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Int Chin J Dent 2009; 9: 61-68.

Cariogenic biofilms can develop secondary caries within a weektime in an undisturbed condition in vitro indicating high caries
risk
Fahimeh Hayati, DDS,a Ayako Okada, DDS, PhD,a,b Junji Tagami, DDS, PhD,a,c,d andKhairul Matin, BDS, PhDa,da

Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyob
Medical and Dental University, Department of Translational Research, School of Dental Medicine,cTsurumi University, Yokohama, Global Center of Excellence Program; International Research Centerdfor Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, and Support
Program for Improving Graduate School Education, Tokyo Medical and Dental University, Tokyo,
Japan
Purpose: This in vitro study assessed the potency of cariogenic biofilms in developing secondary caries inhuman extracted teeth.
Materials and Methods: Cavities (3x2x2 mm3) were prepared in the blocks of mid-buccal human extracted
molars. Cavities were filled with Clearfil AP-X composite with (SE-Bond) or without (No-bond) application ofan adhesive. Biofilms were formed on both groups of specimens using three species of cariogenic bacteria
inside an oral biofilm reactor for 20 hours and specimens were further incubated separately for 7 days or 30 days
dividing into two groups. Developed secondary caries lesions were examined using fluorescence microscopeand scanning electron microscope. Sizes of the lesions were measured by using image analysis software. Data
were statistically analyzed by one-way ANOVA and Turkey’s HSD methods.
Results: The secondary caries were visible at resin-enamel interfaces in all cases including the 7-day specimens.
Photomicrographs and data on image analysis clearly showed that the lesion size was smaller in SE-Bond sample
compared to No-bond samples, which was statistically significant (p<0.05).
Conclusion: It is suggested that sucrose supplemented biofilms of mutans streptococci have the potency indeveloping secondary caries lesion within a week time indicating high caries risks in case plaque-biofilms left
undisturbed in the oral cavity. (Int Chin J Dent 2009; 9: 61-68.)
Key Words: cariogenic biofilm, mutans streptococci, resin composite, secondary caries.

Introduction
Human dental plaque biofilm is a very complex microbial community that forms on the hard tooth tissues aswell as the restorative materials.1-3 Caries development is a dynamic process between demineralization of dental
hard tissue that eventually results in cavitations and this development depends upon acids and enzymes producedby bacteria present in dental plaque biofilms.4 Dental plaque biofilms may harbor many bacteria that are
involved in the development of disease conditions such as secondary caries and demineralization process ofmarginal enamel and dentin.5,6 Streptococcus gordonii is regarded as an early colonizer of tooth surface, while
Streptococcus mutans and Streptococcus are considered to be involved in latter stages of the dental biofilm
colonization.7 Bacterial adhesion to the surface of composite resins and other dental restorative materials plays
an important role of secondary caries formation.8
Secondary caries is the same as a primary caries located at the margin of a restoration, also known as therecurrent dental caries.

It is reported that most suspected cariogenic groups of bacteria such as mutans

streptococci were widely present and could have an important role in the development of secondary caries
around amalgam restorations.9 Another possibility of secondary caries development has been mentioned asadhesive systems are not capable of eliminating microbes when they colonize in the micro-space between thetooth and the restorative material.10
Artificial mouth models for biofilm formation can benefit in vitro experiments on primary and secondary61

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caries with regard to the microbial and antimicrobial properties of restorative materials or problems resulting
from insufficient marginal adaptation of restorations.11 Information is scarce on the bacterial community formedon resin composites that may produce secondary caries and how much time the pathogens may take to produce
such a lesion, are important issues to investigate. With a view to obtain more information on these subjects, anoral biofilm reactor (OBR) was used in this study to grow multi-species cariogenic (mutans streptococci)
biofilms.12-14 Therefore, the purpose of this study was to assess the potency of cariogenic biofilms formed inside
an OBR on developing secondary caries in human extracted teeth.

Materials and Methods
Tooth specimens
Forty caries-free human maxillary third molar teeth were used in this in vitro study.

