**Ozone Dosage is the Key Factor of Its Effect in Biological Systems**

Tatyana Poznyak, Pamela Guerra Blanco, Arizbeth Pérez Martínez, Isaac Chairez Oria and Clara-L. Santos Cuevas

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76843

### **Abstract**

The applications of ozone are not only restricted to environmental remediation or industrial areas. This gas has been applied in medicine to treat several diseases, where positive effects- have been confirmed by many clinical studies. According to the European Medical Society- of Ozone and the National Center of Scientific Investigation in Cuba, it has not been possible to validate ozone's effectiveness by traditional analytical methods. Thus, this investigation proposed evaluating the effect that ozone has on biological substrates (murine models- with induced carcinogenic tumors, inflammation, and wounds), studying the variations that- ozone (dissolved in physiological solution or ozonated vegetable oils) provokes over the- total unsaturation of lipids (TUL), and by using the so-called method double bond index- (DB-index), make a correlation with the dynamic reactions obtained by several analytical- methods according to each experimental stage considered in this study.-

**Keywords:** ozone therapy, cancer, ozonated oils, inflammation, wound healing, total unsaturation, double bond index-

### **1. Introduction**

Ozone is a gaseous molecule formed by three oxygen atoms; it has a blue color (when dissolved in water) with a strong acrid aroma and a molecular weight of 48mg/mole. The ozone molecule has a cyclic structure with a distance between atoms of 1.25°A.-It has a solubility of 49/100ml of water (at 0°C), that is 10 times greater than the oxygen solubility (4.89/100ml of water) [1].

© 2018 The Author(s). Licensee IntechOpen. Distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/), which permits use, distribution and reproduction for non-commercial purposes, provided the original is properly cited.


\*The ozone toxicity on the respiratory system should not be extrapolatedto the circulatory system due to the differences in biochemistry and the metabolic regimen [3].

**Table 1.** Toxic effects of ozone in gaseous phase\*.

The excessive emissions of NO, NO2, CO, CH4, H2SO4, among others, have favored the increase in ozone concentration in the tropospheric space, above 0.1 ppmv. The reactions between the chemical compounds abovementioned give rise to the so-called photocatalytic smog, which has become the main toxic substance for lungs, eyes, nose, and skin. There are several symptoms that could appear according to ozone concentration that exists in the air (**Table 1**) [1, 2].

### **2. Ozone in medicine**

According to the European Ozone Medical Society and the National Center for Scientific Research in Cuba, among others, the following diseases can be treated with ozone: abscesses, acne, AIDS, allergies, anal fissures, arthritis, asthma, cancerous tumors, cerebral sclerosis, problems in the circulatory system, cirrhosis of the liver, corneal ulcers, cystitis, diarrhea, fistulas, boils, gangrene, gastric ulcers, intestinal disorders, glaucoma, hepatitis, herpes, hypercholesterolemia, colitis, mycosis, and osteomyelitis [2].

Unlike research on the application of ozone at the industrial level, studies in the medical field are scarce. Moreover, the studies describing the interactions of ozone with substances of biological origin and their kinetic implications have not described the entire reaction mechanisms. One of the most important premises in the application of ozone for medical aspects establishes the induction of an extraordinary and temporary response of the body systems associated with peroxidation of lipids and the antioxidant scheme of the organism [1–3].

However, it is suspected that the oxidative effect of ozone causes different effects on the- immune system, sympathetic and parasympathetic systems among others. It is well known- that the presence of compounds derived from oxidation reactions in the human body produces a cascade of biochemical reactions that has been clearly explained but not associated- with the presence of ozonation-derived byproducts. This condition occurs in many events that compromise the health of the human being, such as deep wounds, appearance of neoplasms, and so on. However, the mechanism of reactions through which the cascade ofbiochemical reactions occurs is not entirely known with certainty [3–5]. Even though it is- accepted that ozone (under the adequate dosing strategy) produces a significant number- of benefits in the human body because it is dissolved in oxygen, it increases oxygenation- in the blood, improves circulation, stimulates oxygenation in tissues, and so on, it has not- been established what are the mechanisms that generate such important advantages from the clinical point of view [1, 2].

### **3. How ozone acts and how its toxicity can be avoided**

Oxygen is essential for life; nevertheless, this gas has long-term negative effects. Reactive- oxygen species (ROS) are formed during cellular respiration. The hydroxyl radical OH˙- is the most destructive ROS for enzymes and deoxyribonucleic acid (DNA). The aging- process and metabolic disorders (arteriosclerosis, diabetes, cellular degeneration, etc.) can- be worsened by the presence of ROS.-The application of an excessive ozone dose used in- medical therapy may aggravate the ROS effect on the body. This process can be prevented- if there are proper control methods in ozone dosage, regardless of the medical ozone technique [1].

