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Asian Pac J Dent 2011; 11: 1-8.

An in vitro study to assess the efficacy of OH- ion superadded
alkali-ion water in inhibiting caries and secondary caries
Ryoichiro Uchida, DDS,a Khairul Matin, BDS, PhD,a and Junji Tagami, DDS, PhDa,ba

Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School of Medicalband Dental Sciences, and Global Center of Excellence (GCOE) Program; International ResearchCenter for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University,
Tokyo, Japan
Purpose: This study assessed the caries and secondary caries prevention efficacy of OH- ion superadded
alkali-ion water (AW) using an in vitro model.
Materials and Methods: Class 1 type cavities were prepared on bovine teeth blocks and they were immediately
filled with Clearfil AP-X composite with application of an adhesive system. Biofilms were formed on thespecimens using three species of cariogenic bacteria inside an oral biofilm reactor for 20 hours and specimens
were further incubated separately for 7 days. During the incubation period the specimens were lightly rinsed
with three pH levels of AW (AW-H: 11.5, AW-M: 10.5, and AW-L: 9.5) as the experimenting functional waters
and control solutions (tap water; TW: 7.5 and heart infusion broth; HI: 7.3) dividing into five groups. The
progression of the developed caries and secondary caries lesions were examined using a fluorescencemicroscope. Sizes of the lesions were measured by using an image analyzing software. Data were statisticallyanalyzed by one-way ANOVA and Turkey’s HSD methods.
Results: The caries and secondary caries were visible mainly in enamel at the edge of resin restorations in all
specimens. Photomicrographs and data on image analysis clearly showed that the lesion size was smaller in AW
samples compared to control samples, which was statistically significant when compared between AW-H andTW (p<0.05).
Conclusion: It was suggested that AW can be used to inhibit the progression of caries and secondary caries byadjusting the pH levels as required. (Asian Pac J Dent 2011; 11: 1-8.)
Key Words: alkali-ion water, glucan, secondary caries, Streptococcus mutans

Introduction
Biofilm-dependent oral diseases that include dental caries and periodontal diseases involve number of oral
pathogens.1,2 Among many types of bacteria that participate in the formation of cariogenic biofilms mutans
streptococci, namely Streptococcus mutans and Streptococcus sobrinus, are acidogenic and considered to be theprimary organisms that are responsible for dental caries.1-4
In recent years, resin composite restorative materials are becoming the most common indwelling medical
devices used to restore decayed functional tooth structures and tooth restoration is a widely accepted dental
clinical procedure.5,6 Especially in minimally invasive restorations, direct resin composites have been widelyused in clinical practice.7 These types of resin composites also play a major role in operative, aesthetic, as wellas prosthodontic treatments. However, one of the most frequently occurring reasons for replacement of direct
resin composite restorations is secondary caries.8-11

Secondary caries around restorations which require

replacement of the restoration is a major concern in dentistry.12 One of the major causative factors for thedevelopment of secondary caries is bacterial biofilm formation on the tooth structures and resin composite
surfaces followed by invasion along the interface by damaging the tooth structures.13 That may also cause as aresult of wear or distortion of the restorations.
An artificial biofilm-induced secondary caries model has been established recently14,15 by using an oral
biofilm reactor (OBR) 9,16,17 which has been used to study oral biofilm formation on tooth structures and different
dental materials in vitro by simulating the human oral environment.18 The study model was established toprovide a better understanding of the secondary caries development around resin composite restorations and to1

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assess the efficacy of various antibacterial agents on caries or secondary caries inhibition. One of those studies
reported that the model secondary caries developed using the OBR were clearly visible by 7-days time.14
Therefore, preclinical information can be obtained using this study model within very limited period of time.
A functional-water, commonly known as “alkaline water/ alkali-ion water (AW)”, being used as drinking
water contains a high concentration of OH- ion and it was reported that AW dissolve cariogenic bacteria induced
water insoluble glucans (WIG).19
At present, there is almost no available research report that evidently demonstrates the efficacy of AW in
preventing caries or secondary caries. With a view to obtain more information on these issues, AW andsecondary caries model were used in this study. Therefore, the purpose of this study was to assess the efficacy
of AW in inhibiting the progression of artificial caries and secondary caries in bovine teeth using an OBR.