Tooth blocks(approximately 5×4×4 mm3) were prepared from the mid-buccal portion of human molars by cutting with a low
speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) under running water as coolant. The convex enamelsurfaces on the outermost buccal slices were reduced up to 0.5 mm by gently polishing on an 800-grit silicone
carbide paper under running water to prepare a flat enamel surface and then polished with water based diamondpaste of 0.25 µm. Finally, the blocks were cleaned with deionized water (Milli-Q water, Japan Millipore Corp.,
Tokyo, Japan) ultrasonically cleaned for 5 minutes to remove the remaining grit left during polishing. The
blocks were rinsed with Milli-Q water three times for two minutes each dried accordingly and enamel surfaces
were coated with nail varnish (Fig. 1).

Fig. 1. Schematic diagrams on the left sequentially show the specimen preparation, cavity preparation and theprocedure of restoration. Diagram on the right represents one of the chambers of the oral biofilm reactor (OBR) in
which the artificial biofilms had been formed in situ on the surface of the specimens. Digital photographs represent
tooth specimens before and 20 hours after biofilm formation.

Restorative materials
A two-step self-etching adhesive materials (Clearfil SE Bond) and a hybrid composite (Clearfil AP-X) wereused in this study. The lot numbers and chemical compositions of the materials are listed in Table 1 according to62

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the information provided by the manufacturer (Kuraray Medical, Tokyo, Japan).
Cavity preparation and composite restoration
Class 1 cavities (3 mm long, 2 mm wide, 2 mm deep) were prepared in specimens using a regular-grit taperedbar (ISO #170, Shofu, Kyoto, Japan), diameter 0.8-1.0 mm that mounted in the milling machine (Cendres &Metaux SA CH-2501, Biel-Bienne, Switzerland) and cavity surfaces were finished with a straight bar (ISO #109,
Shofu) in fixed in the milling machine under water coolant.
The prepared specimens of human teeth were randomly divided into two groups according to filling
conditions; with application of adhesive (SE-Bond) and without any applying any bond (No-bond). Cavities ofthe specimens were filled with a two-step self-etching primer/adhesive system and a-light-cured hybrid
composite (Clearfil AP-X). The cavities were not filled completely, keeping about 0.8 mm deep unfilled spacefor biofilm accumulation at the top, and light-cured for 10 s using a conventional halogen light curing unit
(XL3000, 3M-ESPE, Minneapolis, MN, USA). The cured adhesive surface was then carefully filled with ahybrid restorative (Clearfil AP-X, shade A3) and light-cured for 40 s. In the second group, the other twenty
cavities were also filled with AP-X without application of any bond that remained as No-bond group (Fig. 1).
Table 1.

Materials used in the present study.

Material
Clearfil SE Bond

Batch number
0072140103A

Clearfil AP-X

1079AA

Composition
Primer; HEMA, MDP, Hydrophilic dimethacrylates
Photoinitiator, Water
Bond;
Bis-GMA, MDP, HEMA,
Photoinitiator, Filler
Bis-GMA, TEGDMA, Photoinitiator, Barium glass,
Silanated colloidal silica (filler content 85.0 wt%)

Procedure
Apply 20 s, Gentle air-blow
Apply 15 s, Mild air-blow,
and light-cured 20 s
Light-cured 40 s

Manufacturer; Kuraray Medical Inc, Tokyo, Japan: HEMA, 2-hydoroxyethyl methacrylate; MDP: 10-methacryloyloxydecyl
dihydrogen phosphate; Bis-GMA: bisphenol-A diglycidyl methacrylate; TEGDMA: triethylene glycol dimethacrylate.

Preparation of bacteria suspensions
Suspensions of Streptococcus mutans MT8148 (S. mutans), Streptococcus sobrinus 6715 (S. sobrinus) and
Streptococcus gordonii ATCC10558 (S. gordonii) in phosphate buffered saline (PBS) at an optical density 500
nm (OD500) of 2.5 were prepared from a 16-hour fresh cultures in brain heart infusion broth (Becton Dickinson,
Sparks, MD) after washing three times with PBS. The suspensions were stored at 4˚C with continuous gentle
stirring until use.
Growing biofilm on the specimens inside OBR
Artificial S. mutans, S. sobrinus, and S. gordonii biofilms were grown on the surface of class-1 cavity
with/without adhesive material inside two identical water jacket-encircled chambers of the OBR.

