Zakład Stomatologii Zachowawczej Uniwersytetu Medycznego w Białymstoku ul. M. Skłodowskiej-Curie 24A, 15-276 Białystok
Kierownik: prof. dr hab. n. med. Wanda Stokowska
1 Zakład Diagnostyki Mikrobiologicznej i Immunologii Infekcyjnej Uniwersytetu Medycznego w Białymstoku ul. J. Waszyngtona 15A, 15-274 Białystok
Kierownik: prof. dr hab. n. med. Elżbieta A. Tryniszewska
2 Apteka – Cezary Nowosielski ul. Płk. K. Piłata 20A, 07-300 Ostrów Mazowiecki
Streszczenie
Wstęp: Działanie bakteriobójcze, wirusobójcze i grzy-bobójcze ozonu jest ogólnie znane i wykorzystywane od lat w przemyśle i medycynie. Ozonoterapia stała się nieod-łącznym elementem leczenia zakażeń w takich dziedzi-nach jak chirurgia, dermatologia, kosmetyka i stomatologia.
Ozon jest gazem, dlatego bardzo dobrze przenika do tkanek i przestrzeni nawet niełatwo dostępnych. Dzięki właści-wości głębokiej penetracji i długiej obecności w tkankach po leczeniu uzyskuje się długotrwały efekt bakteriobójczy.
Jednakże działanie rodników tlenowych nie jest obojętne dla organizmu człowieka i z tego powodu należy się posługi-wać odpowiednią procedurą i algorytmem terapeutycznym.
Materiał i metody: Postanowiono potwierdzić działanie przeciwbakteryjne ozonu podawanego za pomocą urządzenia Ozonytron (produkcji Biozonix) wobec modelowych szcze-pów Streptococcus salivarius, pneumonia, pyogenes i agalac-tiae, Enterococcus faecalis, Staphylococcus aureus i epider-midis, wykorzystując dwa opracowane sposoby podawania.
Wyniki: Wykazano statystycznie istotną różnicę w wielkości strefy zahamowanego wzrostu bakteryjnego na wszystkich pożywkach w zależności od czasu działania ozonu. W obecnej pracy potwierdzono działanie bakterio-bójcze ozonu wobec szczepów bakteryjnych najczęściej
izolowanych z jamy ustnej. Ponadto porównano dwa modele stosowania gazu na zakażoną pożywkę.
Wniosek: W badaniach nad stosowaniem ozonu w zwal-czaniu bakterii typowych dla chorób zębów wykazano, że gaz ma działanie utleniające na te bakterie i jest bak-teriobójczy.
H a s ł a: ozonoterapia – stomatologia – działanie przeciw-bakteryjne.
Summary
Introduction: Bactericidal, virucidal, and fungicidal activity of ozone is generally known and has been exploited for years in industry and medicine. Ozone therapy became an inherent element of the treatment of infection in such fields as surgery, dermatology, cosmetics, and dentistry. Ozone is a gas, so it penetrates very well even into such tissues and spaces that are not easily accessible. Thanks to the prop-erty of deep penetration and long ozone presence in tissues after treatment, a long -lasting bactericidal effect is achieved.
The action of oxygen radicals is, however, not neutral for the human organism, therefore the use of an appropriate procedure and therapy algorithms is essential.
100 MAGDALENA WILCZYŃSKA-BORAWSKA, KATARZYNA LESZCZYŃSKA, CEZARY NOWOSIELSKI ET AL.
Material and methods: We decided to confirm the effect of antibacterial activity of ozone applied by means of the Ozonytron device (made by Biozonix) on model strains of Streptococcus salivarius, pneumonia, pyogenes, and aga-lactiae, Enterococcus faecalis, Staphylococcus aureus and epidermidis, using two application patterns developed by us.
Results: We found a statistically significant difference in the size of zones of inhibited bacterial growth on all media depending on the time of action of ozone. In the present paper, the bactericidal activity of ozone in relation to bac-terial strains that are most frequently isolated from the oral cavity was confirmed. What is more, two models of appli-cation of the gas on the infected medium were compared.
