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Comparative Evaluation of the Effects of Ozone,

Diode Laser, and Traditional Cavity Disinfectants on
Microleakage
a a a b c a
Şifa Güneş , Emrullah Bahsi , Bayram İnce , Hakan Çolak , Mehmet Dalli , İzzet Yavuz ,
d a
Cafer Sahbaz & Suzan Cangül
a
Department of Restorative Dentistry, Dicle University Dental School, 2100 Diyarbakir,
Turkey
b
Department of Conservative Dentistry, School of Dentistry, Ishik University, 44001 Arbil,
Iraq
c
İzmir Katip Çelebi University, İzmir, Turkey
d
Afyon Kocatepe University Dental School, Afyon, Turkey
Accepted author version posted online: 24 Feb 2014.Published online: 14 Apr 2014.

To cite this article: Şifa Güneş, Emrullah Bahsi, Bayram İnce, Hakan Çolak, Mehmet Dalli, İzzet Yavuz, Cafer Sahbaz &
Suzan Cangül (2014) Comparative Evaluation of the Effects of Ozone, Diode Laser, and Traditional Cavity Disinfectants
on Microleakage, Ozone: Science & Engineering: The Journal of the International Ozone Association, 36:2, 206-211, DOI:
10.1080/01919512.2013.842888

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 Ozone: Science & Engineering, 36: 206–211
Copyright © 2014 International Ozone Association
ISSN: 0191-9512 print / 1547-6545 online
DOI: 10.1080/01919512.2013.842888

Comparative Evaluation of the Effects of Ozone, Diode

Laser, and Traditional Cavity Disinfectants on Microleakage

Şifa Güneş,1 Emrullah Bahsi,1 Bayram İnce,1 Hakan Çolak,2 Mehmet Dalli,3 İzzet Yavuz,1
Cafer Sahbaz,4 and Suzan Cangül1
1
Department of Restorative Dentistry, Dicle University Dental School, 2100 Diyarbakir, Turkey
2
Department of Conservative Dentistry, School of Dentistry, Ishik University, 44001 Arbil, Iraq
Downloaded by [UAA/APU Consortium Library] at 13:23 30 October 2014

3
İzmir Katip Çelebi University, İzmir, Turkey
4
Afyon Kocatepe University Dental School, Afyon, Turkey

Aim: This study aimed to evaluate the effects on
microleakage of the application of ozone gas, laser and tra-
ditional cavity disinfection under in vitro conditions.
Material and Methods: Ninety third-molar teeth extracted
for various reasons were used in this study. All the teeth
were prepared with a standard V cavity on the buccal sur-
face. Then the teeth were randomly allocated to one of
6 groups of 15 teeth; Group 1 benzalkonium chloride, Group
2 chlorhexidine gluconate, Group 3 sodium hypochloride,
Group 4 diode laser, Group 5 ozone gas, Group 6 control
group- no disinfection was applied. Primer was applied to
the cavities, then bond and 10-s polymerization. Clearfil AP-X
was used in the cavities as a hybrid composite and poly-
merized for 20 s. The samples then underwent 1000 thermal
cycles of 30-s application, in baths at temperatures between
5 ± 2 ◦ C and 55 ± 2 ◦ C. All the samples were stirred in
0.5% basic phuxine solution and the sections taken under
stereomicroscope were examined and photographed at 15 ×
magnification. SEM analysis was made and the obtained
results were statistically evaluated with the Kruskal–Wallis
test.
Results: No significant difference was seen between the
groups in respect of the microleakage values of both the
occlusal and gingival edges (p > 0.05). Comparison of the
microleakage values of the occlusal and gingival edges of the
groups determined the least leakage to be in the ozone group
(p > 0.05).
Conclusion: No statistical significance was determined
between the groups. However, the mean least microleakage
was found in the ozone group and the highest amount in the
control group.
Received 6/14/2013; Accepted 8/20/2013
Address correspondence to Şifa Günes, Department of
Restorative Dentistry, Dicle University Dental School, 2100
Diyarbakir, Turkey. E-mail: sifagunesh@windowslive.com
Color versions of one or more of the figures in the article can be
found online at www.tandfonline.com/bose.
Keywords Ozone, Diode Laser, Sodium Hypochloride,
Chlorhexidine Gluconate, Cavity Disinfectant,
Dental Applications
INTRODUCTION
Tooth decay is a microbiological disease resulting from
the destruction of calcified tissues and localized dissolution.
Decay develops as a result of an impairment in the physiolog-
ical balance between tooth mineral and plaque (Deshpande
and Deshpande 2012). Today, in the preparation of cavities,
minimally invasive procedures continue to gain more impor-
tance in the elimination of microorganisms which may remain
in the cavity walls after preparation, rather than the concept
of “widen to protect” as described by Black (Dalli et al.
2012) Due to the potential irritant effects on the pulp tissue
of some chemicals (phenol, thymol, silver nitrate, potassium
cyanide) which were used to disinfect cavities in the past, they
are no longer in use (Meiers and Kresin 1996).
Preparations containing chlorhexidine and benzalkonium
are often recommended in cavity disinfection. Other prod-
ucts recommended for cavity disinfection are chemicals such
as sodium hypochloride (NaOCl 5.25%), hydrogen peroxide
(3%), iodine, potassium iodide and copper sulfate (Meiers and
Kresin 1996). One of the most important properties of sodium
hypochloride is that it is a wide spectrum antimicrobial agent.
Sodium hypochloride has been proven to be effective against
bacteria, bacteriophage, spores, fungi and viruses. Another
important property of sodium chloride is that even at low
concentrations, it is able to show a rapid bactericide effect
(Mohammadi 2008). In addition to antiseptic effects, there are
some toxic effects of NaOCl. As the hybridization obtained in
the adhesive systems by removing collagen from the dentine