Four

specimens from each group were placed on a Teflon holder around a flat bulb pH electrode of the OBR by using
red utility wax (GC, Tokyo, Japan) keeping the top of each cavity open for biofilm accumulation. The Teflon
holder bearing the specimens set through the bottom opening of the chamber by a silicon plug. Pooled sterile
saliva was then poured on the specimen surfaces and incubated for 30 minutes to obtain a coating of salivary
pellicle. The chamber encircled by a water jacket was sealed with another silicon plug fitted with five stainlesssteel tubes (21gauge) so that the chamber itself served as an incubator with a 37ºC inner temperature. The otherends of the five stainless steel tubes were connected to silicon tubes passing through peristatic pumps regulated
by a computer operated controller (Eyela EPC-2000, Tokyo Rika, Tokyo, Japan). One of them was used tocollect the mixed suspension of S. mutans, S. sobrinus, and S. gordonii, two to collect sucrose added HI and the63Hayati et al.

Int Chin J Dent 2009; 9: 61-68.

other two to collect PBS from the prepared stock as described above. All of these liquids form water domes
which are mixed by the force of gravity exerted from the falling liquid drops on the holder and are diffusely
distributed over all of the specimens.
After 20 hours incubation of the biofilm in the OBR chamber, each specimen with artificial biofilms wasremoved from the Teflon holder in the OBR. The specimens with the undisturbed biofilms were then separated
into two groups for further 7 days or 30 days inoculation to observe the progression of the secondary caries
lesions. A 24-well culture plate (Corning Inc., New York, NY, USA) was used to keep all the specimens inseparate wells to incubate at 37ºC and sucrose added HI was supplemented by changing on every alternate day.
Fluorescence microscopy (FM)Morphology of the lesions developed at resin-enamel interface was investigated by using a fluorescencemicroscope (FM) followed by image analysis of the lesions.

In order to detect and measurement of thesecondary caries lesions, after the storage period, the samples were rinsed with PBS buffer and fixed in 4%
paraformaldehyde with 1% glutaraldehyde in PBS for 1 hour. The samples were rinsed with PBS three times for
2 minutes each, and finally rinsed with Milli-Q water three times for 20 minutes each and dried. Following theseprocedures, the samples were embedded in a self-curing epoxy resin (Epon 815, Nissin EM, Tokyo, Japan),subsequently ground to expose in the longitudinal direction with 800-grit silicon carbide paper under runningwater and finished with diamond pastes down to 0.25 µm particle sizes. The samples were cleaned with Milli-Q
water applying mild ultrasonication in between each step for 10 minutes and finally dried.
Secondary caries lesions at the interfaces were evaluated with an inverted fluorescent microscope (Olympus
CKX41, Olympus Imaging Corp., Tokyo, Japan) and imaged using a DP70 system with the aid of DP Manager
Software (Olympus). In order to observe blue fluorescent by using BP460-490C filter, that produced green
micrographic pictures on the PC. Images were taken at ×40 and ×100 magnifications after setting a scale bar
each time. By using the DP70 system and Manager, maximum length and maximum width were directly
measured. Later, area of the lesions was measured using ImageJ V.1.34 software15 utilizing the scale bar oneach image as reference for calibration.
Scanning electron microscopy (SEM)
The morphology of lesions and structural changes at the interface of enamel and resin were investigated byusing an SEM (S-3400NX, Hitachi, Tokyo, Japan). The polished specimens were dried accordingly andsputter-coated with 300 nm of gold using a SC-701AT (Elionix, Tokyo, Japan) and observed with a SEM. The
experiments were repeated three times in order to establish reproducibility.
Statistics analysis
All numerical data was analyzed using the Statistical Package for the Medical Science (SPSS Ver.11 forWindows, SPSS Inc., Chicago, IL, USA) for statistical procedures. The number of specimens was four in eachgroup (n=4) and the experiments were repeated three times under the same conditions to ensure reproducibility.
The data for maximum width, maximum length and area of secondary caries lesions were analyzed by one-way
analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test with a confidence level of95%.