Conclusion: Research on the use of ozone in combat-ting bacteria typical for dental diseases has shown that the gas has an oxidizing effect on these bacteria and is bactericidal.
K e y w o r d s: ozone therapy – dentistry – antibacterial activity.
Introduction
Ozone is an allotropic form of oxygen, one of the most powerful oxidants whose disinfecting properties were known and used in industry and medicine as long ago as the end of the 19th century. Its powerful bactericidal, virucidal and fungicidal activity has been confirmed already by numer-ous researches [1, 2]. One of the major ozone effects on cell metabolism is its impact on NADH and NADPH coenzymes, giving rise to their oxidation (glycolysis, glucogenesis, syn-thesis and β -oxidation of fatty acids, citric acid cycle, elec-tron transport chain). The ozone effect is manifested in all three metabolic pathways, i.e. the ones involving carbohy-drates, proteins and fats. Gram -negative bacteria proved to be less sensitive to the gas than Gram -positive bacteria [2, 3, 4]. The latest research has proven the effectiveness of ozone in fighting infections with anaerobic flora in places that are not easily accessible for other antiseptics [5]. In recent years, ozone therapy has become widely used in den-tistry (treatment of primary and secondary caries, dental root caries, initial fissure caries and even tooth hypersen-sitivity) [6, 7]. The therapeutic role of ozone in cases men-tioned above is connected with the antibacterial activity of the gas in relation to the most caries -causing bacteria (Streptococcus, Lactobacillus, Actinomyces), as well as with the stimulation of remineralization of demineralized tissue, particularly combined with fluoride therapy [8, 9, 10, 11, 12].
At the present time, modern ozone -generating devices allow using this antiseptically acting gas in infected root canals during endodontic treatment [13]. Due to frequently com-plicated morphology and hindered access of the oxygen, the root canal environment of teeth with infected or dead pulp fosters the development of a wide bacterial spectrum.
Incomplete removal of bacteria and their metabolites leads
in consequence to serious complications in the form of inflammatory changes in the alveolar bone, cysts, abscesses and even phlegmons in cavities of the cranial and facial region [14]. The methods of root canal preparation and dis-infection are constantly being developed and modernized, however, in spite of the use of frequently strong antiseptics (sodium hypochlorite 2.5–5.25%, chlorhexidine, antibiot-ics, calcium hydroxide) distant complications still consti-tute between 23 and 47%, depending on the medicaments and the canal preparation method used [15, 16, 17]. In the side canals, the root delta and the dentinal tubules survive strains which are capable of further multiplication. That is why the most effective and least toxic agent is still being sought. Ozone may prove to be the medicament that meets these requirements, particularly due to the state of aggrega-tion and the perfect penetraaggrega-tion deep into the tissues. How-ever, it should not be forgotten that the effects of free radi-cals on the human organism during therapy are not neutral, which is why the preparation of a proper gas application
pattern determining the minimum application time with the best therapeutic effect may turn out to be very important.
The aim of our paper was to evaluate the effect of anti-bacterial activity of ozone applied by means of the Ozonytron device (made by Biozonix) on prepared model strains Strep-tococcus salivarius, pneumonia, pyogenes, agalactiae, Ente-rococcus faecalis, Staphylococcus aureus and epidermidis using two application patterns developed by ourselves.
Material and methods
A device with a set of 11 glass probes which, unlike the devices most frequently used so far, does not generate ozone externally, but decomposes oxygen to the form in statu nascendi precisely at the place of application, was used in the test. The probes, having different shapes of tips allowing precise access to a specific operating area, gener-ate an electromagnetic field of high frequency penetrating deep into the tissues up to 10 cm. A single ozone applica-tion takes 30 seconds.
The gas was applied using a selected probe (supplied by the manufacturer) on substrates with model strains: Strep-tococcus salivarius ATCC 13419, StrepStrep-tococcus pneumo-niae ATCC 49619, Streptococcus pyogenes ATCC 123440, Streptococcus agalactiae ATCC 12403, Streptococcus san-guinis ATCC 10556, Streptococcus mutans ATCC 35668, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228.
