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JDRXXX10.1177/0022034518789898Journal of Dental ResearchEarly Caries in an In Vivo Model

Research Reports: Biomaterials & Bioengineering

Journal of Dental Research
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Early Caries in an In Vivo Model: Structural © International & American Associations
for Dental Research 2018

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DOI: 10.1177/0022034518789898
https://doi.org/10.1177/0022034518789898
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D.T. Yucesoy1, H. Fong1, C. Gresswell1, S. Saadat1,2, W.O. Chung2,

S. Dogan3, and M. Sarikaya1,2,4

Abstract
The utilization of rat models in cariology research has made substantial contributions to decipher mechanisms of caries formation and
to develop preventive treatments. The existing rat models still have potential for improvement toward establishing a more accurate
standard caries protocol to utilize in testing and/or developing new dental technologies. The current caries-scoring methods rely on
optical microscopy–based techniques, which necessitates formation of highly advanced lesions. Moreover, models that facilitate the
implementation of cariogenic bacteria by shifting the balance of oral flora through desalivation and/or antibiotic treatment create
a nonnatural environment. Furthermore, there is a paucity of detailed structural and mechanical characterization on the resulting
carious lesions. The purpose of this study was to develop a rat model that induces formation of mild carious lesions and to provide
comprehensive structural and mechanical characterization. With this aim in mind, an in vivo model promoting progression of mild lesions
was established with specific pathogen-free Sprague-Dawley rats. Cariogenic bacteria, Streptococcus mutans, was implemented into the
oral flora without the use of antibiotics or desalivation surgery. During caries formation, progression of the infection was monitored by
quantifying the relative abundance of S. mutans in oral flora with quantitative real-time polymerase chain reaction. A significant increase
in colonization efficacy of S. mutans was detected during cariogenic challenge (P < 0.01). The resulting carious lesions were analyzed by
conventional light optical and scanning electron microscopy. A detailed structural and morphological characterization on fissure caries
with different degrees of severity was provided. The changes in the morphology and demineralization state of the sound and carious
tissues were quantified by energy-dispersive X-ray spectroscopy, and local mechanical properties were acquired with nanoindentation.
The principles laid out in this work can be utilized in cariology research and developed into a standard protocol for future studies.

Keywords: caries detection, electron microscopy, demineralization, saliva, nanoindentation, Streptococcus mutans

