Introduction
Disinfection of the root canal system is essential for successful endodontic treatment.[1 ] Root canals are usually infected by multiple bacteria.[2 ]-[7 ]
In general, 2.5% sodium hypochlorite and 2% chlorhexidine are used as irrigating solutions
in endodontics.[8 ] Sodium hypochlorite has been used as an irrigant because of its broad antimicrobial
spectrum and ability to dissolve necrotic tissue remnants [9 ] while 2% chlorhexidine has been used as an irrigant due to its broad antimicrobial
activity, substantivity, and low cytotoxicity.[10 ] An alternative that is currently being considered for disinfection of the root canal
system is ozone therapy. Ozone is applied to oral tissues in the forms of ozonated
water, ozonated oil, and oxygen/ozone gas either alone or in combination to treat
dental disease. Ozone has a high oxidation potential, being 1.5 times more effective
than chloride as an antimicrobial agent against several microorganisms, and it can
also stimulate blood flow and immune response.[11 ] An increasing number of irrigants have been proposed in an attempt to achieve optimal
irrigation. However, none of the available irrigants showed all the necessary requirements.[12 ]
No published studies have assessed microbial load reduction in the root canal system
with a combination of reciprocating instrumentation and irrigation with ozonated water.
Reciprocating instrumentation shapes the root canal system rapidly, thus enabling
high-flow, large-volume irrigation, while the biocompatibility and properties of ozonated
water make it an interesting alternative irrigant. Within this context, the present
study sought to evaluate the antimicrobial efficacy of 2.5% sodium hypochlorite, 2%
chlorhexidine, and ozonated water on biofilms of Enterococcus faecalis , Streptococcus mutans , and Candida albicans in mesiobuccal root canals of mandibular molars. The null hypothesis was that equivalent
reductions in biofilm formation would be achieved with the use of 2.5% sodium hypochlorite,
2% chlorhexidine, and ozonated water as irrigants.
Materials and Methods
Sample selection
Sixty permanent mandibular molars were selected among teeth donated by patients at
dental clinic. Written informed consent was obtained from all patients before donation.
The study was approved by Research Ethics Committee (protocol number: 1.841.252).
The sample size was calculated based on a pilot data set using analysis of variance
(ANOVA) with statistical power of 0.80 and alpha of 0.05. A sample size of 15 specimens
per group was required.
The inclusion criteria were as follows:[13 ],[14 ] complete root formation, no previous of endodontic treatment, no pathological external
and/or internal root resorption, no root cracks or fractures, root length ≥ 15 mm,
distinct foramina for the mesiobuccal and mesiolingual canals, root curvature 25°–40°
(severe) (Schneider 1971), and anatomical canal diameter compatible with a size #15
K-file.
Tooth preparation
The teeth were radiographed in the buccolingual direction, and the degree of curvature
of the mesiobuccal root canal was determined.[15 ] The crowns were removed with double-sided diamond discs (Microdont, São Paulo, SP,
Brazil), and root length was standardized to 15 mm. The mesial root length was determined
with a digital caliper (Starrett, Itu, SP, Brazil). The orifice of the mesiolingual
and distal canals was sealed with light-curing resin (Z250 XT; 3M, Deutschland, Germany).[14 ]
The working length was determined visually by inserting a size #15 (Dentsply Maillefer,
Ballaigues, Switzerland) into the root canal until its tip was visible at the apical
foramen under operating microscope visualization (8X). The file was then withdrawn
1 mm to determine the working length. Before contamination, the canals were manually
instrumented with size #10 and #15 K-files (Dentsply Maillefer, Ballaigues, Switzerland)
until reaching the working length and irrigated with 5 mL of distilled water (Dinâmica,
Campinas, SP, Brazil). The apical foramen and the external surfaces of all roots were
sealed.[16 ] The specimens were sterilized in an autoclave (Sercon, Mogi das Cruzes, SP, Brazil)
at 121°C for 15 min.