206 Ş. Güneş et al. March–April 2014

 structure is inhibited, it is not suggested for use in disinfecting
cavities (Pascon et al. 2009).
Various studies have shown the effectiveness of chlorhexy-
dine on both Gram (+) and Gram (-) including aerobic
and anaerobic bacteria, bacterial spores, lipophyllic viruses,
yeasts, fungi, and Enterococcus faecalis. Chlorohexidine,
which has a bacteriostatic effect at low concentrations and a
bactericide effect at high concentrations, can show the effect
of being able to be absorbed by tooth tissue and mucous
membrane by long duration of release on these surfaces
(Kanisavaran 2008). Other advantages can be said to be that
it shows no irritant effect on hard tissues of the tooth and the
taste and smell can be tolerated by the patient (Kanisavaran
2008; Ryan 2010).
Benzalkonium chloride is a disinfectant with both clean-
ing and antiseptic effects of detergent origin. Previous studies
of benzalkonium chloride have shown it to have a strong
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antibacterial effect on micro-organisms such as Streptococcus
mutans, Streptococcus salivarius, Actinomyces viscosus,
Lacidophilus, and Staphylococcus aureus. Therefore, it has
been reported that this preparation is suitable for use in the
elimination of residual micro-organisms in the cavity before
restoration (Mosca et al. 2006; Turkun et al. 2005).
Recently, other methods used in cavity disinfection are
laser and ozone gas technology (Nogales et al. 2008; Saini
2011). Stimulated emission means that the light of laser radi-
ation is strengthened. Stimulation with an external factor of
the medium laser environment, which is known to be a solid,
liquid or gas-filled orb, results in the formation of a laser
beam. Diode lasers are ideal for use in dental surgeries as they
are portable and of small design. Other advantages are that
they do not occupy much space, can be easily moved and are
relatively cheap (Coleton 2008).
In the treatment of peri-implantitis, diode lasers can show
a bactericide effect without leading to any changes in the
implant surface (Coleton 2008; Yukna 2011). The absorption
rates of diode lasers into the tissues are enamel 5%, dentine
30%, decay tissue 35%, soft tissue and blood 60%, and the
response rates are enamel 15%, dentine 20%, decay tissue 3%
and soft tissue/blood 20% (Yukna 2011).
The first time ozone was used on the body surface was
to evaluate the effect on various lesions. Nowadays there are
areas of use in the treatment of many diseases such as intesti-
nal diseases, primarily ulcerative colitis, bacterial diarrhea,
migraine and AIDS (Nogales et al. 2008). Ozone eliminates
the cell walls and cell pockets of bacteria by oxidization. This
oxidization effect makes ozone a highly effective bactericidal
agent. Because of this strong oxidative effect, the protein layer
protecting the decayed tissue is removed and the biomolecules
required for the life and spread of bacteria are oxidized
(Nogales et al. 2008).
There are various studies evaluating the effects of using
ozone on decay lesions. In vitro studies have reported that
the use of ozone significantly reduced the number of micro-
organisms in pit, fissure, and root decay and in occlusal decay
lesions that have not yet developed cavitation (Brazzelli et al.
Ozone, Diode Laser, and Cavity Disinfectants on Microleakage
2006). In the same study, it was also reported that 10 s
of ozone application reduced the number of Streptococcus
mutans and Streptococcus sobrinus by a significant amount
(Brazzelli et al. 2006).
Microleakage is defined as the passage of bacteria, oral
fluids, molecules, and ions between the cavity walls and the
restoration material applied to the cavity (Arslan et al. 2012;
Larson 2005). The prevention of leakage between the sur-
faces of the tooth and restoration is of great importance in
terms of the success of the restoration and its clinical life
(Larson 2005). The ideal restoration material provides accept-