Introduction cariogenic bacteria levels in the oral cavity. Their presence in

Animal models have made a substantial contribution in eluci-
dating the etiology of caries and led to the development of
diagnostic and preventative products (Loesche 1986; Zhang et
al. 2016). A major issue of in vivo procedures, however, is the
inconsistency of the parameters and factors that induce caries
formation (Bowen 2013). In particular, the lack of details about
the cariogenic diet and feeding conditions (Guggenheim et al.
1966; Navia et al. 1969; Firestone et al. 1982), infectious
organism (Willcox et al. 1989; Klinke et al. 2011), age of ani-
mals (Hietala et al. 1997; Bao et al. 2015), and housing condi-
tions (Schuster et al. 1978) often limits the reproducibility of
these procedures (Bowen 2013).
Colonization of cariogenic organisms is essential for caries
formation. Although this can be achieved by direct inoculation
of cariogenic bacteria (Han et al. 2017), the removal of salivary
glands and antibiotic treatment are still commonly utilized pro-
cedures (Zhang et al. 2016; Free et al. 2017; Liu et al. 2018).
Despite rapid colonization, these procedures often disrupt the
balance of oral microbiota and create a nonnatural environment
predominated by particular bacterial species (Kumar and Mason
2015). Another common challenge is the limited detection of
high levels in the oral flora is an indication of caries formation
(van Houte 1994; Tanzer et al. 2001). Accurate detection and
quantification of cariogenic organisms in animals are therefore
crucial. Bacterial species in the current models are detected by
agar plating. These methods, however, do not allow accurate
quantification due to the biased growth of bacteria under in
vitro conditions (Yoshida, Suzuki, Nakano, Kawada, et al.
1
GEMSEC, Genetically Engineered Materials Science and Engineering
Center, Department of Materials Science and Engineering, University of
Washington, Seattle, WA, USA
2
Department of Oral Health Sciences, University of Washington, Seattle,
WA, USA
3
Department of Restorative Dentistry, University of Washington,
Seattle, WA, USA
4
Department of Chemical Engineering, University of Washington, Seattle,
WA, USA
A supplemental appendix to this article is available online.
Corresponding Author:
M. Sarikaya, Department of Materials Science and Engineering, University
of Washington, 327 Roberts Hall, Seattle, WA 98195-2120, USA.
Email: sarikaya@u.washington.edu
2 Journal of Dental Research 00(0)
2003). Quantitative real-time polymerase chain reaction
(qPCR) assay is an alternative approach to conventional agar
plating for early detection and real-time monitoring of cario-
genic organisms in saliva (Yoshida, Suzuki, Nakano, Oho, et al.
2003). Implementing qPCR to a rat model could therefore
facilitate quantifying the cariogenic state of bacteria during the
progression of caries.
Another common challenge is the limited resolution of the
conventional optical microscopy–based caries detection and
scoring techniques (Keyes 1958; Larson 1981; Bowen et al.
1986; Han et al. 2017). Due to this limitation, most animal
models were developed to promote formation of relatively
advanced carious lesions (Hafström-Björkman et al. 1991;
Hietala et al. 1993). Caries formation starts with initial demin-
eralization and then progresses into a reversible early-stage
lesion that eventually leads to an irreversible lesion with cavi-
tation (Cummins 2013). Therefore, therapies that facilitate
arresting and remineralizing early lesions need to be promoted
to preserve the form and function of the tooth (Pretty 2006;
Yen and Yelick 2011). The key to this vision is to modify cur-
rent caries formation and scoring protocols so that new thera-
pies targeting early caries can be tested in vivo. At this point,
presenting data only by conventional scoring may be incom-
plete or misleading. The caries scores should be supported by
complementary methods that offer more explicit analyses
(Pretty and Ellwood 2013). Scanning electron microscopy
(SEM) provides the resolution to detect and quantify subtle
changes in the morphology and demineralization state of the hard
tissues (Hietala et al. 1993; Angker et al. 2004; Reynolds et al.
2008). Furthermore, nanomechanical testing (e.g., nanoinden-