The root canals were contaminated with standard strains of E. faecalis (ATCC 29212; LabCenter, Campinas, SP, Brazil), S. mutans (25175; LabCenter, Campinas, SP, Brazil), and C. albicans (10231; LabCenter, Campinas, SP, Brazil)). The bacterial suspension was prepared
in a tube containing 10 mL of sterile saline (Dinâmica, Campinas, SP, Brazil), matched
to a 10 McFarland standard.[14 ],[17 ]
A 20-μL aliquot of the final suspension was injected into each root canal using a
0.3-cc insulin syringe (Ultrafine BD, São Paulo, SP, Brazil). Specimens were stored
in 24-well cell culture plates (CoStar, New York, NY, USA) in an incubator (Fanem
Ltda, São Paulo, SP, Brazil) at 37°C in a 5% CO2 atmosphere for 21 days.[14 ],[16 ]-[18 ]
The viability and purity of the microorganisms within the canals were checked weekly
by random sampling of two specimens using sterile paper points.[14 ],[16 ],[19 ]
Bacterial samples were collected using sterile paper points (Dentsply Maillefer, Ballaigues,
Switzerland) compatible with the anatomical diameter of the root canal before and
after instrumentation.[14 ],[19 ] The paper point was inserted into the canal for 30 s and immediately placed in a
test tube containing 5 mL of BHI broth (Acumedia Manufacturers, Lansing, MI, USA).
Canal instrumentation
Instrumentation was performed using the WaveOne Gold (WOG) single-file reciprocating
system (Dentsply Maillefer, Ballaigues, Switzerland), powered by an X-Smart Plus electric
motor (Dentsply Maillefer, Ballaigues, Switzerland) set to operate in the WaveOne
mode, with a slight pressure in the apical direction of up to 3–4 mm.
Instrumentation was performed according to the manufacturer’s instructions. A size
#15 (Dentsply Maillefer, Ballaigues, Switzerland) was used to confirm the path of
the canal to the foramen. The glide path was expanded by at least 0.16 mm using a
ProGlider file (Dentsply Maillefer, Ballaigues, Switzerland) until the working length
was reached. Before the first cycle of WOG file, a ProGlider file (Dentsply Maillefer,
Ballaigues, Switzerland) was applied in one in-and-out brushing motion at a speed
of 300 rpm with light apical pressure, set at 2 Ncm for torque control. The Primary
WOG 25/.07 file was the file that best fitted to the canal and was used to the full
working length. The Primary 25/.07 and ProGlider files were single use.
Three cycles of instrumentation were performed. Each cycle consisted of three short
in-and-out brushing motions.[17 ]
The canals were irrigated using a 5-mL plastic syringe (Ultradent Products Inc., South
Jordan, UT, USA) with a 0.55 × 20 mm hypodermic needle (24G; Ultrafine BD, São Paulo,
SP, Brazil), inserted up to the middle third of the canal (10 mm). All canals were
instrumented by the same operator.
The specimens were randomly divided (www.random.org.br ) into four groups (n = 15) according to irrigating solution: SH: 2.5% sodium hypochlorite (compounded
– Farmácia Art Med, Jundiaí, SP, Brazil); CH: 2% chlorhexidine solution (compounded
– Farmácia Art Med, Jundiaí, SP, Brazil); O3 : ozonated water (40 μg/mL); and control: double-distilled water.
In all groups, before instrumentation, samples were collected for viable bacterial
counts. After use of the ProGlider file (Dentsply Maillefer, Ballaigues, Switzerland)
and during instrumentation (each cycle), canals were irrigated with the solutions
corresponding to groups SH, CH, O3 , and control, for 3 min, using 5 mL (10) of the irrigating solution, for a total
of 20 mL irrigant. After this protocol, a new sample collection was performed for
viable bacterial counts.
Preparation of ozonated water
An ozone generator was used (Philozon, Camboriu, SC, Brazil). Sterile double-distilled
water was cooled at 14°C and kept under refrigeration until use. The ozone gas was
adjusted in the generator to the concentration required for this experimental model
(40 μg/mL).[20 ]
Specimen culture
The specimens were diluted, seeded, and cultured. Total viable bacterial count was
determined using a colony counter. Counts were given as colony-forming units (CFU).
Statistical analysis
Antimicrobial efficacy was evaluated as bacterial growth (in CFU/mL) and percentage
biofilm reduction. Percentage reduction of biofilm was calculated using the following
formula:
BCB−100,X,(BCB−BCA)−X%
BCB, biofilm count before instrumentation; BCA, biofilm count after instrumentation.