Notice that, in the ozone-oxygen mixture, the former is not equilibrated with ozone, because- ozone reacts immediately with a certain number of molecules in biological fluids, mainly antioxidants, proteins, carbohydrates, and specifically the polyunsaturated fatty acids (PUFAs) [3].

The reaction kinetics and the sequence of such reactions are uncertain, and it has been briefly described [1, 6–8]. The subsequent formation of byproducts that may be responsible for the clinical effects of ozone and the accumulation of final products must be controlled in order to avoid some of the undesirable side effects of the therapies based on ozone.-

It is widely accepted that the main reactions of ozone with biological molecules are executed according to the following stages [1]: (1) ozone reacts with ascorbic acid, uric acid,- sulfhydryl groups (SH−) from proteins, and glycoproteins generating ROS, which trigger- several biochemical stages in the blood exvivo. The ROS are neutralized 0.5–1.0min later- by the antioxidants of the system and (2) ozone reacts with the double bonds (>C=C<) of- arachidonic acid and triglycerides in the plasma, which produces a molecule of hydrogen peroxide (H<sup>2</sup> O2 ) and two aldehyde molecules known as lipid peroxidation products- (pPOL).-

According to these stages, it is possible to claim that not ozone but ROS and pPOL are the compounds responsible for the multiple biochemical reactions that occur in the cells of the body, in particular, in particular, the second reaction, which has been characterized as the key factor of the therapeutic effects of ozone. In this way, the study of the ozonation byproducts formation improves the understanding of the clinical effects, which is helpful to choose the better ozone's application scheme in a medical treatment.

As soon as ozone dissolves in biological fluids, it reacts with PUFAs, and then the concentration of hydrogen peroxide increases. However, with a similar rate, it begins to diminish as the molecule diffuses quickly toward erythrocytes, leukocytes and platelets, while several antioxidative processes are performed. Due to the presence of enzymes such as GSH-Px and GSH, the intracellular concentrations of hydrogen peroxide are reduced within the plasma and the intracellular fluid [1, 3].

The activity of the pPOL, under prolonged therapies, can give rise to the regulation of- antioxidant enzymes, the appearance of oxidative proteins, and the release of stem cells,- which is considered a crucial factor to explain some effects of ozone applications as medical- therapy [1].

### **4. Ozone application pathways**

 The therapeutic indications of ozone are based on the theory that at low concentrations of this gas (in the gaseous phase), some significant phenomena occur within the cell. It has been proved that at concentrations of 5–10mg/L or lower, there are therapeutic effects with a wide margin of safety in the patient. At present, concentrations ranging from 5 to 60mg-L−1 are accepted for the medical application of ozone [9].

### **4.1. Direct methods**

*Rectal insufflation:-*The gaseous mixture is introduced to human body by the rectum, and it is absorbed in the bowels.

*Intramuscular injection:-*In this technique, 10mL of gaseous mixture are injected in the buttocks of the patient.

*Major and minor autohemotherapy:-*This technique has been used since 1960. The minor autohemotherapy requires 10mL of blood, which is put in contact with the gaseous mixture to finally return it to the human body. The major autohemotherapy requires 50–100mL of blood, which is put in contact with the gas for a few minutes to then return it to the patient.

*Ozone bag:-*A plastic bag is placed around the treated area. The gaseous mixture is pumped into the bag, then the gas is absorbed by the human body through the skin. This is one of the methods where the reaction occurs in two stages, one is absorption by the skin and the second is the direct reaction of the skin compounds with ozone. That implies a combined model that involves mass transfer of ozone and its reaction with the bio-substrate [1, 2, 9].

### **4.2. Indirect methods**

*Ozonated water:* Ozonated water is used to wash wounds, skin burns and skin infections.-

*Intra articular injection:-*The ozonated water is injected directly between the joints and is used for the treatment of arthritis and rheumatism.

*Ozonated oil:* The ozonated oil is applied as an ointment for long times with low ozone doses [1, 2, 9].

### **5. Control methods for ozone's therapeutic applications**

Oxidative stress is the main concept that explains ozone's therapeutic effect over the human body. Ozone's paradoxical effect as a promoter of antioxidant response capable of regulating oxidative stress is common in the animal kingdom. This fact suggests that the adequate ozone's dose, besides the oxidation induced in biological subjects, may enhance the antioxidant response of the living organism. This is a critical factor issued by the immunological system to overcome infections, ischemia, and cellular regeneration [3].