tation) provides invaluable information about the local
mechanical properties of hard tissues to correlate the function
to the structure obtained from the SEM analysis (Fong et al.
2003; Dogan et al. 2018).
In this study, the 2-pronged aim was 1) to develop a rat
model that promotes slow progression of mild caries with a
strict monitoring of the colonization level of Streptococcus
mutans by qPCR and 2) to establish a structure-function rela-
tionship by interrogating the local morphology, mineral con-
tent, and mechanical properties of early fissure caries with
light microscopy, SEM, and nanoindentation. The principles
laid out here can be applied to other in vivo cariology models
and developed into a standard protocol for future caries treat-
ment and remineralization studies.
Materials and Methods
Animals
Thirteen-day-old specific pathogen-free Sprague-Dawley rats
(14 female and 14 male, Rattus norvegicus, Crl:SD code 400;
Charles-River) were obtained with 7 dams and housed in plas-
tic cages (Allentown Inc.) on corncob bedding with access to
food and water ad libitum. Prior to shipping, rats were screened
by vendor for the absence of specific pathogens (see Appendix
MM1). Animals were handled in accordance to guidelines of
the National Research Council’s Guide for the Care and Use of
Laboratory Animals. All experimental procedures were
approved by the Institutional Animal Care and Use Committee
of University of Washington. The study is reported in accor-
dance with the ARRIVE guidelines (Kilkenny et al. 2010).
Bacteria Maintenance and Culturing
S. mutans UA159 (American Type Culture Collection 700610)
was utilized as a cariogenic bacterium and cultured according
to instructions described previously (Yucesoy et al. 2015; see
Appendix MM2). Before inoculation, S. mutans was grown to
a midlog phase. Next, cells were harvested at 2,000 × g for 5
min and resuspended in fresh brain-heart infusion broth.
S. mutans Infection and Caries Formation
When pups were aged 14 d (day 5), the dams were infected
with S. mutans by pipetting 0.1 mL of a cell suspension con-
taining ~109 colony-forming units into their mouths. Four pups
were kept as a negative control. The infection procedure was
repeated for 5 consecutive days (days −5 to −1) to establish
colonization of S. mutans in dams so that vertical transmission
of infection to pups would occur.
The pups were weaned when aged 19 d (day 0) and distrib-
uted by sex (4 animals per cage). Next, pups were infected
directly with S. mutans for 4 consecutive days by pipetting 0.1 mL
of a S. mutans suspension containing ~109 colony-forming
units into their cheek pouches. During the inoculation period
(days 0 to 3), access of pups to the bedding was restricted by
placing a stainless-steel mesh on top of the bedding. All ani-
mals were fed with cariogenic MIT-200 powdered diet (w/w,
67% sucrose, 20% lactalbumin, 3% cottonseed oil, 6% cellu-
lose, 1% vitamin mix No. 40060, and 3% mineral mix No.
70191; Harlan Labs; Michalek et al. 1975) and filter-sterilized
10% sucrose water (w/v) ad libitum.
Monitoring S. mutans Infection
S. mutans colonization in pups was monitored by colony count-
ing and qPCR. On day −5, saliva samples were collected from
randomly selected 12 rats. Animals were anesthetized with iso-
flurane, and the saliva accumulated under their tongue and on
cheek pouches was collected. Saliva samples were streaked
onto selective mitis salivarius–bacitracin agar plates supple-
mented with 1% potassium tellurite and 0.2 U/mL of bacitracin
(selective for mutans group streptococci) and incubated at 37
°C for 48 h under anaerobic conditions (Saravia et al. 2013).
Resulting colonies were enumerated (see Appendix MM2).
For qPCR, oral swabbing did not generate enough sample
for accurate analysis; therefore, extracted teeth were used for
sample collection. Animals (3 animals per each day) were ran-
domly selected on days 0, 6, 12, and 25 and sacrificed by CO2
asphyxiation (n = 3). Dissected jaws were suspended in 5 mL
of sterile Dulbecco’s phosphate-buffered saline (Gibco) and
sonicated for 15 s. Cells were harvested at 13,000 × g for
2 min, and genomic DNA was isolated with Wizard DNA
Purification Kit (Promega) and utilized as a template (see
Early Caries in an In Vivo Model 3

