One of the main goals of an endodontic therapy is to eradicate infection from the root canal. No matter how meticulously a chemo-mechanical treatment is performed, clinicians sometimes fail to achieve this treatment goal, since residual microorganisms seem to be the main cause of treatment failures (1). Sodium hypochlorite (NaOCl) is today the most common antimicrobial irrigation solution used for chemo-mechanical preparation of root canals (2). However, this endodontic irrigant can evoke a cytotoxic response in contact with periapical or oral mucosal tissues (3).
Ozone was used in root canal disinfection therapy to enhance the antimicrobial effect (2, 4, 5). Some researchers (2) used the ozonated NaOCl solution, while others used ozonated water (4, 6-9) and gaseous ozone (7-9). The antimicrobial effect of tested agent could be different regarding the organization of tested microorganisms: bacteria predominantly grow as either planktonic cells which are unattached and live freely in suspension, or as microorganisms embedded in biofilms (8, 10).
Ozone, which is an allotropic triatomic form of oxygen, is a powerful oxidant that can be produced in various types of ozone generators. In 1896, Nikola Tesla patented the first high frequency ozone generator in the USA. The mode device of high frequency ozone generator Ozonytron (Biozonix, München, Germany) that was used in this study is based on Tesla’s principle.
Ozone acts biochemically in different ways. It inactivates bacteria, viruses, fungi, yeast and protozoa. Ozone enhances circulation, stimulates the oxygen metabolism and forms peroxides. It can dissolve malignant tumors and activate the immune system (5).
After being ozonized, sodium hypochlorite (NaOCl) solution becomes an even more effective oxidant. Ozone oxidizes the cell walls of microorganisms and destroys them. It speeds up the dissolution activity and reduces the time required for a routine root canal therapy (2). Chang et al. (11) used ozone in a root canal treatment. It was delivered from the HealOzone unit. They used a technique aimed to combine ozone with NaOCl during chemo-mechanical root canal disinfection. Ozone dramatically improved the predictability of root canal therapy. Moreover, the authors of the study stated that ozone could shorten the time required for treatment (2).
Ozone is effective when prescribed in sufficient concentration, used for adequate time and delivered correctly into root canals after the traditional cleaning, shaping and irrigation has been completed (12).
The aim of this study was to evaluate in vitro the antimicrobial efficacy of ozone delivered to infected root canals of extracted teeth by special KP syringe of Ozonytron - a high frequency ozone generator (Biozonix, München, Germany).
MATERIAL AND METHODS
Preparation of samples
Forty-five extracted human single rooted teeth (upper incisors and upper and lower canines) stored in sterile isotonic saline solution were selected according to size and shape similarities. Teeth have been extracted for medical reasons in several dental offices in Zagreb, Croatia. Patients agreed to donate their teeth for purposes of this experiment and genuine informed consent was obtained from research participants after the decision of the Research Ethics Committee was made on June 28th, 2013. After mechanical cleaning, and sterilizing in Euroklav 23 V-S (Melag, Berlin, Germany) at 120° C and 300 kPa, the teeth were stored again in a sterile isotonic saline solution in atmosphere of 100% humidity and at 370 C.
Clinical crowns of the specimens were sectioned at the cemento-enamel junction using a diamond bur TR-11 ISO199/016 (MANI, INC., Utsunomiya, Tochigi, Japan) with water-cooling spray. All teeth were subsequently resected 13 mm in length from the apex to ensure that all specimens have the same length and embedded in acrylic Palavit G resin (Heraeus Kulzer GmbH, Wertheim, Germany). Tooth canals were instrumented using the ProTaper rotary instrumentation techniqu222e using conventional sequence (S1, SX, S1, S2, F1, F2, F3) according to the manufacturer’s instruction at 300 rpm (Maillefer, Ballaigues, Switzerland). The teeth were divided in one experimental group (35 teeth) and two control groups (positive and negative control groups, five teeth each).