Results were analyzed using Biostat 4.0 (Sociedade Civil Mamirauá, Belém, Pará, Brasil).
All data obtained in CFU/mL were log10 transformed. The log10-transformed and percentage biofilm reduction data were tested
for normality using the Shapiro–Wilk test. All data were found to be normally distributed
and therefore analyzed using one-way ANOVA followed by Tukey’s multiple comparison
test at the 1% significance level.
Discussion
The experimental procedure of biofilm analysis of this study was based on the studies
of Machado et al .,[17 ] Cord et al .,[14 ] Ghinzelli et al .,[21 ] and Pinheiro et al .[22 ] The culture method to evaluate antibacterial activity was based on the study of
Alves et al .,[23 ] who indicated that the culture method can be used effectively in ex vivo studies
to test the antibacterial efficacy of treatment protocols and it is equivalent to
polymerase chain reaction. Serial dilutions and sowing in BHI agar for counting CFU
were performed according to Le Goff et al .[24 ]
The microbial reduction produced by 2.5% sodium hypochlorite (98.07%) may be explained
based on the observations of Rutala and Weber,[25 ]-[27 ] who suggested that, when combined with water, sodium hypochlorite produces hypochlorous
acid, which contains active chlorine. Chlorine exerts its bactericidal action through
the irreversible oxidation of sulfhydryl groups of essential bacterial enzymes, disrupting
the metabolic function of bacterial cells.[8 ] Sodium hypochlorite may also have a deleterious effect on bacterial DNA, which involves
the formation of chlorinated derivatives of nucleotide bases. In addition, there are
reports that sodium hypochlorite may induce bacterial membrane disruption.[28 ] According to Estrela et al .,[29 ] the tissue dissolution capacity of sodium hypochlorite is based on the reaction
with fatty acids and lipids, which are transformed into soap and alcohol. Hypochlorous
acid and hypochlorite ions lead to amino acid degradation and hydrolysis.
Chlorhexidine reduced bacterial counts by 98.31% after instrumentation probably due
to its ability to adsorb to dentin, acting on bacterial cell walls and cytoplasmic
membrane, resulting in the loss of osmotic balance and leakage of intracellular material.[30 ] Its antimicrobial activity has residual effects ranging from 7 days [31 ] to 12 weeks. Chlorhexidine substantivity is facilitated by its viscosity, which
keeps the solution in contact with the canal walls and dentinal tubules.[32 ] According to Estrela et al .,[29 ] the antimicrobial activity of chlorhexidine may be explained by the interaction
between its cationic nature and the anionic compound on the bacterial surface (phosphatase
groups of teichoic acids in Gram-positive bacteria and lipopolysaccharides in Gram-negative
bacteria). According to Gomes et al .,[32 ] low concentrations of chlorhexidine allow low-molecular-weight substances to be
released, resulting in bacteriostatic effects. At high concentrations, it has bactericidal
effects due to precipitation and/or coagulation of the cytoplasm.
The microbial reduction promoted by ozonated water (98.02%) is in agreement with the
study of Nogales et al .,[20 ] who stated that ozone was a promising agent for endodontic treatment. Ozone’s oxidizing
power is exerted specifically on polyunsaturated fatty acids of the bacterial membrane,
increasing oxygen delivery to tissues and modulating the immune system, thereby improving
and accelerating tissue repair. According to Goztas et al .,[33 ] the advantages of ozone in the aqueous phase include lack of mutagenicity, rapid
microbicidal effects, and ease of handling. The authors also reported that ozonated
water shows no cytotoxicity and is highly biocompatible compared with other antimicrobial
agents. Ozone may have other clinical applications in addition to root canal irrigation,
unlike hypochlorite and chlorhexidine, which have no other therapeutic uses in dentistry;
this would justify the financial investment in ozonation equipment.
The control group also showed a significant microbial reduction after instrumentation
(72.98%). This result was probably due to instrumentation using the WOG reciprocating
system. The design and gold wire alloy in the system make the file more flexible and
more efficient in terms of cutting efficiency compared with other nickel–titanium
systems. These features increase the capacity to auger debris out of the coronal third
of the canal.[34 ]