Appendix MM3). The reaction parameters,

oligonucleotide primers, and probes used in
qPCR are listed in the Appendix Table.

Caries Assessment with Optical

Microscopy
Caries scoring was performed with the mod-
ified Keyes’s method (Keyes 1958; Larson
1981). Appendix MM4 details the chemi-
cals and procedures used in caries scoring.
Figure 1.  Schematics of experimental timeline of bacteria inoculation, sucrose challenge,
infection monitoring during caries formation, and the characterization procedures used. Arrows
SEM and EDXS Analysis pointing up indicate days of bacteria sampling. Inoculation days of dams and pups are depicted
with curved arrows. EDXS, energy-dispersive X-ray spectrometry; SEM, scanning electron
SEM and energy-dispersive X-ray spec- microscopy.
trometry (EDXS) characterization was car-
ried out in a JSM 6100 (JEOL) at 15 kV, as described previously
(Dogan et al. 2018; see Appendix MM5). Line profiles were
obtained from different regions with ImageJ and mean profiles
calculated (n = 5). Full-width half-maximum values of nega-
tive peaks appearing at the dentin-enamel junction (DEJ) were
determined after background correction with OriginPro 8
software.
Nanoindentation Tests
Nanoindentation measurements were carried out on hemisec-
tioned teeth with an Ubi nanoindentation system (Hysitron) as
described previously (Dogan et al. 2018; Gibson et al. 2008;
Oliver and Pharr 1992) (see Appendix MM6). The maximum
indentation depth for all measurements was kept at 120 ± 10
nm. Results were averaged over 40 measurements.
Statistics
Caries scores are expressed as mean ± SE. Statistical analysis
was performed on the relative abundance of S. mutans with the
Microsoft Excel Worksheet, and the statistical significance of
mean values was determined by a Student’s t test, with P <
0.01 considered significant.
Results
Here, we report the development of a rat model that induced
formation of mild carious lesions with real-time monitoring of
S. mutans infection. Detailed morphology, mineral content,
and local properties analyses of early fissure caries were done
with optical microscopy, SEM, and nanoindentation (Fig. 1).
Selection, Weight Gains, and General
Health of Animals
Female and male rats were used to primarily eliminate any pos-
sible potential sex bias in caries formation, as discussed by
clinical studies (Lukacs and Largaespada 2006). All animals
thrived throughout the experiment, and no significant differ-
ence in their weight gains was observed.
Monitoring S. mutans Infection
Mitis salivarius–bacitracin agar plates that were inoculated
with day −5 saliva samples (before bacteria inoculation)
showed no evidence of S. mutans growth. The relative abun-
dance of S. mutans in mouth flora at day 0 (weaning day) was
0.00112% ± 0.00005%, demonstrating that the vertical trans-
mission from dams to pups was successful (Fig. 2A). The
relative amount S. mutans increased gradually and reached
0.00671% ± 0.00008% at day 6. The saturation point of
S. mutans colonization was observed at day 12 with 0.01304%
± 0.00012%. Afterward, the relative abundance of S. mutans
remained relatively stable at 0.01281% ± 0.00015% without a
significant change (P = 0.42, t test) in comparison with day 12
(n = 3).
Caries Assessment with Optical Microscopy
The carious lesions established on buccal/lingual (smooth),
morsal, sulcal, and proximal sites were scored according to
penetration depth of murexide as enamel or slight, moderate,
or extensive dentin-type lesions. Representative images of
average carious lesions obtained after 50 d of postinfection and
resulting mean caries scores per animal (12 animals, n = 12)
are shown in Figure 2B to 2E and 2G, respectively. Darker
regions that are visible on buccal surfaces before (Fig. 2B) and
after (Fig. 2C, D) staining were scored as enamel-type lesions.
On fissure/sulcal and proximal sites, dentin-type lesions
were more prominent, possibly due to the more favorable
microenvironment for colonization (Fig. 2E). Especially in the
distal fissures of first and second molars, lamination of dentin
was visible, indicating the severity of lesions. Moreover, in the
middle fissure of the first molar and mesial fissure of second
molar teeth, enamel loss was very apparent. No carious lesion
formation was observed in noninfected rats with murexide
staining (Fig. 2F). Overall, enamel-type caries with a slight
4 Journal of Dental Research 00(0)