able insulation by adapting to the cavity walls. When this is
not provided, negative effects such as marginal colouring and
fracture, secondary decay, corrosion, sensitivity, and inflam-
mation of the pulp may be seen in the teeth resulting from
microleakage. The main reasons for microleakage have been
shown to be adaptation problems of the restoration material
and contractions made while attaching to the tooth structure.
This study aimed to examine the effects on microleakage of
the application of ozone gas, diode laser and traditional cavity
disinfection under in vitro conditions.
MATERIAL AND METHODS
Ninety human wisdom teeth extracted for orthodontic and
periodontal reasons were used in this study. The teeth were
stored in distilled water at room temperature until the study
was to be conducted. All the teeth were prepared with stan-
dard Class V cavities on the buccal surfaces using a cylindrical
diamond drill (Diatech, Coltene/Whaledent AG, Switzerland)
under water cooling. Each cavity was prepared as mesio-distal
with a width of 3 mm, occlusal-gingival width 2 mm and depth
of 2 mm. The gingival edges were extended to 1 mm below the
enamel-cement border. Bizotaj was made on 0.5–1 mm width
of all the enamel edges of the cavities. Then the teeth were
randomly allocated to one of 6 groups of 15 teeth.
Following cavity disinfection, as described below for each
of the 6 groups, primer, bond and composite produced by
the same company were applied to all the groups. Firstly,
primer (Clearfil protect bond, Kuraray Medical Inc., Japan)
was applied to the class V cavities with a single-use brush
and 20 s passed. Solvent was removed using low-pressure air
spray. Then the bond (Clearfil Protect Bond, Kuraray Medical
Inc. Japan) was applied and spread over the whole cavity
wall with slight air spray and was polymerized using an LED
(Light Emitting Diode-Elipar Freelight, 3M ESPE, Germany)
light source at 1000 mW/cm2 for 10 s. Clearfil AP-X (A2)
(Kuraray Medical Inc., Japan) was then applied as a hybrid
composite restoration material and was polymerized with an
LED light source for 20 s.
Group 1 (Benzalkonium chloride): Benzalkonium chloride
was applied with a single-use brush to all the prepared
cavities. After waiting 60 s, it was dried with an air spray.
Then primer, bond and composite were applied in the
manner described above.
March–April 2014 207
 Group 2 (Chlorhexidine gluconate): Chlorohexidine
gluconate was applied with a single-use brush to all the
prepared cavities. After waiting 60 s, it was dried with an
air spray. Then primer, bond and composite were applied
in the manner described above.
Group 3 (Sodium hypochloride): Sodium hypochloride was
applied with a single-use brush to all the prepared cav-
ities. After waiting 30 s, it was dried with an air spray.
Then primer, bond and composite were applied in the
manner described here.
Group 4 (Diode laser): Diode laser (smart 980-5, DEVA
laser) was applied to the prepared cavity surface for 30 s.
Then primer, bond and composite were applied in the
manner described here.
Group 5 (Ozone): Ozone (Prozone, W&H, Bürmoos,
Austria) was applied to the prepared cavity surface with
an appropriate applicator for 6 s. Then primer, bond and
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(Ultimate 500K, NSK, Japan). The surfaces of the sections to
be examined under microscopy were rubbed down with water
sandpaper.
The gingival and occlusal leakage values of the section sur-
faces obtained under stereo-optic microscopy (Olympus SZ
40 SZ-X7, Olympus Corporation, Tokyo, Japan) were exam-
ined at 15× magnification by an independent researcher and
the scores were recorded (Figures 1 and 2). The degrees of
marginal leakage were defined and a scoring system was used
similar to that used by Demirci et al. (2010).
Microleakage Scores
0. No stained leakage
1. Stained leakage to as far as half the cavity walls
2. Stained leakage on the entire cavity wall
3. Stained leakage on the cavity walls and base
4. Stained leakage partially or completely reaching the