Most of the technical methods employed to control the medical application of ozone are- based on the measurement of oxidative stress. Some of the typical methods employed to- measure the oxidative stress include the quantification of reactive species by electronic- paramagnetic resonance; the analytical determination of antioxidants and measurement of- total antioxidant capacity; the detection of oxidized biological markers, such as the lipid- peroxidation products (pLPO), malondialdehyde, 4-hydroxynonenal, isoprostane, and oxidized proteins; as well as the measurement of damaged DNA [10]. While these methods measure key species associated with the oxidative stress, they present some inconveniences- regarding their own analytical limitations, mainly the sensitivity and the time required to- complete the analysis.

Nowadays, ozone's medical application remains empirical. According to the "Madrid Declaration on Ozone Therapy," each patient responds differently to the controlled oxidative stress induced by ozone treatments; thus, ozone's administration must be developed in a progressive way, that is, starting with small doses and progressively increasing them [9].

### **5.1. Determination of the total unsaturation**

 The determination of the total unsaturation (TU) of organic compounds is a technique developed by Russian researches in the middle of the last century [11]. It is a useful tool based on ozone's reactions, particularly, with the double bonds (>C=C<). Ozone reacts selectively with different compounds; one of the most specific reactions of ozone takes place with unsaturated organic compounds [12]. The reaction rate constants of ozone with all the >C=C-<bonds are similar, regardless of the structure of the compounds that contain them [13]. Through the TU technique, it is possible to determine the TU of the lipids in the biological substrates, and the ones contained in vegetableoils, due to their composition,which make TU a suitable technique to control ozone's therapeutic applications.-

By this technique, the ozone reactive substrate contained in one sample can be quantified in a precise way (±1%) and in a short time of analysis (1–3min) [11, 14, 15]. The TU determination consists of the measurements of ozone necessary to react with certain samples diluted in chloroform. Afterwards, this quantity is compared with ozone consumed in its reaction with a standard sample of known concentration, as well as the stoichiometric of its reaction with ozone. Ozone's consumptions are obtained from the area of the characteristic plot of ozone concentration versus time, called ozonogram. The detailed procedure can be reviewed in [15, 16]. The mathematical formula used to calculate TU is:-

$$\text{TU} = \frac{\mathbf{C}\_{\text{ST}} \times V\_{\text{ST}} \times \mathbf{S}\_{\text{s}} \times V\_{\text{SCL}}}{\mathbf{S}\_{\text{ST}} \times V\_{\text{s}} \times W\_{\text{s}}} [\text{wJ}] \left[\frac{\text{mol}\_{\text{s} \times \text{c} \times \text{c}}}{\mathbf{S}\_{\text{sample}}}\right] \tag{1}$$

where CSTis the concentration (mol L-1) of the standard solution, VST and V<sup>S</sup> are the volumes (ml) of the standard and the sample, respectively, SSTand SS are the ozonogram areas for the standard and samples, respectively, while VSOL and WSare the solution volume (ml) and weight of the sample (g), respectively.-

### *5.1.1. Determination of the ozonation degree of vegetable oils by TU-*

Vegetable oils are composed mainly of fatty acids (free and as esters in triglycerides); the unsaturated ones constitute the principal substrate that reacts with ozone. In addition, vegetable oils contain minor compounds (unsaponifiable matter), which include sterols, polyphenols, pigments, antioxidants, as well as characteristic compounds extracted from seeds. These compounds are also reactive with ozone because they contain double bonds and some other oxidable elements. The products of ozonated oils constitute a mixture of peroxides (isoozonides, hydroperoxides, poly peroxides) with therapeutic action. The formation of these species is described by Criegee mechanism. The type and their yield depend on reaction conditions [12, 13, 17, 18]. **Figure 1** shows the reaction pathway of ozone with the unsaturated compounds of oils.

 TU quantifies ozone mass that reacts with an oxidable sample. Notice that this method considers that the stoichiometry of the reaction of ozone with >C=C-<is 1:1. Then, the TU quantifies the oxidizable substrate by ozone that is contained in the analyzed sample. Thus, the ozonation degree of oils can be easily obtained, as the percentage of all compounds in oils that react with ozone (major and minor compounds). The importance of a reliable determination of the ozonation degree is related to the therapeutic effects of ozonated oils, which, in turn, strongly depend on the type of oil and its ozonation degree.