Materials and Methods
SolutionsThree different pH levels of AW: AW-H (pH11.5), AW-M (pH10.5) and AW-L (pH9.5) were produced using
a water electrolysis device (TK7705, Panasonic Electric Works Co., Ltd., Osaka, Japan). Tap water (TW) and asolution of Heart Infusion broth (HI, Becton Dickinson, Sparks, MD, USA) with sucrose (1% final
concentrations) were used as a control solution. The pH level of TW was 7.5 and that of HI was 7.3 on average.

Fig. 1. Schematic diagrams sequentially show the specimen preparation, secondary caries induction andobservation using a fluorescence microscope (FM). Class-1 type cavity prepared, immediately restored with resin
composite followed by mutans streptococci biofilm formation inside an OBR and then rinsed 3 times daily with thesolutions during final 7-day incubation period. Zone of measuring the maximum width, the maximum length andarea of a lesion is indicated.

Tooth block preparation
Bovine lower incisor teeth were used to prepare square shaped dentino-enamel blocks in this study. Tooth
blocks (approximately 5×4×4 mm3) were prepared from mid-labial parts by cutting with a low speed diamond2

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saw (Isomet, Buehler, Lake Bluff, IL, USA) under running water as coolant. The convex enamel surfaces on theoutermost 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 diamond paste 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).
Cavity preparation and composite restoration
Class 1 cavities (3 mm long, 2 mm wide, 2 mm deep) were prepared in specimens using a diameter 0.8-1.0
mm regular-grit tapered bur (ISO #170, Shofu, Kyoto, Japan) mounted in a milling machine (Cendres & Metaux
SA CH-2501, Biel-Bienne, Switzerland) and cavity surfaces were finished with a straight bur (ISO #109, Shofu)
in fixed in the milling machine under water coolant. Cavities of the specimens were filled with a two-step
self-etching primer/adhesive system and a-light-cured hybrid composite (Clearfil AP-X). The cavities were notfilled completely, keeping about 0.8 mm deep unfilled space for biofilm accumulation at the top, and theadhesive was 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 a hybrid restorative (Clearfil
AP-X, shade A3) and light-cured for 40 s (Fig. 1).
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 to
the information provided by the manufacturer (Kuraray Medical, Tokyo, Japan).
Table 1.

Materials used in the present study

Material

Lot number

Composition

Procedure

Manufacturer

ClearfilMega Bond00927A

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

Apply 20 s, Gentle air-blow

Kuraray Medical Inc.,Tokyo, Japan

ClearfilAP-X

01371A
01213A

Apply 15 s, Mild air-blow,
and light-cured 10 s
Light-cured 40 s

Kuraray Medical Inc.

HEMA, 2-hydoroxyethyl methacrylate; MDP, 10-methacryloyloxydecyl dihydrogen phosphate; Bis-GMA, bisphenol-A diglycidyl
methacrylate; TEGDMA, triethylene glycol dimethacrylate

Preparation of bacterial suspensions
Suspensions of Streptococcus mutans MT8148 (S. mutans), Streptococcus sobrinus 6715 (S. sobrinus), andStreptococcus 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, USA) 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 each of the Class-1 cavity filled withresin composite material. 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 forbiofilm accumulation. The Teflon holder bearing the specimens set through the bottom opening of the chamber

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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 siliconplug fitted with five stainless steel tubes (21gauge) so that the chamber itself served as an incubator with a 37ºC
inner temperature. The other ends of the five stainless steel tubes were connected to silicon tubes passing
through peristatic pumps regulated by a computer operated controller (EYALA EPC-2000, Tokyo Rika, Tokyo,
Japan). One of them was used to collect the mixed suspension of S. mutans, S. sobrinus, and S. gordonii, two tocollect sucrose added HI and the other two to collect PBS from the prepared stock as described above. All ofthese 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 was removed from the Teflon holder in the OBR. The
specimens with the undisturbed biofilms were inoculated for further 7 days to observe the effects of AWs ininhibiting 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 in separate wells to incubate at 37ºC and sucrose added HI was
supplemented by changing on every alternate day.
Rinsing with the solutions
During the 7-day incubation period in HI, specimens were separately rinsed with AW-H, AW-M, AW-L, TW
and HI (n=4 in each group) for 20 s three times daily. In this study, a Water Bath Shaker (Taitec, Personal-11,
Tokyo, Japan) was used with a moderate stroke of 10 Hz for 20 s three times with an interval of 1 minute each tosimulate the gargling condition of an adult.
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 incubation 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 BP460-490C filter was used and that produced green

