in alloxan-induced type 1 diabetic rats. A: The mean caries (C) scores in
the maxillary molars and mandibular molars of alloxan-induced diabetic rats and nondiabetic rats by soft X-ray examination. Significantly
different from the diabetic F0 group (**P < 0.01). B: The mean ABR scores in the maxillary molars and mandibular molars of alloxan-induced
diabetic rats and nondiabetic rats by soft X-ray examination. Significantly different from the diabetic F0 group (**P < 0.01). C and D: Soft X-ray
images of dental caries and ABR in alloxan-induced diabetic rats. M1, the first molar; M2, the second molar; M3, the third molar. C: The mandible
of a fluoride-untreated rat (the DM F0 group). Dental caries with a focal radiolucent area (arrowheads) are observed in the dental crown. In the
alveolar bone, focal radiolucent areas are detected in the apical area adjacent to the carious molars (arrows). D: The mandible of a fluoride-
treated rat (the DM F50 group) with intact molars and normal alveolar bone. Scale bars = 2 mm.

Fluoride Suppresses Periodontal Lesions and Dental
Caries in Type 2 Diabetic db/db Mice
Fluoride treatment also macroscopically suppressed dental
caries in the diabetic db/db mice; however, this effect was
milder than that in the diabetic rats. Caries development
tended to be largely prevented by fluoride treatment, and
the mean score and incidence of lesions in the db/db F50
group were significantly lower than those in the db/db F0
group. No dental caries were observed in the db/+ F0 group,
although some molars (,10%) were affected with dental
caries in the db/+ F25, db/+ F50, and db/+ F100 groups
(Fig. 3A and Supplementary Table 5).
Histopathologically, fluoride treatment mildly suppressed
the development of dental caries and reduced the incidence
of lesions in the db/db F0 group to almost half of that in the
db/db F25 and db/db F50 groups. In mice, the incidence of
AP was almost the same as that for MP, and the incidence
of these two lesions in the db/db F25 and db/db F50 groups
was approximately half of that in the db/db F0 group, as
was the case with the incidence of caries. GV was equally
observed in almost 40% of each group of nondiabetic db/+
mice, whereas the incidence of GV was almost doubled in
the db/db F0 group. The incidence of GV in the db/db F25
and db/db F50 groups was slightly suppressed as the rate of
dental caries decreased; however, the incidence of GV was
not reduced as much as it was in nondiabetic mice with few
dental caries (Fig. 3B).
In fluoride-untreated diabetic db/db mice, ABR and MP
were inevitably accompanied by moderate caries (++) with
P, AP, and moderate GV (++) (Fig. 3C–E). In contrast,
ABR and MP were not detected in any region around the
noncarious molars regardless of the presence or absence of
diabetes (Fig. 3F–H). In addition, ABR and MP remained in
the fluoride-treated diabetic db/db mice and were always
found together with AP (Fig. 3I–K and Supplementary
Tables 6 and 7).
DISCUSSION
The current study showed that suppurative AP that originated
from crown caries (Fig. 4A) and periodontal inflammation,
including GV, MP, and ABR, which is pathognomonic for
PD (Fig. 4B), concurrently developed in periodontal tissues
around carious molars in the diabetic rodents. In addition,
diabetes.diabetesjournals.org Nakahara, Ozaki, and Matsuura 2871

Figure 2—Fluoride histopathologically suppresses periodontal lesions and dental caries in alloxan-induced type 1 diabetic rats. A: The incidence
of histopathological findings in alloxan-induced diabetic rats and nondiabetic rats. Significantly different from the diabetic F0 group (**P < 0.01).
C, caries. B and C: The histopathological features of periodontal lesions and dental caries in alloxan-induced diabetic rats. B: Periodontal
inflammation adjacent to a carious molar in a fluoride-untreated diabetic rat (the DM F0 group). MP, GV, and ABR (asterisks) are accompanied by
moderate caries (arrowhead) with AP (arrow). The inflammatory cells in the periodontal tissue are adjacent to the area of apical inflammation. C:
Normal periodontal tissues are adjacent to an intact molar in a fluoride-treated diabetic rat (the DM F50 group). AP and MP are not observed in
any region around the noncarious molars. Scale bars = 400 mm.
2872 Diabetes Induces Caries but Not PD in Rodents Diabetes Volume 66, November 2017

Figure 3—Fluoride suppresses periodontal lesions and dental caries in type 2 diabetic db/db mice. A: The mean caries (C) scores for the
maxillary molars and mandibular molars of diabetic db/db mice and nondiabetic db/+ mice by macroscopic examination. Significantly different
from the db/db F0 group (*P < 0.05; **P < 0.01). B: The incidence of histopathological findings in diabetic db/db mice and nondiabetic db/+
mice. Significantly different from the db/db F0 group (*P < 0.05; **P < 0.01). C–K: Histopathological features of periodontal lesions and dental
diabetes.diabetesjournals.org Nakahara, Ozaki, and Matsuura 2873