showed no cavitation on enamel. The uni-

form contrast of dentin underneath indi-
cated no significant demineralization
activity (Fig. 3A2–A4). The murexide-
positive enamel-type lesion located on the
proximal surface of the second molar
(Fig. 3B2), however, showed a cavitation
about ~80 µm in depth. In contrast, 2 fis-
sure lesions that were scored as extensive
and moderate dentin lesions based on
staining (Fig. 3B3, B4) displayed superfi-
cial cavitation on enamel with an almost
intact dentin underneath.
SEM classification on fissure caries is
demonstrated in Figure 4, with 4 represen-
tative specimens—3 of which exhibited
fissure caries with different degrees of
severity and 1 with no caries. SEM images
of the caries-free control sample (Fig.
4A1), recorded in secondary electron
imaging mode, showed no caries-like
structural features. Moreover, the uniform
contrast through the enamel and dentin in
the BSE images and EDXS maps indi-
cated that there was no demineralization.
The narrow crack with smooth edges is
more likely due to the drying process dur-
ing sample preparation.
In the enamel-type lesion (Fig. 4B1),
the enamel layer at the lateral site of the
fissure became thinner due to demineral-
ization while the underlying dentin was
still intact. The uniform contrast through
the dentin in the BSE image of Figure 4B2
and EDXS calcium and phosphorus maps
(Fig. 4B3, B4) indicated that the mineral
content was identical and that demineral-
ization was restricted to enamel.
In the moderate dentin-type lesion
(Fig. 4C1), enamel cracks were more
Figure 2.  Monitoring of S. mutans infection progression in rats and optical analysis of carious
prominent lateral to the fissure. The long
lesions with caries scores. (A) Relative abundance of Streptococcus mutans in oral flora (y-axis) on crack starting from the enamel and pro-
different days (x-axis) quantified by quantitative real-time polymerase chain reaction. *P < 0.01 vs. gressing into the dentin implies the pres-
day 0. **P < 0.01 vs. day 6. #No significant difference vs. day 12. Representative carious lesions ence of more severe demineralization as
formed at day 50 (B) before and (C, D) after murexide staining. Representative hemisectioned
teeth images from (E) infected and (F) noninfected (control) animals. Surface lesions are visible compared with the enamel lesion in
before and after staining, while fissure carious lesions dominate cross-sectioned images of teeth Figure 4C. The dark gray area adjacent to
obtained from infected animal. (G) Mean caries scores per rat (12 animals, n = 12) categorized the enamel (Fig. 4C2) implies lesser min-
as enamel (E) and slight (Ds), moderate (Dm), or extensive (Dx) dentin-type lesions based on eral content versus the rest of dentin. The
Keyes’s scoring. Arrows indicate molar fissure regions where caries are present in infected rats
(E) but not in the noninfected control group (F). decreases in calcium and phosphorus
contents in this particular demineralized
dentinal involvement were developed more prevalently than area were confirmed with EDXS (Fig. 4C3, C4). Here, dentin
moderate and extensive dentinal lesions (Fig. 2G). is still intact without any delamination, classifying it as moder-
ate dentin-type lesion.
In the last set of images shown in Figure 4D, the enamel on
SEM and EDXS Analysis
one side of the fissure (highlighted with arrow) is lost com-
Backscattered electron (BSE) images of fissures after murexide pletely, and the underlying dentin is delaminated. The loss of
staining are demonstrated in Figure 3. The murexide-negative underlying dentin in this particular area was about 100 µm in
fissures and proximal sites on the second molar (Fig. 3A1) depth, indicating extensive dentin lesion.
Early Caries in an In Vivo Model 5

The dark gray regions adjacent to the

DEJ were further analyzed by line profiling
(Fig. 4A2–D2, dotted line), and the change
in the contrast by full-width half-maximum
analysis provided the average width of the
crack. The calculated values for caries-free,
enamel, moderate, and extensive samples
were 16.15 ± 1.25 µm, 2.50 ± 1.25 µm,
10.56 ± 1.27 µm, and 9.32 ± 1.25 µm,
respectively (Fig. 4A5–D5). The differ-
ences between the peak minimum and
average background were 2.98, 9.47, 5.70,
and 55.45 (gray values, arbitrary unit),
respectively. The increase in the calculated
peak minimum-average background values
compared with the caries-free sample
reflects the extent of the demineralized
region due to cariogenic activity.

Nanoindentation Tests
Local nanomechanical properties of sound
and carious tissues were determined with
nanoindentation (Fig. 5). The measured
hardness and elastic modulus of the sound
enamel were 3.27 ± 0.38 GPa and 68.7 ±
6.5 GPa, respectively. In carious enamel,
about a 50% reduction in hardness (1.29 ±
0.28 GPa) and elastic modulus (34.41 ±
6.4 GPa) values was observed in compari-
son with the sound enamel. A similar
decreasing trend in hardness and elastic
modulus values was also observed in cari-
ous dentin (0.82 ± 0.08 GPa and 21.7 ± 1.6
GPa) in comparison with sound dentin
(1.27 ± 0.17 GPa and 33.1 ± 3.1 GPa).