micrographic pictures on the PC. Images were taken at ×40 and ×100 magnifications after setting a scale bar
each time. Maximum length (from the edge of the restoration to deepest point of the lesion) and maximum
width (from the edge of the restoration to widest point of the lesion) were directly measured using the DP70
system and Manager. Later, area of the lesions was measured using ImageJ V.1.34 software20 utilizing the scalebar on each image as reference for calibration. Only this part of a lesion that remained adjoined with resin
considered as the secondary caries lesion (Fig. 1).
Statistical analysis
All numerical data was analyzed using the Statistical Package for the Medical Science (SPSS Ver.11 for

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Asian Pac J Dent 2011; 11: 1-8.

Windows, 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 images clearly displayed development of caries and secondary caries like lesions in all groups withremarkable morphological patterns loosing green fluorescence and appearing brighter (Fig. 2). Lesions entirely
were remarkably large in TW and HI specimens although the incubation was only for 7 days. On individual
observation of each of the FM image followed by group-wise comparison and data on image analysis (Fig. 3) it
appeared that the secondary caries like lesion size was smaller in AW-H and AW-M treated specimens compared
to AW-L, TW, HI treated specimens. AW-H displayed statistically significant differences (p<0.05) with TW
and HI specimens (Fig. 3a and 3b).
Demineralization along the cavity wall extended most (length of the lesion) in TW specimens, and that
extension was shortest in AW-H specimens (Fig. 3b). However, there were no statistical significant differences
when compared between the groups. The width of the demineralized enamel lesion extended most in HI
specimens compared to all other specimens; only in AW-H and AW-M specimens showed significantly smaller
lesion size (p < 0.05) compared to TW and HI specimens (Fig. 3b).

Fig. 2. Fluorescent photomicrographs represent typical secondary caries lesions from each group. A ‘Control’
specimen without any lesions and the experimental specimens; HI and TW (upper panel), AW-L, AW-M and AW-H
(lower panel) are seen. Large-sized lesions (indicated by large red arrows) are seen in HI and TW specimens. In
AW-L, AW-M and AW-H specimens the size of the lesions appeared to be reducing with increase of pH level(indicated by small red arrows).

Discussion
Currently, a good number of electrolyzed waters including AW are either being used or being investigated for
medical and dental purposes.21

AWs that were produced by electrolyzing tap water in the present study5

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contained high concentrations of OH- ion which showed resemblances with NaOH;22 specifically indisintegrating the WIGs of cariogenic biofilms as reported previously.19 In this study, the direct effects of three
pH levels of AW after application on a secondary caries model were investigated. Both morphologically andnumerically AW displayed the efficacy of inhibiting the progression of enamel demineralization and that wasmore effective with the increase of pH level. It is unlikely to achieve remineralization of enamel lesions once
induced by the acid attack from the biofilms on application of AW according the experimental conditions in thisstudy. In contrary, there would be inhibitory factors that either prevented or slowed down the progression ofdemineralization of the enamel on first place.

Fig. 3.
abGraphs represent the size of the secondary caries lesions; a) area and b) maximum length and maximum width.
Horizontal lines indicate no significant differences, otherwise found significantly different according to one-way
ANOVA and Tukey’s HSD test (p<0.05), n=4 in each group. Horizontal lines indicate no significant differences.

