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Evaluation of antibacterial activity of visible light-responsive
TiO2-based photocatalyst coating on orthodontic materials against
cariogenic bacteria
Tomomi Sudo, DDS (1), Susumu Imai, PhD (2), Tsuyoshi Ochiai, PhD (3,4), Hiroyuki
Kanzaki, DDS, PhD (1), Yusuke Morita, DDS (1), Nobuhiro Hanada, DDS, PhD (2), andYoshiki Nakamura, DDS, PhD (1)(1) Department of Orthodontics, School of Dental Medicine, Tsurumi University, (2) Department ofTranslational Research, School of Dental Medicine, Tsurumi University, Yokohama, (3) Photocatalyst
Group, Kanagawa Academy of Science and Technology, Kawasaki, and (4) Photocatalysis
International Research Center, Tokyo University of Science, Noda, Japan
Purpose: The objectives of this study were to evaluate antibacterial activity of visible light-responsive
photocatalytic titanium dioxide (Vis-TiO2) to cariogenic bacteria.
Materials and Methods: In all analyses, brookite Vis-TiO2 was coated on stainless steel surfaces, which were
illuminated with visible light (405 nm; LED-generated). Antibacterial activity against cariogenic bacteria wasassessed by colony-forming unit (CFU) assay, radial diffusion assay, and bacterial viability analysis.
Results: CFU assay demonstrated that the number of colonies of both bacteria on coated plates with illumination
reduced to around 10 %. The coated materials produced clear zones in culture petri dishes of both cariogenic
bacterial species. Fluorescence microscopy revealed that most of the bacteria on the coated plates with
illumination was identified as dead. In flow cytometry analysis, more than 68% of S. mutans and more than 91%
of S. sobrinus were killed on the coated plates with illumination. Vis-TiO2 has strong bactericidal effects on bothcariogenic bacteria after illumination. Water-insoluble glucan on the coated plate with illumination was
significantly less formed.
Conclusion: The visible light-responsive photocatalytic titanium dioxide coating on orthodontic materials has
strong antibacterial activity, which is based primarily on bactericidal activity. The Vis-TiO2 coating is useful for
dental caries prevention without any adverse effect to the human body.
(Asian Pac J Dent 2016; 16: 15-22.)
Key Words: antibacterial, bactericidal activity, dental caries, orthodontic material photocatalysis, visible light

Introduction
Dental caries is one of the most prevalent diseases in the world [1], and also one of the serious side effects
during orthodontic treatment [2]. The caries around the orthodontic brackets, which are developed by enamel
demineralization, have been a critical problem in daily orthodontic treatment [3]. It was caused by prolonged
accumulation of bacterial biofilm on the enamel surfaces around the orthodontic brackets. Most of the biofilm is
initially formed on the surface of orthodontic bracket and spread on the enamel surface around the bracket.
Therefore, it is clinically important to inhibit the formation of biofilm on the orthodontic bracket [4].
The biofilm is formed by cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus [5-10].
Therefore, it has been clinically important to inhibit the activity of cariogenic bacteria. However, it is difficult
for the orthodontic patients to clean the brackets with complex forms on their teeth during orthodontic treatment;such cleaning requires a great deal of compliance by the patients.
There have been several studies on the prevention of dental caries. One potential strategy is to coat theorthodontic brackets with antibacterial reagents such as photocatalytic TiO2 and to irradiate them with ultraviolet
light (UV) [11,12]. The release of reactive oxygen species (ROS) from the photocatalytic TiO2 inhibits thegrowth of cariogenic bacteria [13]. However, the use of UV irradiation is dangerous, since UV is harmful to oral
mucosa and skin [14].

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Recently, LED light has been shown to have the potential to activate photocatalytic TiO2 [15-17]. Therefore,
the purpose of this study was to evaluate the antibacterial activity of visible light-responsive photocatalytic TiO2
against cariogenic bacteria with the activation by LED visible light which has no adverse effect to the humanbody.