During cleaning and shaping, tooth canals were irrigated with 2.0 mL of sterile saline solution between each reamer, using a syringe (Becton Dickinson S.A., Huesca, Spain) and a 27-gauge needle (BD Drogheda, Spain). After completion of tooth preparation, all canals received a final irrigation with 10 ml sterile saline solution. The teeth were plasma sterilized along with all plastic disposable material (Sterrad 200, Johnson &Johnson, USA) and all further manipulations were performed aseptically in laminar flow.
Before starting the experiment, sterility of the samples was determined. Root canals were filled with 0.02 ml of sterile Brain-Heart infusion broth (BH) (BBL Brain Heart infusion broth, Becton Dickinson and Company, Sparks, USA, Le Pont de Cleux, France) using syringe (Becton Dickinson S.A., Huesca, Spain). Canal orifices were sealed with sterile paraffin oil (MDSS, Hannover, Germany) and covered with sterile microscope cover slide (Vitrognost, cover glass, Biognost, Zagreb, Croatia) and each specimen was placed in plastic tube containing 3 ml of sterile BHI. Plastic tubes were sealed with paraffin oil and incubated (incubator: Termo-medicinski aparati, Dugo Selo, Zagreb, Croatia) for 24 h at 35 şC under athmospheric conditions (13).
After incubation, a sterile paper point (ProTaper F2, Maillefer, Ballaigues, Switzerland) was dipped into the root canal for 30 seconds and rolled back and forth on a blood agar. The paper point was subsequently placed in a 3 ml sterile BH for further 48-hours incubation at 35 şC. After that, BH was placed on a blood agar and incubated at 35 şC for 18-24 hours. Also, BH from plastic tubes adjacent to the outer surface of specimen was incubated on a blood agar at 35 şC for 18-24 hours. All root canals and plastic dishes remained sterile during testing.
Microorganisms used in this study were: Enterococcus faecalis ATCC 29212 (LGC-ATCC, Wesel, Germany), Staphylococcus aureus ATCC 25922 (LGC-ATCC, Wesel, Germany), Staphylococcus epidermidis = Coagulase – negative staphylococcus (CNS) (aerobic bacteria isolated from mouth swabs). The log phase of bacteria grown on a blood agar plate (Enterococcus faecalis, Staphylococcus aureus, CNS) were used for suspension preparation. Suspension was prepared in Brain-Heart Infusion Broth with concentration of 108 CFU (Colony Forming Units). After vortexation (Techo Kartell, Noviglio, Italy), 0.01 ml of each suspension was inoculated by calibrated loop (aerobic incubation at 350 C for 24 hours) on a blood agar. Equal volumes (1 ml) of suspensions were mixed forming a final suspension for inoculation.
Preparation of the bacterial suspension and inoculation
A total of 0.02 ml of bacterial suspension was inserted into root canals by syringe. The root canals were sealed with sterile paraffin oil and covered with sterile microscopic cover slide. Subsequently, the teeth were submerged into 3 ml of BH broth in plastic tubes. The plastic tubes were sealed with paraffin oil and incubated for 24 h at 35 şC under atmospheric conditions.
Ozone gas delivery
Ozone was generated and applied by special KP syringe of high frequency ozone generator Ozonytron (Biozonix, München, Germany). KP syringe is a specific type of syringe that contains a centrally positioned simple glass tube filled with noble gas surrounded with titanium mash. Ozone was generated from aspirated atmospheric air by dielectric barrier discharge in space between the glass tube and titanium mash (14). According to the manufacturer, KP syringe contains a reservoir of 2.2 ml for ozone production in concentration of 525 ppm and is suitable for disinfection and treatment of root canals. A glass tube slides inside the hollow plunger during the process of instillation thus pushing the ozone enriched air mixture through the tip of the syringe into a prepared root canal.
Before being ozonized, root canals were dried using sterile paper point (ProTaper F2, Maillefer, Ballaigues, Switzerland).
The root canals were ozonized using a KP syringe and a 27-gauge needle (BD Drogheda, Spain). KP syringe was prepared for application according to the manufacturer’s recommendation and applied for 5 seconds at the speed of ≈ 0.4 ml/sec.
Sixty seconds after the ozone application, the root canals were refilled with 0.02 ml of sterile BH using syringe.