Tested strains were multiplied on Columbia Agar substrate with 5% sheep blood (Emapol). The culture was grown for 18–24 hours at a temperature of 37°C in oxygen atmosphere,
then bacterial suspensions for the experiments in a 0,85%
NaCl solution with the density of 0.5 according to McFar-land scale were prepared. The investigation of antibacte-rial activity of ozone was carried out using two application models. In each model, the suspension of examined bacteria
OZONE IN DENTISTRY: EXPERIMENTAL STUDY 101 was cultured with a cotton swab on the surface of solid
TSA (Tryptic Soy Agar, Oxoid). The nozzle of the probe was placed in the middle of the plate about 1 mm above the substrate surface, and the Ozonytron device was switched on. The cultures were grown at a temperature of 37°C in oxygen atmosphere over 24 hours. The reading of the test consisted in measuring (in millimeters) the diameter of the zone where the growth was completely inhibited and the zone where the bacterial growth in the amount of 102–103 cells/mL was maintained. All determinations were carried out three times. The first model of application (1) consisted of a continuous action on the substrate surface with cultured bacterial suspension; the probe generated an electromagnetic field during 30 seconds, 1 minute, 2 minutes and 3 minutes, respectively. The second model of application (2) consisted in the effect on the substrate „in pulses”. Each pulse lasted 30 seconds, and a 30 -second break followed. Four pulse pat-terns were carried out: only 30 seconds, 30 seconds twice (1 minute of application with a 30 -second break), 4 times
30 seconds (2 minutes of application with 30 -second breaks), 6 times 30 seconds (3 minutes with 30 -second breaks). The significance of differences between the mean values for both models of application was evaluated statistically using the Student’s t -test assuming the significance level of 0.05.
Results
The comparison of results of the test has revealed a sta-tistically significant difference in the size of zones, where the bacterial growth was inhibited, on all substrates depending on the time of action of ozone (tables 1 and 2). With both models of application (1) and (2), an increase of the growth inhibition zones in direct proportion to the extension of the exposure time has been proven. In the first model, the com-parison of zones with complete growth inhibition after 30 seconds vs after 1 minute showed a statistically significant difference (p < 0.05) – table 1 – A. As the exposure time
T a b l e 1. First model of ozone application T a b e l a 1. Pierwszy model podawania ozonu
Bacterial strain Szczep bakerii
Zones of inhibited bacterial growth (mm) Strefy zahamowanego wzrostu bakteryjnego (mm) A) completely inhibited bacterial growth
A) całkowicie zahamowany wzrost bakteryjny B) bacterial growth in the amount of 102–103 cells/mL B) wzrost bakteryjny w ilości 102–103 komórek/mL
30 s 1 min 2 min 3 min 30 s 1 min 2 min 3 min
Streptococcus salivarius 7.7 10.3* 14.3 16 12.7 18.7* 22.3 25.3
Streptococcus mutans 7.6 11.3* 13.6 16.3 13 16.7* 21.7 24
Streptococcus sanquis 7 11.7* 14 15.6 12.3 15.3* 22 25
Streptococus pneumoniae 8.3 10.3* 11.7 14.3 16.3 20.7* 24.3 27.7
Streptococcus pyogenes 8 11.3* 12.7 15 13.3 16.7* 18.7 23.7
Streptococcus agalactiae 6.7 10* 12 13.3 15 21.3* 24.7 28.7
Enterococcus faecalis 5.7 9* 10.7 12 13.7 19.6* 25.7 28.3
Staphylococcus aureus 6 8.3* 9.7 11.7 11.3 16* 19.3 25.3
Staphylococcus epidermidis 7.7 10.7* 13 14.3 12.3 18* 21.7 26.3
* all zones after 30 s vs zones after 1 min, p < 0.05 / wszystkie strefy po 30 s. względem stref po 1 min, p < 0,05
T a b l e 2. Second model of ozone application (ozone pulses) T a b e l a 2. Drugi model (pulsacyjny) podawania ozonu
Bacterial strain Szczep bakerii
Zones of inhibited bacterial growth (mm) Strefy zahamowanego wzrostu bakteryjnego (mm) A) completely inhibited bacterial growth