### *5.1.2. Double bond index (DB index)-*

The DB index is the term used to extend the TU application to biological substrates. It is obtained from the measurement of ozone that has reacted with the double bonds of lipids, previously extracted from biological fluids or tissues [15]. With the determination of the total unsaturation of lipids in plasma and cell membranes, it is possible to evaluate changes in

**Figure 1.** Reaction pathway of ozone with unsaturated compounds.

lipid metabolism [11, 14]. The DB index is strongly related with the level of oxidative stress in subjects, since the lipids involved in the measurement correspond to those remaining after the oxidative stress mechanism [15].

The DB index determination corresponds to the procedure described for TU measurement. For liquid samples, such as blood plasma, the mathematical expression DB index is calculated according to:

$$\text{DB} - \text{index} = \frac{\mathbb{C}\_{\text{SI}} \times \mathbb{V}\_{\text{SI}} \times \mathbb{S}\_{\text{s}} \times \mathbb{V}\_{\text{s} \text{in}}}{\mathbb{S}\_{\text{SI}} \times \mathbb{V}\_{\text{s}} \times \mathbb{V}\_{\text{j}^{\text{in}}} \times \mathbb{K}} \text{[}\text{:u]} \tag{2}$$

 where CSTis the concentration of the standard solution (mol L-1), VST, and V<sup>S</sup> are the volumes (ml) of the standard and the sample, respectively, SSTand SS are the ozonogram areas for the standard and samples, while VSOL and VPLare the volume (ml) of solution and the volume (ml) of blood plasma from sample, respectively; K is a correlated coefficient equal to 10−7mL/mole. C.u means "conditional unit" (1 c.u-=-1 × 10−5 mole D.B./mL) [15].

The DB index, measured in healthy subjects, has been determined showing its impendence of age or sex, excluding children ≤1year and aged people ≥60years. Apparently, only diseases or pathologies produced changes in this value [14]. The DB index of lipids (blood plasma and erythrocytes) for healthy European and Mexican population are listed in **Table 2**.

Some reported clinical cases illustrated the prognostic and diagnostic criteria of the DB index- changes. Among the diseases where the DB index has been successfully used as a preclinical tool,- the oxidative process of pneumonia in children was precisely characterized by this method [11]. In- this study, and inverse correlation between the DB index and the LPO activity was observed. The- authors concluded that the DB index evolution can describe the disease evolution accurately [11].

Another relevant case of the DB index application in medical procedures corresponds to its application to evaluate the therapy effectiveness of burned patients [19]. Depending on the burned magnitude, the reported DB index values of different patients ranged from 34 to 287c.u. The evolution of the DB index correlated for each subject with its personal damage and its treatment effectiveness. The authors concluded that the changes in the DB index were observed before the clinical manifestations, showing its potential as a prognostic tool for clinical practice [19]. Some other diseases where the DB index has been related with the evolution of the illness and the effect of the treatment include cancer [20], diabetes [14], exposition to hexavalent chromium [21], as well as inflammatory processes [22]. In these cases, the DB index resulted in a powerful tool to adjust the therapeutic treatment, according to the individual needs of the subjects under treatment.-


**Table 2.** DB-index value for European and Mexican healthy population [15].

### **6. In vivo studies of the TU and the DB index application**

### **6.1. Direct applications: cancer**

The methodology proposed in this section evaluated the implantation of C6 cells in an animal (murine) model [23, 24]. The oxygen and the ozone dissolved in the saline solution were dosed by an intraperitoneal pathway in athymic mice (Balb/CNu/Nu). To evaluate the effect of ozone dosage on the tumor implanted in mice, the measurements of the DB index were carried out on the lipid fraction of plasma of blood, erythrocytes, tumor, and liver.

 **Ozonated physiological solution (NaCl 0.9%):** Physiological solution (0.9% NaCl) was used as carrier media for the oxidant agent (ozone or oxygen). The ozone concentration in oxygen was 4.6-±-0.2mg-L−1that corresponds to its concentration of 1.15-±-0.2mg-L−1 of ozone in the saline solution. Considering the volume of the injected physiological solution (90μL), only **0.103 µg** of ozone was injected to mice in treatments.-

 **Therapeutic protocol:** This experiment considered four groups (n-=-6) of athymic nude mice with C6 glioma that practically have the same tumor size (74.60-±-21mm<sup>3</sup> ). The oxidant agents were dissolved in the physiological solution, and they were administrated into the mice by intraperitoneal injection (90μL) [25]. The treatment period length was 15 days. The number and the frequency of injections were different for each treated group. In the first and second groups, the injections (oxygen for the first and ozone for the second) were carried out every second day (7 times); the third group was treated with ozone every fifth day (three times). Then the mice were sacrificed, and the samples of blood and tissues were selected to determine the DB index.-