composite were applied in the manner described here. pulp

Group 6 (Control Group): No disinfection procedure was
One tooth was selected at random from each group. These
applied to the prepared cavity surface. Then primer, bond
teeth were covered with Pt in an ion covering unit (BAL-TEC
and composite were applied in the manner described here.
SCD 050, Capovani Brothers Inc., Scotia, NY, USA). Then the
The disinfectants used, the duration of application and the
morphology of the surface between the resin-tooth hard tissue
manufacturers are shown in Table 1.
was examined under a scanning electron microscope (LEO
EVO 40 (LEO Ltd., Cambridge, UK) in the Scanning Electron
Before the finishing procedures, all the samples were Microscope Research Laboratory at Inonu University Science
stored at 37 ◦ C for 24 h in an incubator (Nüve Incubator and Technology Research Centre and photographs were taken
EN 500, Ankara, Turkey). The finishing procedures were thus (not shown in this article).
made 24 h after completion of the restorations with a diamond
finishing drill (Diatech Coltene/Whaladent AG, Switzerland)
under water and the restorations were polished with aluminum
oxide covered discs (Sof-Lex, 3M ESPE, St. Paul, MN, USA).
After a further 24 h at 37 ◦ C in an incubator, the samples were
then subjected to 1000 thermal cycles (30 s application time)
in baths ranging in temperature from 5 ± 2 ◦ C to 55 ± 2 ◦ C.
Then the root tips of the samples were closed with com-
posite resin and the teeth were coated with two coats of
acid-resistant nail varnish in such a way as to leave the varnish
1 mm outside the margins of the restoration. All the samples
were stirred in 0.5% basic phuxine solution and stored in the
incubator at 37 ◦ C for 24 h. Then the teeth were washed in
running tap water to remove excess stain.
With the teeth in buccco-palatinal/lingual direction and
the restorative material centered, sections 0.2 mm in thick-
FIGURE 1. (a) Microleakage in the ozone group; (b) Microleakage
ness were cut under irrigation with a diamond separator in the control group.
(Horico, Diamond Instruments, Germany) and micro-motor

TABLE 1. Cavity Disinfectants, Duration of Application and Manufacturers

Groups Cavity disinfectant Duration of application (s) Manufacturer

Group 1 Benzalkonium chloride 60 Zefiran (Turkey)
Group 2 Chlorhexidine gluconate 60 Ceraxidin-c (IMICRYL, Turkey)
Group 3 Sodium hypochloride 30 VISION (ADD, Turkey)
Group 4 Diode laser 30 Smart980-5 (DEKA Laser, Italy)
Group 5 Ozone 6 Prozone (W&H, Austria)

208 Ş. Güneş et al. March–April 2014

 FIGURE 2. (a) SEM image of ozone group; (b) SEM image of control group.