A) Całkowicie zahamowany wzrost bakteryjny B) bacterial growth in the amount of 102–103 cells/mL B) wzrost bakteryjny w ilości 102–103 komórek/mL
30 s 1 min
2 × 30 s 2 min
4 × 30 s 3 min
6 × 30 s 30 s 1 min
2 × 30 s 2 min
4 × 30 s 3 min 6 × 30 s
Streptococcus salivarius 7.7 12.7* 17 18.3 12.7 21.7* 25.3 28
Streptococcus mutans 7.6 12.7* 16.3 17.7 13 19.3* 26.3 28.7
Streptococcus sanquis 7 13.3* 17.3 19 12.3 21* 25.7 30
Streptococus pneumoniae 8.3 13* 14.3 16.3 16.3 22.7* 23.3 27.3
Streptococcus pyogenes 8 14.3* 15.7 17.3 13.3 17.7* 21.7 26.3
Streptococcus agalactiae 6.7 10.7* 13.3 15.7 15 21.3* 25.7 30.7
Enterococcus faecalis 5.7 9.3* 12.3 12.7 13.7 19.7* 26 28.7
Staphylococcus aureus 6 9.3* 10.7 14.3 11.3 19.3* 23.3 26.7
Staphylococcus epidermidis 7.7 12* 14.3 16.7 12.3 20.7* 23.3 27.3
* all zones after 30 s vs zones after 1 min (2 × 30 s), p < 0.05 / wszystkie strefy po 30 s. względem stref po 1 min (2 × 30 s), p < 0,05
102 MAGDALENA WILCZYŃSKA-BORAWSKA, KATARZYNA LESZCZYŃSKA, CEZARY NOWOSIELSKI ET AL.
increased to 2 and 3 minutes, the zones increased propor-tionally in size. The increase of the diameter of zones where the bacterial growth remained at a small level of 102–103 cells/mL was similar with extending time of application from 30 seconds to 1 minute (table 1 – B). In the second model, the pulse exposure, also a gradual increase of zones with bacterial growth inhibition (table 2) with increasing number of pulses was revealed. Such a state concerned both the complete inhibition zone (table 2 – A) and the zone where isolated growths in the peripheries were observed (table 2 – B). A separate issue is the comparison between two application models that we made. It turns out that in the exposure pattern in pulses of 30 seconds each with breaks, the zones with bacterial growth inhibition were significantly larger than in the pattern of continuous ozone action with a specified duration (fig. 1), and what is more, this applied to all model strains tested.
Discussion
In our study, we have confirmed ozone antibacterial action towards strains of bacteria isolated from the oral cavity. Moreover, it turned out that time and the way of gas application itself are of great importance. We used two experimental models of exposure to ozone, from which the pulsatory one has proved to be considerably more effec-tive. Hence, it appears that the elaboration of various algo-rithms for the ozone use, i.e. particular ones depending on a method of its obtainment, so as to the least possi-ble dose would exert the most powerful therapeutic effect, has a relevant practical justification. In our experiment we have demonstrated that the gas application based on -lasting pulses, retaining intervals for consecutive expo-sure, may exert a better antibacterial effect against model strains used in the study rather than continuous exposure.
This surprising result is difficult to be accounted for, in particular bearing in mind the fact that there are no reports on the comparison of various methods of ozone applica-tion with the use of Ozonytron device. Pulses resulted in
a considerably larger field of destroyed bacteria than the field obtained by means of a several minute continuous application. The effect of ozone pulsatory dosing repeated in cycles is therefore more comprehensive. This may stem from the mechanism of ozone-destroying effect on bacteria.
Binding a strong oxidant to biomolecules of bacterial cell membranes containing cystine, cysteine, methionine and histidine leads to the termination of bacterial vital functions only after a few seconds. An interval during the exposure may allow for a deeper ozone penetration into an infected tissue. A deep ozone penetration into a tissue resulting in a long -lasting bactericidal effect after a few second appli-cation was recorded by Lynch et al. and Mills et al. [18, 19].