Figure 4—Diagrammatic representation of the lesion process. A: Caries-forming process. B: PD-forming process. C: Periodontal inflammation
surrounding a carious molar in a diabetic rodent. D: Periodontal inflammation surrounding a noncarious molar. E: A scheme of the expected
process of periodontal inflammation surrounding carious molars in the diabetic (DM) rodent model. F: The PD-forming process in an animal
model for human PD with experimental manipulations. AB, alveolar bone; AF, apical foramen; D, dentin; E, enamel; F, fluoride; G, gingiva; P,
dental pulp; PL, periodontal ligament.

the prevalence of periodontal inflammation was highly cor-
related with that of dental caries, and MP was frequently
found with carious inflammation (Fig. 4C). These results
suggest that carious inflammation might have a strong effect
on adjacent periodontal tissue in diabetic animals. However,
we could not definitively distinguish PD-derived inflammation
from carious inflammation around the dental root. As a result
of fluoride treatment, the cariostatic effect on teeth (14,15) in
diabetic rodent models was obvious, and the progression of
dental caries was markedly suppressed.
In the diabetic rodent models, fluoride treatment dras-
tically attenuated the incidence and severity of periodontal

caries in diabetic db/db mice. The boxes in C correspond to D and E. The boxes in F correspond to G and H. The boxes in I correspond to
J and K. C–E: Periodontal inflammation adjacent to a carious molar in a fluoride-untreated diabetic db/db mouse (the db/db F0 group). C: Severe
caries with P. P is associated with AP. D: AP with ABR. Apical inflammation expands to the marginal area. E: MP and moderate GV are ac-
companied by caries with P and AP. F–H: Normal periodontal tissue adjacent to an intact molar in a fluoride-treated diabetic db/db mouse
(the db/db F50 group). F: Intact molar and normal periodontal tissue. G: Normal apical tissue. H: Normal gingiva and marginal tissue. I–K:
Apical and marginal inflammation adjacent to a carious molar in a fluoride-treated diabetic db/db mouse (the db/db F25 group). I: Moderate
caries accompanied by pulpal and apical inflammation. J: AP and surrounding ABR. K: MP with ABR is found together with apical inflammation.
Scale bars = 100 mm.
2874 Diabetes Induces Caries but Not PD in Rodents
inflammation in addition to preventing dental caries. ABR
and MP persisted together with enhanced GV, and PD ap-
parently occurred in some caries in fluoride-treated diabetic
rodents. However, the lesions inevitably led directly to AP,
followed by P, and were completely consistent with the
incidence of dental caries. Fluoride has no inhibitory effect
on oral bacteria (18), and there is no report involving the
effect of the fluoride on periodontal inflammation in hu-
man and experimental animals at present. It is highly prob-
able that fluoride could not suppress the progression of
dental caries once cariogenic bacteria penetrate the tooth
surface. Moreover, ABR was usually detected in diabetic
animals at the apical area adjacent to the carious molars
(Fig. 4C), although this lesion is characterized by a decline
in the alveolar bone crest in human PD (Fig. 4B); the site of
ABR completely differed between patients with diabetes with
PD and diabetic rodents. Thus, these facts strongly suggest
that carious inflammation may expand from the apex of a
dental root and progress rostrally, thereby affecting the mar-
ginal periodontal tissue when carious inflammation penetrates
the apical foramen (Fig. 4E). PD-derived inflammation might
not have developed in our type 1 and type 2 diabetic animal
models.
The establishment of animal models for diabetic PD is
necessary because diabetes is becoming an epidemiologically
important risk factor for PD in humans (1). Human PD is
initiated by GV and progresses apically along the periodon-
tal ligament, causing progressive ABR (Fig. 4B) (19). Similar
PD progression is confirmed in nondiabetic rodents sub-
jected to ligature placement, gingival inoculation with peri-
odontal pathogens, or lipopolysaccharides (20), whereas GV
surrounding noncarious molars did not progress further
and cause MP and ABR associated with long-term hypergly-
cemia in our 2 diabetic animal models (Fig. 4D). These facts
suggest that a certain threshold stimulus including above-
mentioned experimental manipulation that causes the de-
struction of gingival tissue may also be necessary for the
development of the clinical state of diabetic PD (3) (Fig. 4F).
Moreover, it is certain that hyperglycemia causes dental
caries in diabetic rodents (9–13,21) and that carious inflam-
mation leads to mistakes in interpreting the development
of PD in diabetic animals. Protection against dental caries is
an indispensable tool for research on PD using diabetic an-
imals, and caries inhibition by fluoride treatment is a simple
but effective tool for this type of research. In conclusion,
long-term hyperglycemia naturally induces dental caries but
not PD in the studied type 1 and type 2 diabetic rodents.
Acknowledgments. The authors thank the Kumamoto Laboratory of the
Nonclinical Research Center of LSI Medience Corporation for conducting the rat experi-
ments. The authors also thank Nana Shako, Laboratory of Pathology, Faculty of
Pharmaceutical Sciences, Setsunan University, for help with the mouse experiments.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. Y.N. participated in the study conception and
design, performed the experiments, analyzed data, and wrote the manuscript. K.O.
participated in the study conception and design, participated in the experiments
Diabetes Volume 66, November 2017
and data interpretation, and reviewed and edited the manuscript. T.M. participated in
the study conception and design, participated in data interpretation, and reviewed
and edited the manuscript. T.M. is the guarantor of this work and, as such, had full
access to all the data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
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