S. mutans and Streptococcus sobrinus (S. sobrinus) are the micro-organisms most strongly associated withdental caries.4 Their cariogenicity is largely determined by their ability to adhere to tooth surfaces and the
production of water insoluble glucan (WIG) resulting in subsequent formation of dental plaque or biofilm.23,24
These two species of mutans streptococci produce at least seven extracellular glucosyltransferases (GTFs) thatconvert sucrose into glucan.25 S. mutans also produce acid that may cause demineralization. Streptococcus
gordonii (S. gordonii) is another species that also plays some role in the synthesis of WIG.26
Mutans streptococci promote tooth decay through the ability to adhere, production of extra-cellular matrix(biofilms) and production of large amounts of acids.

Biofilm formation occurs through the synthesis ofwater-soluble and water-insoluble glucans via the catalyzing activity of the glucosyltransferase enzymes(GTF).23,25 Glucan is believed to be the major factor contributing to the ability of mutans streptococci to adhere
to the tooth surface and for aggregation of the bacterial cells within a biofilm.

The ability of mutans

streptococcal GTF to synthesize WIG from sucrose is an important virulence factor in initial caries development,
potentially by increasing the colonization ability of infecting cariogenic bacteria and by influencing the diffusion
characteristics of dental plaque.24 Synthesis of WIG is known to be one of the most important virulence
properties of cariogenic bacteria that contributes to the development of mature biofilm and the potency of AW
on disintegrating WIG were understood in a previous study.19 Therefore, the 20-hr biofilms formed on therestored specimens in the present study might have been disintegrated similarly on AW application, so that theexisting bacteria were not capable enough to produce acids. Also, AW itself may have contributed in changing
the acidic atmosphere to a non-acidic one by delivering the available OH- ions.

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AW is being used as drinking water that contains a high concentration of OH- ion usually produced by theelectrolysis of ordinary tap water. The Japanese Ministry of Health and Welfare has certified that alkaline
electrolyzed water assists in the alleviation of gastrointestinal disorders, acidosis, chronic diarrhea, and poor
digestion in 1960.27,28 Hence, the possibility of intra-oral use of AW for caries and secondary caries prevention
thought to be wide open and efficacy of OH- ion superadded AW was evaluated in this study.
Since the prevalence of primary caries is on the decline worldwide, secondary caries remains an unresolved
problem in restorative dentistry. It was expressed that extensive experimental and clinical researches are needed
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.12,13 The development of an OBR made it
possible to create an in vitro secondary caries model induced by cariogenic streptococci on the enamel wall atcomposite resin interface. In the present study, similar types of carious lesion formation on the exposed tooth
surfaces around the composite resin restorations were observed as reported previously.14 The caries lesions wereclearly visible at the interface of restorations by FM in all specimens due to the loss of green fluorescence inboth enamel and dentin. For instance, no detectable lesion was visible in a control specimen without biofilm
formation (Fig. 2). 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.29 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. Expectedly, the sizes of secondary caries lesions in AW-H and AW-M groups were smaller than thatof AW-L, TW and HI groups. That might also be because OH- ion can interfere the adhesion of biofilm on resin
composite and enamel surfaces.
In this study, the results indicated that AW with pH levels above 11.5 found to be significantly effective ininhibiting the progression of demineralization. AW with 10.5 pH level was also found to be effective. The
results suggested that higher OH- ion concentrations may have disintegrated more glucan by a mechanism
similar to that of NaOH.30 In addition, it can be considered that more pathogenic bacteria along with thedisintegrated WIG may have been detached by AW than that of TW or HI resulting in less acid production andsmaller sized lesion production. Therefore, it is anticipated that simple gargling with alkali-ion water may
effectively prevent dental caries and secondary caries in human oral cavity. It is suggested that AW can be used
to inhibit the progression of caries and secondary caries and also as prophylactic therapy by adjusting the pH
levels as required.
Acknowledgment
This work was supported by the Global Center of Excellence Program; International Research Center for Molecular Science in Tooth andBone Diseases at Tokyo Medical and Dental University, Tokyo, Japan.

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Correspondence to:Dr. Khairul Matin
Cariology and Operative Dentistry, 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
Copyright ©2011 by the Asian Pacific Journal of Dentistry.

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Accepted December 4, 2010.
Online ISSN 2185-3487, Print ISSN 2185-3479