After ozonation, a microbiological examination was performed using sterile paper point (ProTaper F2, Maillefer, Ballaigues, Switzerland) which was dipped into BH inside the root canal and removed after 30 seconds. Subsequently, the paper point was dipped in BH + inactivating agent SCDLP medium (Peptone, sodium chloride, Potassium dihydrogen phosphate, glucose, Lecithin, Polysorbate 80, water, pH after sterilization 7,2) according to the industrial standard method ISO 20743:2007(E). After vortexation, the paper point was removed and two ml of extracted fluid were diluted in log 10 steps with BH + inactivating agent. 0.1 ml of the solution diluted up to 10 -1 to 10 -5 was applied to a blood agar with sterile pipette.
Blood agar plates were incubated for 24 h at 35 şC. Subsequently, the BH from plastic tubes adjacent to the outer surface of a specimen was incubated on a blood agar at 35 şC for 18-24 hours. All plastic dishes remained sterile during testing.
The colonies grown on plates were identified using standard microbiology testing (bile esculine, catalase, DNA-ase, coagulase testing).
The control of bacterial growth was performed along with the control of concentration in each experiment in parallel with inoculation of canals. Experimental specimens were plasma sterilized between cycles along with all plastic disposable material. All sterility testing assays were performed as described above in the text. The enumeration of microorganisms was performed in dilution containing 30-200 colonies, and concentration counts were expressed as log10 (CFU +1).
Ten specimens in control groups (five in the positive control group and five in the negative control group) were prepared and inoculated with microorganisms in the same manner as those belonging to the experimental group. The specimens in the negative control group were not treated at all. The root canals in the positive control group were irrigated with 2.0 mL of 2.5% v/v NaOCl for 5 minutes (8). The measurement of bacterial growth was performed, as well as the measurement of concentration, in the same manner as in the experimental group.
Analysis regarding a total reduction of microorganisms:
KP syringe mode of ozone application showed a statistically significant reduction in a total number of microorganisms 745±1.38 (89.3%), (p=0.0001), compared to the baseline number of microorganisms. In the positive control group, the reduction of total number of microorganisms was also statistically significant but it was not 100% (68.7%). In the negative control group, the number of microorganisms remained unchanged.
Analysis regarding the type of microorganisms:
Reduction of each tested microorganism was statistically significant regarding the baseline value (p=0.0001) (Table 1).
|Group||Staphylococcus aureus microorganisms reduction||Staphylococcus epidermidis microorganisms reduction||Enterococcus faecalis microorganisms reduction|
|KP syringe inside canal||7.87±0.98 (94.0%)||7.97±0.88 (88.6%)||6.74±1.10 (79.7%)|
In the positive control groups, the reduction of the number of each microorganism was statistically significant regarding the baseline count, but it was not 100% (Staphylococcus aureus 77.0%, Staphylococcus epidermidis 53.5% and Enterococcus faecalis 62.5%).
Regarding the number of Staphylococcus aureus, and the number of Staphylococcus epidermidis, there were significantly less surviving microorganisms in experimental groups after ozonization compared to the positive controls.
After ozonization, there was no difference in the number of Enterococcus faecalis between the positive control (2.5% NaOCl) and the experimental group.
Regarding the number of sterile specimens, 20% specimens were contaminated with Enterococcus faecalis, 71% specimens with Staphyloccocus aureus and 77% specimens were contaminated with Staphyloccocus epidermidis after ozonization (Table 2).
In this in vitro study, the antimicrobial efficacy of ozone, generated in KP syringe by Ozonytron unit (Biozonix, München, Germany), was evaluated in a mixed suspension of three different species of microorganisms found in the infected root canals of extracted teeth: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis.
In recent time, there have been several studies reported in the literature dealing with the effect of different modes of ozone therapy on endodontic pathogens, including the most prevalent species Enterococcus faecalis (15-18). Enterococcus faecalis, a gram-positive facultative anaerobe, is of particular interest since it is the most prevalent bacterial species found in retreatment cases. Enterococcus faecalis can survive as monoinfection in 33% of cases and is quite resistant to calcium hydroxide (19, 20). Its role in root canal treatment failures is well studied along with the current concepts of treatment (21-24).