RESULTS DISCUSSION
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The Shapiro–Wilks test was applied to determine normal With increased aesthetic expectations in recent years,
distribution of occlusal and gingival variables. According to developments in adhesive dentistry have accelerated. These
these results, in the comparison of groups where data did not developments in adhesive dentistry have enabled restorations
show normal distribution, the Kruskal–Wallis variance anal- to be made with a minimum loss of tooth hard tissue and for
ysis method of nonparametric tests was applied. The highest these restorations to be changed in a positive way for an indi-
microleakage values obtained from the occlusal and gingival vidual’s aesthetic appearance. Despite these developments,
edges resulting from this examination are shown in Table 2. an excellent connection between tooth tissue and adhesive
When the occlusal and gingival edge microleakage values of material has still not been provided.
the restorations were examined statistically, a statistically sig- During polymerization of dimethacrylate based composites
nificant difference was seen in both occlusal (p > 0.05) and there is contraction at a rate of approximately 2–6%. Micro
gingival (p > 0.05) microleakage values. gaps may develop between the tooth and adhesive material as
On examination of the occlusal edge data, the lowest leak- a result of polymerization contraction in the restorative mate-
age was seen in the ozone gas group and the highest leakage rial. Microleakage occurs with the passage of bacteria, ions,
value was determined in the control group. Similarly, when and fluids from these gaps. The penetration of bacteria to
the gingival edge leakage values were examined, the lowest the unsuccessfully bonded surface as a result of microleakage
leakage was seen in the ozone gas group and the highest causes secondary caries in the restoration and failure of the
leakage value was determined in the control group. restoration (Mjor and Toffenetti 2000).
Those bacteria that have not been totally eliminated after
mechanical cleaning lead to failure of restorations can be
TABLE 2. Occlusal and Gingival Edge Leakage Values Obtained as a
Result of the Statistical Evaluation
counted as another factor. However, much attention has been
paid to isolation during restorative treatment, the continuing
Groups N Mean Rank presence of bacteria in the cavities continues the prolifera-
tion of the smear layer by continuing enzymatic activities.
Occlusal Bzk Chl 30 92.63 Even after mechanical cleaning of decay, it is recommended
Chlorhex 30 96.60 that these bacteria which may continue to be present in the
Diode 30 86.25 cavity walls and tooth hard tissue, are eliminated chemically.
Hypochloride 30 103.58 Residual bacteria may play a role in pulp sensitivity, pulp
Ozone 30 69.40 inflammation and the formation of secondary caries (Brandt
Control 30 94.53 and de Wet 2006; Mjor 2005). Therefore, it is important to dis-
Total 180 infect the cavity with an antibacterial agent before placement
Gingival Bzk Chl 30 90.53 of the restorations (Sharma et al. 2009).
Chlorhex 30 103.42 One of the potential problems that may develop after cavity
Diode 30 72.43 disinfection is the impairments in the bonding of hydrophylic
Hypochloride 30 103.30 resin to the dentine (Sharma et al. 2009; Sharma et al. 2011).
Ozone 30 79.98 On the other hand there are views that re-wetting dentine
Control 30 93.33 primer with a hydrophylic structure which has been applied
Total 180 before cavity disinfection will create a better bond (Carrilho