Discussion
Figure 3.  Hemisectioned molar teeth obtained from noninfected rats: (A1) optical and (A2–
An in vivo caries formation model pro- A4) backscattered electron images. The second molar showing a uniform contrast along enamel
moting mild carious lesions in rats was and dentin: (A2) proximal and (A3, A4) fissure surfaces. Hemisectioned molar teeth obtained
developed in this study. The resulting cari- from infected rats: (B1) optical and (B2–B4) backscattered electron images. (B2) An E-type
ous lesions were then analyzed in detail lesion on proximal surface of the second molar. (B3, B4) Dx and Dm type of carious lesions in
fissure sites. White arrows indicate comparable locations of the molars from the control (A2 and
via structural imaging and nanomechani- A4) and infected (B2 and B4) groups.
cal tests to develop an in-depth under-
standing of the nature of carious lesions. To ensure prominent of the colonization efficacy of S. mutans in oral flora and
caries formation, emphasis was placed on the age of animals, thereby enabled assessment of the success of the inoculation
infection procedure, and diet. In contrast to the previously procedure in real time.
established models, S. mutans colonization was ensured by a Following the Larson’s (1981) scoring method, carious
prior inoculation of dams for vertical transmission of S. mutans, lesions were initially evaluated with murexide staining. While
as well as direct inoculation of pups without the use of antibiot- murexide staining was useful in locating the carious lesions
ics. Moreover, during the inoculation process, pups’ access to (Fig. 2), it was limited in revealing the details of the caries.
their bedding and any chewable objects were eliminated so that This limitation, however, was alleviated by SEM imaging and
the possible tooth-cleaning effect of chewing was prevented nanoindentation testing. For example, in the light optical image
(Navia and Harris 1969; Schuster et al. 1978). Furthermore, the in Figure 3B1, although all the regions (fissures and proximal)
implementation of qPCR to the rat model facilitated monitoring were murexide positive, it was unclear how advanced the
6 Journal of Dental Research 00(0)

Figure 4.  Hemisectioned rat molars: (A1–D1, first row) secondary electron imaging and (A2–D2, second row) backscattered electron images and
associated (A3-D3, third row) calcium (Ca) and (A4–D4, fourth row) phosphorus (P) energy-dispersive X-ray spectrometry maps. (A5–D5, fifth
row) Line profile analysis along the dotted line in A2 to D2 (row 5). Black arrows indicate crack positions on line graphs. Each sample was stained
with murexide before cross-sectioning and scanning electron microscopy analysis. Murexide staining analysis demonstrated no caries on the first
sample (column A), while others showed enamel (column B) and moderate (column C) and extensive (column D) dentin-type lesions. Elemental
concentrations on energy-dispersive X-ray spectrometry maps were shown in an increasing order with black, blue, green, and red dots. Yellow arrows
point out the crack positions on each image, while the white arrows highlight the demineralized regions adjacent to dentin-enamel junction (DEJ).