Wilczińska-Borawska et al. (18) tested the application of ozone through electrical discharge of a glass probe (using a selected probe supplied by a manufacturer). The researchers used the modes which were different from the modes used in this study. In their study, the first mode was a continuous application of ozone gas and the second mode was ozone application through short-lasting pulses, retaining intervals for consecutive exposure. The authors have documented antimicrobial activity against several strains of bacteria isolated from the oral cavity such as Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. Our results also confirmed the antimicrobial effect of ozone produced by KP syringe of Ozonytron device on Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. Some recent studies (15, 16) confirmed the antimicrobial effect of ozone gas on Enterococcus faecalis; however, a complete sterilization was not achieved.
Similar results were obtained by Eik et al. (25). They studied the effect of ozone on periodontopathogenic species and noticed that four potentially „superinfecting“species, Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae and Candida albicans, can survive for at least part of their life cycle.
Case et al. (15) reported that exposure to O3-enriched air for a total period of 2 min resulted in a 71.6% reduction in viable CFU of Enterococcus faecalis in biofilm as compared with the control group.
In our study, KP syringe was more effective in killing Staphylococcus aureus and Staphylococcus epidermidis, sterilizing 71% and 77% of tested specimens, compared to 20% sterilized specimens in the Enterococcus faecalis group. In our study, the concentration of ozone produced by KP syringe was 525 ppm. Schneider (26, 27) reported that concentration of ozone ranging between 300 and 800 ppm produced by Ozonytron device has bactericidal effects because the lethal border of the ozone concentration for bacteria is 296 ppm.
Another study performed by Hems et al. (8) tested the antibacterial efficacy of ozone, produced by custom-made bench generator, against Enterococcus faecalis in both broth and biofilm cultures. Ozone had an antibacterial effect on Enterococcus faecalis suspended in fluid, but a relatively low effect when embedded in biofilms. Lynch (28) and Huth et al. (9) stated that Hems et al. (8) used an extremely low dose of ozone in their experiments (concentration of ozone in water mentioned in their paper was only 0.68 ppm).
Huth et al. (9) assessed the antimicrobial efficacy of aqueous (1.25-20 µg mL-1) and gaseous ozone (1-53 g m-3) against endodontic pathogens Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa in suspension and in a biofilm model. Concentrations of gaseous ozone down to 1 g m-3 almost and aqueous ozone down to 5 µg mL-1 completely eliminated the suspended microorganisms as did NaOCl and chlorhexidine. Hydrogen peroxide and lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose- and strain-dependently effective against the biofilm microorganisms. Although our experiments were different from those made by Huth et al., the results of this study are in agreement with their results regarding the dose- and strain-dependence of ozone antimicrobial activity. In our study, only one KP syringe dose was applied (2.2ml). It is most likely that better antimicrobial efficacy could be achieved by establishing the optimal dosage of ozone by KP syringe. Kaptan et al. (16) reported positive effects of topical gaseous ozone in recurrent doses in eradication of Enterococcus faecalis biofilm from the root canals. They noticed that ozone had a greater antimicrobial effect if combined with 2% NaOCl. Ozone therapy can be utilized as primary therapy or to support other types of therapies (29). The use of ozone during and after chemo-mechanical treatment, as well as the application of ultrasonic devices and laser, can result in a purposeful method of improving the success of endodontic procedure (30, 31).
Ability of ozone generated by KP syringe of Ozonytron unit, as well as NaOCl, to significantly reduce number of Enterococcus faecalis and two other tested bacteria, is of clinical importance. However, further effort should be made in order to evaluate optimal doses of ozone and an optimal time exposure. Inconsistent data on the most adequate time of ozone application and concentration to use against endodontic pathogens have been proposed in the literature.
In conclusion, the application of ozone by KP syringe of Ozonytron unit significantly decreases the absolute count of microorganisms, as well as the count of each type of bacteria separately. KP probe was statistically more effective compared to NaOCl as positive control for Staphylococcus aureus and Staphylococcus epidermidis. The absolute count of Enterococcus faecalis was decreased without statistically significant difference among the tested groups and the positive control, respectively. Among the three types of bacteria in suspension, KP probe had the lowest effect on Enterococcus faecalis.