Materials and Methods
Sample preparation
The materials used in this study consisted of stainless steel disks (5.0 mm diameter x 3.0 mm thick) and squareplates (100.0 x 100.0 mm). The surface roughness (Ra) were measured using a confocal laser scanningmicroscope (OLS 3000, Olympus Co., Tokyo, Japan). Ra of the stainless steel was Ra 2.14 ± 0.097 µm, which isthe same as that of orthodontic brackets (Metal bracket, Densply Sankin, Tokyo, Japan: Ra 2.12 ± 0.174 µm).
A dip-coating method (using 3 wt% TiO2 brookite-rich sol; NTB-13, Showa Denko, Tokyo, Japan) was used
to apply visible light-responsive photocatalytic titanium dioxide (Vis-TiO2) to the surfaces of the materials [18].
The coated materials were heated at 100˚C for 15 min to modify the TiO2 nanoparticles onto the surface. After
heating, the excess coating agent was removed in the ultrasonic device. Then during drying the temperature wasmaintained under 100˚C in order to prevent the inactivation of brookite TiO2. Ra after coating was 2.00 ± 0.154µm, indicating that there was no statistical difference before and after coating. All materials were sterilized with
ethylene oxide gas before use.
The illumination device used in this study consisted of an array of 90 small LED illuminator bulbs (Fig. 1),
and emitted visible light at 405 nm (SPL-90-CC, Revox, Sagamihara, Japan). The distance between LED andbacteria in all of this study was 35 mm, and the intensity of the light was 180 Lx.
Bacterial strains
Cariogenic bacteria Streptococcus mutans ATCC 25175 (S. mutans) and Streptococcus sobrinus ATCC 33478 (S.
sobrinus) were used in this study. Both bacteria were inoculated into 4 mL of Tryptic Soy (TS) broth (Becton,
Dickinson and Company, Sparks, MD, USA) and were cultured at 37˚C for 16 h.
Colony-forming unit (CFU) assay
Bacterial cells from the culture medium were washed with PBS and the aliquots (1.5 mL) of the each bacterial
suspension were poured on the surface of the coated and non-coated square plates on ice, and light illumination
was performed for 2 h. After illumination, bacterial cells were harvested by centrifugation (1,750×g, 4˚C, 20min). Each bacterial cell pellet was resuspended in 1 mL PBS and subjected to serial 10-fold dilutions in PBS.
The dilutions of each bacteria were inoculated on MS agar (Difico Mitis Salivarius Agar (semi-selective medium
for streptococci); BD Biosciences, Franklin Lakes, NJ, USA) in petri dishes with spiral plating equipment (Eddy
Jet, IUL SA, Barcelona, Spain), and petri dishes were incubated under anaerobic conditions in an
AnaeroPack-Anaero box (AnaeroPack System, Mitsubishi Gas Chemical Co. Inc, Tokyo, Japan) at 37˚C for 48 h.
The number of colonies was calculated in accordance with the spiral plater instruction manual.
Radial diffusion assay
Radial diffusion assays [19] were performed as follows. The coated and non-coated disks (5.0 mm) mentioned
above were placed on petri dishes. TS agar medium (6 mg TS and 100 mg agarose in 10 mL distilled water) was
sterilized by autoclaving and cooled to 50°C. Bacterial cells collected by centrifugation from TS culture medium
were added in sterilized TS agar medium at a cell density of OD540 = 1.0 (corresponding to 100 µL or 300 µL of