carious lesions were in the 3 regions, shown by arrows. The
SEM imaging revealed that the extent of enamel cavitation was
the most severe in Figure 3B2 whereas the enamel was mostly
intact in Figure 3B3. In all cases, SEM revealed a dark band at
the DEJ, indicating severe demineralization along the bound-
ary between enamel and dentin tissues. However, the dark
bands seen here are relatively narrow, within 20 µm, about an
order of magnitude smaller than the width obtained by murex-
ide staining (Fig. 3B1). This suggests that the severity of den-
tin-type caries might not have been as extensive as indicated
with the staining visualized by optical microscopy. Since den-
tin is rich in organic content (i.e., collagen) and porous, the
stain could have possibly penetrated the healthy dentin. It
should also be pointed out that fissure cracks are occasionally
created artificially on enamel during subsequent cleaning and
staining steps, which involves drying of the tooth specimen. In
such cases, the stain would penetrate through the artificially
created fissure cracks and possibly cause false-positive caries
scores. It is further noted that demineralization along the DEJ
was prominent in Figure 4C2 and 4D2, a common observation
of fissure caries by SEM analysis. The accompanying EDXS
maps, indeed, confirmed a decrease in mineral content within
the DEJ. Therefore, the demineralized region along the DEJ
could be used as a specific feature in SEM and EDXS analyses
to specify demineralized areas (Fig. 4D1–D4) and differentiate
them from the drying artifacts (Fig. 4A1–A4).
The carious enamel and dentin along the DEJ were further
quantified for mechanical properties by localized nanoindenta-
tions in discrete regions. As shown in Figure 5C to 5E, the cari-
ous enamel and dentin had elastic modulus and hardness values
Early Caries in an In Vivo Model 7

Figure 5.  Nanomechanical characterization of carious and healthy dentin and enamel. (A, B) Light optical images of indentation sites. Average
indentation curves (force-depth) of (C) enamel and (D) dentin. (E) Mean hardness (H) and elastic modulus (E) values of carious and sound dentin and
enamel tissues. (F) Indentation footprints of indenter tip on carious and healthy dentin and enamel tissues.

significantly lower than those corresponding to the sound tis-
sues. Some carious regions, however, resulted in rougher sur-
faces after polishing, which prevented complete nanoindentation
measurements on the entire surface. Nanoindentation in this
work was nonetheless shown to be a highly reliable tool in pro-
viding further insights into the nature of the carious versus sound
tissues in examining the degree of demineralization quantita-
tively. Taken together, the accuracy of overall caries score can be
enhanced by SEM imaging with EDXS elemental analysis and
nanoindentation. Incorporating these analytical techniques in
caries scoring can help eliminate false-negatives or false positives
that are prevalent in data relying solely on optical imaging.
In summary, an in vivo model was developed promoting
slow progression of mild caries in rats. S. mutans colonization
was facilitated via vertical transmission. Besides the conven-
tional infection-monitoring and caries-scoring methods, more
accurate modalities were implemented. First, unlike the con-
ventional semiquantitative agar-plating procedures, qPCR was
utilized to accurately quantify bacterial infection during caries
formation. Second, in addition to murexide-based caries detec-
tion and scoring methods, SEM and EDXS analyses were car-
ried out on carious and sounds tissues that provided detailed
structure, morphology, mineral content, and elemental compo-
sitional data. Third, nanoindentation tests quantitatively pre-
sented local mechanical property variations of carious lesions
due to demineralization. In conclusion, the in vivo model
reported in this work can be utilized in cariology research and
may develop into a standard protocol for testing new caries
prevention and treatment procedures.
Author Contributions
D.T. Yucesoy, H. Fong, C. Gresswell, S. Saadat, W.O. Chung, S.
Dogan, M. Sarikaya, contributed to conception, design, data anal-
ysis, acquisition, and interpretation, drafted and critically revised
the manuscript. All authors gave final approval and agree to be
accountable for all aspects of the work.
Acknowledgments
The authors gratefully acknowledge financial support from the
Proof of Concept Grant funded by Life Sciences Discovery Fund
(Washington State) and Spencer Funds from Restorative Dentistry
(University of Washington). The funders had no role in study
design, data collection and analysis, decision to publish, or prepa-
ration of the manuscript. Authors are grateful to Prof. Jason M.
Tanzer (University of Connecticut) for his helpful discussions on
rat caries model and Prof. Junlan Wang (University of Washington)
and her student Zhou (Joe) Yang (University of Washington) for
the access of their nanoindentation system. The authors declare no
potential conflicts of interest with respect to the authorship and/or
publication of this article.
8 Journal of Dental Research 00(0)
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