Additionally, it is known that there are bacterial defensive mechanisms against a strong multi -oxidant driven forma-tion of intracellular free radicals. Naturally, they are toxic to the cell. Intracellular enzymes, i.e. catalase and superox-ide dismutase, may partially inhibit the synthesis of radicals inside the cell. It is evident that bacteria deficient in these enzymes are more sensitive to ozone action. Perhaps, a long exposure triggers the aforementioned defensive mechanisms, while short but frequent pulses block them. Furthermore, the nature of ozone production using Ozonytron device is worth paying attention to. During the procedure, a -frequency electromagnetic field is generated. It features an ability to penetrate through the air and fluids, giving rise to the splitting of oxygen molecule bonds precisely at the site of action of a glass plasmatic probe. During intervals between several-second applications, exchange of the air content surrounding just the probe and the irradiation field (a new oxygen batch) may take place. This air portion is affected by a re -generated electromagnetic field. Ozone is a gas revealing a tendency for fast degradation in a directly proportional way to ambient temperature. Generating ozone for future use is impossible. During an ozonolysis, the ozone degrades to an instable ozonide that, in turn, immediately decomposes to carbonyl and a dual ion. Of course, the results of our observation require a revision because it is not a good methodological approach to transfer data obtained under laboratory conditions to the clinical ones. Neverthe-less, this can be helpful in the verification of some ozone use regimens recommended by the manufactures of ozone generation devices for therapeutic purposes. Based on the conducted experiment, we were able to test differences among the methods of ozone application with avoidance of any patient’s exposure. According to Baysan and Lynch, a 20 second ozone application time yielded a 0.041 ppm ozone concentration, while maximum European standard is 0.12 ppm ozone in a room sustaining for one hour [20]. Given that, the use of ozone in short pulses is safer and more effective.
Another aspect of our paper is to prove the antibacterial effect of ozone in relation to all strains tested by us. Our results confirm clinical reports and observations concern-ing the beneficial effect of conservative caries -preventive treatment [9, 10, 11, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27].
A particular recommendation of the ozone therapy is
Fig. 1. Antibacterial activity of ozone in two application models; first model of application (1), second model of application (2)
Ryc. 1. Przeciwbakteryjne działanie ozonu przy dwóch sposobach podawania: pierwszy model podawania (1), drugi model podawania (2)
Streptococcus salivarius – (1)
Streptococcus sanquinis – (1) 30 s.
1 min 2 min 3 min
OZONE IN DENTISTRY: EXPERIMENTAL STUDY 103 a consequence of the rapidity and complete painlessness
of the method. Stimulation of the remineralization effect, hardening of already carious tissue and the disinfection effect in the not easily accessible fissures of permanent teeth – the consequence of all these factors is that ozone is more and more frequently and readily used in the treatment of young patients. A separate aspect of the antibacterial activ-ity of ozone is the possibilactiv-ity to use this antiseptic for the purpose of disinfecting infected canals using the method of antiseptic preparation. The antiseptics used so far may cause early complications, complications in the course of treatment (pain after penetration outside of the physiological orifice of the canal, air accumulation, necrosis of the apical periodontal tissue) as well as late complications (periodon-titis and inflammation of the alveolar bone). At the present time, no other medicaments apart from liquids or suspen-sions (hydrogen peroxide solution, sodium hypochlorite, calcium hydroxide solution, antibiotic mixes) are used, and they have a limited ability to penetrate the canals, prima-rily due to the state of aggregation and the intermolecular interactions. Perhaps the use of gas in canals, especially in those with a difficult anatomy, with extended root delta and numerous side branches, in C canals, will bring bet-ter clinical effects due to a betbet-ter penetration deep into the tissue, the root dentinal tubules and remaining therein even over a few weeks as a reservoir of available oxygen [18, 19, 27].
Conclusions
To sum up, our experimental study has revealed a strong oxidative effect of ozone towards the following bacterial strains: Streptococcus salivarius, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Strep-tococcus sanguinis, StrepStrep-tococcus mutans, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epi-dermidis. Furthermore, we have demonstrated that the way of ozone application is important in terms of the efficacy of antibacterial action of this gas. 30-second pulses have proved to be more effective compared to a constant several minute application. Therefore, the elongation of continu-ous irradiation time does not enhance the ozone antibacte-rial effect. The above mentioned conclusions based on our experimental study, however, need a further revision.
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