Ozone, Diode Laser, and Cavity Disinfectants on Microleakage March–April 2014 209
 et al. 2007; Stanislawczuk et al. 2009). In studies examining
the effects on the bonding support (dentine bonding systems)
of cavity disinfection, it has been revealed that different results
may be obtained depending on the contents of the cavity dis-
infectant, the dentine bonding system used, the restorative
material selected and the procedure applied (Sacramento et al.
2012).
Chlorhexidine gluconate is frequently selected as a cav-
ity disinfectant because of its antibacterial effect and high
affinity (Ersin et al. 2008; Turkun et al. 2004). In a study
evaluating the microleakage effects of 3 different cavity
disinfectants (consepsis, tubulicid red, ora-5) on Class V
cavities, Türkün et al. (2004) found that consepsis, which
contains chlorhexidine, and tubilicid red, which contains
benzalkonium, had no negative effect on microleakage. The
results of the current study conform with these findings
in that no significant difference was observed between the
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occlusal and gingival edges for each disinfectant applied in
the restorations made with an antibacterial bonding agent.
In a study of cavity disinfectants under in vitro conditions,
Ercan et al. (2009) reported that hydrogen peroxide and 0.2%
chlorhexidine, used in gel form and 2% solution had no neg-
ative effect on bonding in self-etch adhesive systems. Sung
et al. (2004) used canal irrigation solutions containing sodium
hypochloride and chlorhexidine, as cavity disinfectant and
there was shown to be no negative effect on the formation of
microleakage.
As another means of cavity disinfection, diode laser was
also used in the current study. Previous studies have reported
that although laser application had no negative effect on
microleakage, a reduced fluid flow within the tubule was seen,
associated with blockage developing at the tubule opening as a
result of the application. Lasers are known to eliminate resid-
ual bacteria by removing the smear layer. Therefore lasers can
be used in cavity disinfection (Kouchi et al. 1980; Lee et al.
2006).
In a study by Kouchi et al. (1980) it was shown that
S. mutans may be found in the dentine even at a depth of
1050–1150 µm. In dentine of 50 µm thickness, the efficacy
of chlorhexidine gluconate falls to 54% but diode laser has
been reported to eradicate 90.8% of micro-organisms at 5 W
and 97.7% at 7W.
Say et al. (2004) made restorations using two different
bonding agents (Optibond solo and One-step bond) after
applying 1% benzalkonium chloride and 2% chlorhexidine
gluconate to the cavities. The study concluded that no negative
effect of the disinfectants had been shown.
There is increasingly widespread use of ozone gas as a
natural disinfectant. Together with a powerful antibacterial,
antiviral and antifungal effect, the property of wound healing
has widened the area of use of ozone. Obtained by fragmen-
tation of the oxygen found in the air, ozone returns to its raw
material form of oxygen following disinfection. Thus, leaving
no residue or waste after disinfection gives ozone an unri-
valled property as a disinfectant (Azarpazhooh and Limeback
2008).
210 Ş. Güneş et al.
In a study evaluating the antimicrobial effect of ozone on
S. mutans and S. sobrinus, a 99% success rate was seen in
respect of eliminating micro-organisms from the application
of ozone for both 10-s and 20-s periods on teeth extracted for
initial root surface decay (Baysan et al. 2000). In the current
study, the least microleakage was seen in the ozone group.
In a study examining whether or not there were any changes
in the bonding strength of composite resins between enamel
and dentine following ozone application, the conclusion was
reached that ozone had no negative effect on bonding strength
(Cadenaro et al. 2009).
In an in-vitro study, Huth et al. (2006) researched the toxic
effects on the epithelium inside the mouth of a 1-minute
application of ozone and compared these effects with those
of chlorhexidine gluconate (0.2%, 2%), sodium hypochloride
(5.25%, 2.25%) and hydrogen peroxide (3%). According to
the study results, in contrast to the various toxic effects shown
by the disinfectants, ozone displayed no toxic effect on the live
cells of the fluid (Huth et al. 2006). Nagayoshi et al. (2004)
reported a decrease in the number of E. faecalis and S. mutans
following the application of ozone to contaminated dentine
tubules. In the same study, similar results were shown from
sonication together with ozone and the application of 2.5%
NaOCl.
In the current study, 1000 thermal cycles were applied
to the samples. The number of cycles accepted as sufficient
according to ISO standards is 500-630. In some studies where
the number of thermal cycles applied have been below this
standard in an attempt to replicate the effect of ageing, there
has been the warning that it may not be sufficient (Dallı et al.
2010).
In the current study, basic phuxine was used in the deter-
mination of microleakage scores. As bacteria are of different
sizes, basic phuxine or methylene blue have been advocated
as the most realistic methods to be used in the evaluation of
microleakage (Ferrari and Garcia-Godoy 2002; Hanks et al.
1994).
When the microleakagge scores obtained in the current
study were compared, there was no statistically significant
difference between the groups (p > 0.05). However, it must
be said that the smallest amount of microleakage was seen in
the group to which ozone gas had been applied. In ascend-
ing order of microleakage scores, the groups were ozone,
diode laser, benzalkonium chloride, chlorhexidine gluconate,
sodium hypochloride and finally the most microleakage was
seen in the group where no cavity disinfection had been
applied.
CONCLUSIONS
According to the results of this study, the least
microleakage was observed to be in the ozone group and the
most in the control group. Thus, modern methods of cavity
disinfection were determined to be more successful than tra-
ditional methods of cavity disinfection. When the results of
March–April 2014
 this study are examined, it can be said that microleakage was
reduced by cavity disinfection whereas there was an increase
in microleakage in the restorations where no cavity disin-
fection was applied. According to the results of this in-vitro
study, the application of ozone gas is the most suitable method
of cavity disinfection. However, further in-vivo studies are
required to support these results.
FUNDING
This study was supported by Dicle University Scientific
Research Projects Co-ordination Department.
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