Results
FM observations and image analysis

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FM images clearly displayed development of secondary caries in all groups with remarkable morphologicalpatterns loosing green fluorescence and appearing brighter (Fig. 2). As expected, in FM images and data on
image analysis (Table 2) it appeared that the lesion size was smaller in SE-Bond treated specimens compared tountreated No-bond specimens. Although, at 7-day that difference was very marginal gap formation between
enamel and resin was very difficult to detect in case of SE-Bond (Fig. 2a), but that could randomly be detected in
No-bond specimens (Fig. 2b). The widths of both 7-day and 30-day SE-Bond specimens were significant
smaller compared to 30-day No-bond specimens (p<0.05). Demineralization along the cavity wall extended
more (length of the lesion) in No-bond specimens compared to SE-Bond in human teeth that penetrated deep
significantly in 30 days (p<0.05). The area of the demineralized enamel lesion was smallest in 7-day SE-Bondtreated specimens.

In reverse, lesion area was largest in 30-day No-bond treated specimens that showedsignificant differences when the data were compared (p<0.05). All these data indicate that damage was less in
SE-Bond specimens and was severe in No-bond specimens. The three cariogenic species microcosm plaquebiofilms demonstrated strong acidogenic and acid-tolerating potency by keeping low pH around the tooth surface
for relatively long period of time.

Fig. 2. FM photographs of specimens with a) and without b) adhesive material after 7 days (original
magnification ×100). Red arrows indicate secondary caries lesions in both photographs. A clear lesion mark ofenamel demineralization can be seen in SE-Bond specimen, but almost no gap between enamel and composite
resin can be detected a). Enamel demineralization extended deep along the enamel wall in No-bond specimen
with a gap between enamel and composite resin indicated by black arrow b).

Table 2.

Average sizes (maximum width, maximum length, and area) of secondary caries lesions.

Bond

SE-BondIncubation period

7d

Maximum width (µm)(79±22)No-bond
30 d

a

7d

30 d

b

(110±43)(163±61)a,b(199±42)(202±36)(296±70)a(1.3E+04±
2.7E+03)(1.6E+04±
2.5E+03)(4.2E+04±
8.1E+03)a(86±10)aMaximum length (µm)(178±70)

Area (µm2)(9.9E+03±
5.4E+03)a

All data indicate (Average±SD). Same superscript letters are significantly different according to one-way ANOVA and Tukey’s
HSD test (p<0.05), n=4 in each group.

SEM observations
Presence of secondary caries lesions due to demineralization of enamel particles, adhesive material remain
sandwiched at enamel-resin interface in SE-Bond groups and gaps in No-bond groups were observed on SEM
inspection (Fig. 3). SE-Bond remained bonded on to resin but lost attachment with the enamel by inevitable acid65Hayati et al.

Int Chin J Dent 2009; 9: 61-68.

attacks from the biofilm (Fig. 3a). On the other hand, gap formation between enamel and resin were remarkably
wide in case of No-bond samples. For example approximately gap size in case of human sample of No-bond
was 9.2 µm (Fig. 3b). Also, in 7 day specimens similar demineralization pattern was observed, although lesion
size very small in case of SE-Bond group.

Fig. 3. SEM photographs of specimens with a) and without b) adhesive material after 30 days (Magnification,×250). Enamel demineralization was small in SE-Bond specimens indicated by white arrows and almost no gap
between enamel and composite resin can be detected, arrowheads indicated bonding layer between enamel andresin a). Damage was large in No-bond specimen shown by white arrows with a visible wide gap indicated byblack arrow b).

Discussion
The development of secondary caries around restorative materials is determined by the physico-chemical
properties of the materials (e.g., shrinkage, corrosion, solubility, fluoride content, and permeability) and theirclinical performance; that is, cavity-sealing ability, microleakage of bacteria, fluids, and acidic products andcavity preparation.

The multi-factorial etiology of secondary caries includes age, oral hygiene, bacterialcolonization, salivary composition, fluoride bioavailability, and most of all the quality of restoration including
the properties of restorative materials. It was expressed that extensive experimental and clinical researches areneeded to develop suitable prevention approaches for secondary caries expression and to know the complex
interaction among the restoration, the oral environment and the tooth surface.16 The development of an OBR
made it possible to create an in vitro secondary caries model induced by cariogenic streptococci on the enamelwall at composite resin interface. In the present study, all specimens presented carious lesion formation on the
exposed tooth surfaces around the composite resin restorations. Restorative materials may harbor bacteria to
initiate plaque accumulation and secondary caries formation in the oral cavity and organic acids can be trapped
within the glucan barrier of the mature plaque produced by these bacteria, resulting in a prolonged low pH
around the tooth surface.17