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S. mutans or S. sobrinus culture, respectively). The TS agar medium containing bacterial cells was poured overthe disks in the dishes (achieving a depth sufficient to submerge the disks), and then another aliquot of enriched
TS agar medium (600 mg TS and 100 mg agarose in 10 mL distilled water) was added on the pre-poured TS
agar medium.
After light illumination for 2 h in the air at room temperature, the bacteria in the dishes were incubated in thedark under anaerobic condition at 37˚C for 24 h using an AnaeroPack-Anaero box. After incubation, diametersof the clear zones of bacterial inhibition around the disk materials were measured with a slide calipers.
Antibacterial activity was calculated as follows; Antibacterial activity (mm) = diameter of clear zone (mm) −diameter of disk (mm) [20].
Bacterial viability analysis by fluorescent microscopy and flow cytometry
The viability of bacterial cells on each square plate was evaluated by the fluorescence microscopy (Olympus AX
80; Olympus Co. Ltd., Tokyo, Japan) using the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes,
Carlsbad, CA, USA). Briefly, bacterial cells (S. mutans or S. sobrinus) from the culture medium were washedwith PBS; 50 µL of the suspension of either species were combined with 0.5 µL of the BacLight reagent. This
staining solution contains SYTO 9, a cell membrane-permeable green fluorophore, and PI, a cell
membrane-impermeable red fluorophore. In this fluorescence system, viable bacterial cells exhibit green
fluorescence, while nonviable cells exhibit red fluorescence. The excitation/emission maxima for these dyes areabout 480/500 nm for SYTO 9 stain and 490/635 nm for PI. Bacterial cells were observed by fluorescencemicroscopy using a 470/490 nm excitation cube.
Furthermore, the bacterial cells were quantified by flow cytometry using a BD Accuri C6 flow cytometer (BD,
Bioscience, Franklin Lakes, NJ, USA) [21,22]. The stained bacterial cells were excited using a 488-nm
solid-state laser, and fluorescence was detected using 533/30 BP (FL-1 channel) and 670 LP filters (FL-3channel) for SYTO 9 and PI, respectively. The bacterial fraction (SYTO 9-positive population) was gated on an
SSC/FL1 plot, and fluorescence of PI was monitored in the FL-3 channel.
Effects of Vis-TiO2 coating on cariogenic biofilms
The inhibitory effects of the TiO2 coating on cariogenic biofilm formation were examined on the surfaces of thedisks using separate suspensions of S. mutans and S. sobrinus. The culture medium for biofilm formation was
prepared using TS broth without dextrose, which was supplemented with 0.5% sucrose (Wako, Osaka, Japan)(TSS broth). The pre-cultured suspensions of S. mutans or S. sobrinus were added to TSS broth. Then the steriledisks (n = 5 for each bacterial species) were incubated in 500 µL of the above medium in a 48-well culture plate
under continuous LED light illumination for 24 h at 37˚C.
The disks were retrieved from the medium, gently washed three times with sterile phosphate-buffered saline(PBS; pH 7.4), soaked into 1 mL of 0.5 N NaOH in a glass test tube (Iwaki, Tokyo, Japan) for 10 min tosolubilize water-insoluble glucan (WIG), and the WIG was completely detached from the disks by vortexing.
After picking out the disks, the suspensions in the tubes were centrifuged (1,750×g, 4˚C, 15 min) to pellet of thebacteria. WIG content in the supernatants was analyzed using the phenol-sulfuric acid method [23,24].
Statistical analysis
Statistical analyses were performed using SPSS 22.0 for Windows (SPSS Inc., Version III, Chicago, IL, USA).
Amount of biofilm were compared using Kruskal-Wallis and Mann-Whitney U-tests. The results of radial17

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Asian Pac J Dent 2016; 16: 15-22

diffusion assays, and CFU assays were compared using the Student’s t-test. Differences were considered to besignificant at p < 0.05.

Results
Antibacterial activity by UV-responsive TiO2 has been shown in many studies [3,25-27]. However, there havebeen few studies on the antibacterial activity by Vis-TiO2. In this study we examined whether Vis-TiO2 coatedon the orthodontic materials has antibacterial activity against cariogenic bacteria.

Fig. 1
LED illuminator-bulbs for visible light

Fig. 2Anti-bacterial activity of coated and non-coated disks (n = 5) against S. mutans (a, b)
and S. sobrinus (c, d) with and without illumination by radial diffusion assay.
The asterisks indicate statistically significant differences (p < 0.05).

We examined the effect of Vis-TiO2 on the formation of colony by cariogenic bacteria. CFU assay showedthat the antibacterial activity on the coated materials with visible-light illumination induced a significant
reduction in the number of colony. The number of S. mutans was reduced from 448.8 to 7.3 and that of S.
sobrinus from 417.5 to 8.5, and consequently about 95% of colony by both S. mutans and S. sobrinus was notformed on the coated plates with illumination (Table 1).
Table 1 The CFU of S. mutans and S. sobrinus on coated and non-coated plate materials after illumination for 2 h
S. mutans

S. sobrinus

Non-coated, Light (−)

440.2 ± 12.9

394.7 ± 23.9

Non-coated, Light (+)

448.8 ± 27.9

417.5 ± 28.9

S. mutans

S. sobrinus

Coated, Light (−)

450.4 ± 39.0

420.7 ± 33.5

Coated, Light (+)*7.3 ± 1.0*8.5 ± 0.7

The asterisks indicate statistically significant differences (p < 0.01) compared to non-coated condition in each strain (n = 5).