During the experiment, when there is active biofilm formation in almost

uninterrupted condition the bacteria secreted acids, reduced pH and dissolved minerals of the enamel in similar
caries process like in the oral cavity within one week time. Among several characteristics of S. mutans include a
proclivity for adhesion to the tooth surface;18 synthesizing WIG with sucrose as a substrate, resulting insubsequent formation of plaque19 and reduction of the pH by producing acid,20 which causes demineralization of
the tooth and leads to the carious lesion.
S. mutans is the leading pathogen responsible for the development of dental caries. When sucrose intake is66

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Int Chin J Dent 2009; 9: 61-68.

frequent, S. mutans synthesizes water insoluble glucans catalyzed by glucosyltransferases from dietary sucrose.
Glucans are important in the adhesive interactions of S. mutans with the tooth surface as well as contributing to
the formation of the matrix of cariogenic biofilms. The later strongly adheres to tooth surfaces and aids inpreventing diffusion of bacterial organic acids which in turn leads to decalcification of tooth tissues enamel(dental caries).21 Combination of S. gordonii and S. sobrinus with S. mutans the caries inducing artificial biofilm
appeared to be more natural as they can build up a multispecies microbial community that is much more stable
and resistant to external inhibiting factors. In the oral cavity, S. sobrinus seems to be more acidogenic than theother species of mutans streptococci.22,23
The caries lesions were clearly visible at the interface of restorations by FM in all specimens due to the loss ofgreen fluorescence in both enamel and dentin. For instance, no detectable lesion was visible in a control
specimen without biofilm formation (data not shown), compared to that a typical enamel lesion developed by the
cariogenic biofilm in almost all experimental specimens could easily be detected, which appeared asbright-green rather than pure fluorescent green from the normal enamel. In order to permit accurate quantitative
analyses of the secondary caries size, we measured maximum width and maximum length of the outer lesions
using the FM images at low magnification (×40).
In vitro caries model allows the simultaneous production of primary and secondary caries-like enamel lesions
in a considerable number of specimens, and facilitates the possibility to manipulate and transfer plaque and toothwithout necessarily terminating the experiment.24 During restorative treatment when SE-Bond is applied it
makes a compact interface or bond between enamel and resin inhibiting direct penetration of acid. Also,
SE-Bond forms coating layers on enamel surface of the unfilled cavity that protected the enamel to some extent.
It was clearly understood within one week time that the use of Clearfil SE-Bond systems for bonding ofcomposite resin to enamel contributes to inhibit secondary caries compared to without adhesive application(No-bond). In all groups the length of the lesion was larger than the width indicating easier acid penetration
along the enamel-resin interface rather than widely spreading into the enamel structure.
In the present study, visible marginal gaps between the restorative material and tooth structure observed by
SEM. Results also showed that larger lesions were associated with wider gaps when incubation period was
longer (30 days) and that occurred mainly in all No-bond specimens that findings are in agreement with previous
in vitro studies25 demonstrating that secondary caries lesions develop next to marginal gaps. Expectedly, the sizeof secondary caries lesions in bonded (SE-Bond) groups was smaller than that of un-bonded (No-bond) groups.
That might be because SE-Bond can build up a satisfactory adhesion between composite resin and enamel
inhibiting penetration of acids and can minimize gap formation.
Therefore, it can be suggested that cariogenic biofilm of mutans streptococci can develop secondary caries inhuman teeth within a week time and may cause extensive damage to the tooth structures if left unnoticed which
is an indication of high caries risk for clinical conditions. Further studies on biofilm attachment on restorative
materials and secondary caries development would be beneficial.
Acknowledgment
This work was supported by the Global Center of Excellence Program; International Research Center for Molecular Science in Tooth andBone Diseases in Tokyo Medical and Dental University and a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS, No.
16390544).

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Correspondence to:Dr. Khairul Matin
Cariology and Operative Dentistry, Dept of Restorative Sciences,
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
Fax: +81-3-5803-0195 E-mail: matin.ope@tmd.ac.jp
Accepted December 2, 2009.

Copyright ©2009 by the International Chinese Journal of Dentistry.

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