Antibacterial activity of the Vis-TiO2 was also confirmed by radial diffusion assay [28,29]. This assay
determined the area influenced by the Vis-TiO2. The results demonstrated that no detectable clear zone wasobserved around the coated and non-coated materials without illumination or around non-coated material with
illumination in the S. mutans or S. sobrinus cultured petri dishes (Figs. 2a and 2c). However, the coated materials
with illumination evidently produced clear zones in both S. mutans and S. sobrinus culture petri dishes.

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We further confirmed the antibacterial activity of the Vis-TiO2 by detecting live and dead cells in the cultured
bacterial cells. Fluorescence microscopy revealed that most of bacteria of both species on the non-coated plateswith or without illumination was identified as alive as shown by green fluorescence (Figs. 3a-c and e-g). In
addition, bacterial cells on coated plates without illumination was also alive. On the other hand, the bacteria on
the coated plates with illumination was identified as dead, as shown by red fluorescence (Figs. 3d and 3h). These
results indicate that the Vis-TiO2 with illumination has strong bactericidal effects on both species of cariogenic
bacteria. These results evidently indicate that most of the antibacterial activity of the Vis-TiO2 is based on
bactericidal activity [30], not the inhibition of bacterial growth.

Fig. 3

Fluorescence staining of the populations of S. mutans (a, b, c, d) and S. sobrinus (e, f, g, h) on coated and non-coatedplate materials (n = 5) after illumination for 2 h. (a, e): non-coated, light (−), (b, f): non-coated, light (+), (c, g): coated,
light (−), (d, h): coated, light (+). Original magnification 400×, scale bar 30 µm

Flow cytometry analysis quantitatively confirmed these results of S. mutans (Fig. 4a) and S. sobrinus (Fig.
4b). In each figure bacterial cells on non-coated plates showed fluorescence peak of FL-3 around 105. Similar tothem, bacterial cells on coated plates without illumination showed fluorescence peak of FL-3 around 106. On theother hand, bacterial cells on coated plates with illumination showed the increase of fluorescence in FL-3, whichindicates the increase of dead cells in the samples. The number of FL-3 negative cells was more than 96%,
signifying bacterial cells non-coated on plates remained alive even after 2 h illumination (Table 2). In addition,
the bacterial cells on the coated plate without illumination was also alive. However, more than 68% of S. mutans
and more than 91% of S. sobrinus bacterial cells were FL-3 positive, i.e., killed on the coated plate after 2 h ofillumination.
Table 2 Percentages of live and dead bacterial cells of S. mutans and S. sobrinus on coated and non-coatedplates with illumination in flow cytometry analysis
S. mutans

S. sobrinus

Live (%)

Dead (%)

Live (%)

Dead (%)

Non-coated, Light (−)

97.27 ± 0.0165

2.73 ± 0.0165

98.28 ± 0.0178

1.72 ± 0.0178

Non-coated, Light (+)

96.26 ± 0.0097

3.74 ± 0.0097

98.58 ± 0.0034

1.42 ± 0.0034

Coated, Light (−)

96.66 ± 0.0056

3.34 ± 0.0056

98.57 ± 0.0028

1.43 ± 0.0028

Coated, Light (+)*31.84 ± 0.2850*68.16 ± 0.2850*8.52 ± 0.0335*91.48 ± 0.0335

The asterisks indicate statistically significant differences (p < 0.05) compared to non-coated, light (+) conditions in eachstrain of S. mutans or S. sobrinus (n = 5).

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On the basis of these results, we examined whether the Vis-TiO2 has an inhibitory effect on biofilm formation
through antibacterial or bactericidal activity (Fig. 5). The amount of WIG on the coated disk with illumination
was significantly less than those on the other three disks: non-coated without illumination, non-coated with
illumination, and coated without illumination.

Fig. 4
Flow cytometry analysis of cell
populations of S. mutans (a) and S.
sobrinus (b) after illumination for 2 h(n = 5).
X-axis indicates fluorescenceintensity of FL-3 channel, and Y-axis
indicates the number of cell count at
each fluorescence intensity.
Pink dot curve: non-coated, light (−),
blue dot curve: non-coated, light (+),
green curve: coated, light (−),
red curve: coated, light (+).
Vertical black line indicatesthreshold.

Fig. 5

Amount of WIG in the biofilm formed on coated and non-coated disks. S. mutans and S. sobrinus with or without
illumination (n = 5). The asterisks indicate statistically significant differences (p < 0.05).

Discussion
S.mutans and S.sobrinus play a pivotal role in dental caries and they are inherently difficult to eradicate.
Mechanical tooth cleaning by brushing is the main method for prevention of dental caries in contemporary
orthodontic treatments. This has been performed for a long time, the consensus view is that patients generally areunable to brush well enough to remove plaque around multi-bracket appliances. There have been some studies
on the antibacterial effects of orthodontic adhesive resins [31]. But the surplus of the adhesive resins during thebracket bonding is usually removed from the enamel surface, and consequently the antibacterial effects of theadhesive resins are not expected around the brackets.
Significant reduction of colony was observed in CFU assay (Table 1). The antibacterial activities ofVisible-light responsive nickel-doped TiO2 was investigated in the previous study [32]. In our study, we
examined antibacterial activity of pure brookite-rich TiO2, which showed similar high antibacterial activity.
These results suggest that the Vis-TiO2 in this study has notable ability to inhibit the growth of cariogenic
bacteria.

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The obtained from radial diffusion assay results were very interesting from the point of the antibacterial
activity (Fig. 2). The activity of the Vis-TiO2 was observed on the surface area of the coated disk and in the areasome distance away from the disks. Thus, the Vis-TiO2 has potential antibacterial activity against cariogenic
bacteria not only on the coated surface but also in the area some distance away from the coated surface, which isone of the characteristic features of this study. This is probably due to the release of ROS from the coated disks,
which diffuse around the disks with concentration gradient [33], and consequently inhibit the bacterial growth in
the area away from the surface.
Fluorescent microscopy demonstrated that, the effect of Vis-TiO2 was bactericidal activity (Figs. 3 and 4).
The bactericidal activity is based on the fragmentation of organic materials by the photocatalyst. The TiO2 on theplates generates ROS such as hydroxyl radical (•OH), superoxide radical (O2•−) [34], and singlet oxygen (1O2)[35], under visible LED light illumination. The bactericidal activity of the Vis-TiO2 could be caused by thestrong oxidative activity of ROS.
WIG is the essential component of biofilm, which generates strong adherence property to the surfaces of thematerials and is usually produced by the cariogenic bacteria by polymerization of glucose derived from sucrosein the food [36-40]. The Vis-TiO2 has the strong ability to reduce biofilm formation on the coated disk with
illumination (Fig. 5). These results indicate that the Vis-TiO2 inhibits the formation of biofilm, which result fromantibacterial activity to cariogenic bacteria. This is expected to be advantageous for the prevention of caries
around orthodontic brackets. Vis-TiO2 has potential application for the prevention of dental caries through the
inhibition of biofilm formation not only in orthodontics, but also in dentistry.
The visible light-responsive photocatalytic titanium dioxide coating on orthodontic materials has strong
antibacterial activity, which is based primarily on bactericidal activity. The visible light-responsive
photocatalytic titanium dioxide coating is useful for dental caries prevention without any adverse effect to thehuman body.
Conflict of Interests
The authors declare that there is no conflict of interest regarding the publication of this manuscript.

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Correspondence to:
Dr. Yoshiki Nakamura
Department of Orthodontics, School of Dental Medicine, Tsurumi University
2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Fax: +81-45-573-9599 E-mail: nakamura-ys@tsurumi-u.ac.jp
Copyright ©2016 by the Asian Pacific Journal of Dentistry.

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Accepted March 27, 2016.
Online ISSN 2185-3487, Print ISSN 2185-3479