**Evolution of volume and necrosis of tumor:** The variation of tumor volume for 15 days is shown in **Figure 2**. As we can see, ozone promoted the tumor volume growth compared- with the control group by 10 and 44% every fifth and second day, respectively, both compared with the control group. On the contrary, oxygen inhibits the tumor growth by 30%.- Even when the tumor volume results suggest a better performance in the group treated with- oxygen, the tissue necrosis demonstrate a lower activity of tumor cells in groups treated- with ozone. Furthermore, the microscopically obtained results showed that the ozone dose- influenced the tumor necrosis.-

Some studies have shown that oxygen may inhibit the tumor angiogenesis, which limits the nutrient availability [26], and could be related with slower tumor growth observed in the group treated with oxygen. On the other hand, ozone showed a stressing effect, which was reflected by the accelerated tumor growth. Ozone may induce a pronounced influence on tumor metabolism, particularly in the respiratory cycle and glycolysis, showing a positive influence on oxygen utilization in tumor [27]. These facts may explain the increased rate of tumor growth [28, 29].

Since tumor growth was slower when the ozone dose also was applied every fifth day (compared with ozone applied every second day), and a higher necrosis was observed, we may conclude that ozone dose plays a major role in two observed phenomena, in regulating the

**Figure 2.** Tumor volume increase associated with the dosage strategy showing the control group, pure oxygen and ozone dosed every second and every fifth day (n = 6).-

rate of the tumor growth and in the tissue necrosis. It is important to note that both, the tumor cell activity and tumor necrosis, the significant positive effects of the treatment were achieved under the smaller ozone dose. Considering that tumor necrosis is a positive result of the treatment based on ozonated saline solution, the smaller ozone dose (every fifth day) was the better tested strategy to treat this type of tumor.

**[18F] FDG in tumors with PET/CT: Tumor metabolic activity:** [18F] FDG (2-deoxy-2-[18F] fluoro-D-glucose)-positron emission tomography (PET) and X-ray CT imaging were performed using a micro-PET/CT scanner (Albira, ONCOVISION, Spain). **Figure 3** shows the variation of FDG in the tissue of tumor that was obtained by the image processing analysis corresponding to the set of three planes of exposition (top of the image). In the center of the figure, the acquired PET images (with the gamma camera taken in the same planes) are located. At the bottom, the over-position of both images, tomographic and PET, is shown to correlate the tumor anatomical position with its activity.

**Figure 4** represents the specific areas that demonstrate the tumor activity by color variation. The red color corresponds to larger tumor activity, and, contrary to that, the blue areas describe the regions with smaller or even null activities [30–33]. **Figure 4b** corresponds to the mice dosed with dissolved oxygen, showing an area in red color that is larger than the one detected for the mice of the control group. **Figures 4c** and **d** represent images of the mice dosed with ozone every second and fifth day, respectively. As we can see, under the smaller ozone dose the significant decrease of tumor cell activity is obtained (>80%). It is important to note that in both cases, the tumor cell activity and tumor necrosis, the significant positive effects of the treatment were achieved under the smaller ozone dose.-

**Figure 3.** Example of microPET image obtained when the ozone gas Dosage is administered every fifth day.-

**Figure 4.** 18FDG tumor activity of the considered studied group control (a), only oxygen every second day (b), ozone every second day (c) and ozone every fifth day (d).-

**The DB index variation of plasma, erythrocytes, tumor, and liver:** The measurement of DB index (reactive sites of biological substrates) of lipids of plasma and erythrocytes as well as of tumor tissue and liver was carried out to find the possible correlation with the ozone dose and its effect on the tumor volume and activity. **Figure 5** depicts the DB index of the plasma and erythrocytes from mice after the ozone and oxygen treatment, both compared with the control group. The first fact to note is the values of the DB index of both plasma and erythrocytes, which are very high in comparison with healthy mice: 2.7 × 10−2and 2.35×10−2 with respect to 2.0 × 10−5 and 0.57 × 10−5 mol ml−1. Usually the values of the DB index of lipids of the plasma and erythrocytes are close to each other (0.57 and 0.43-× 10−5 mol ml−1) [15]. These high values of the DB index in mice with this type of cancer indicate that lipid peroxidation was substantially reduced.

**Figure 5.** DB-index values obtained from lipid samples extracted from plasma and erythrocytes (n = 6).-

On the other hand, in mice treated with ozone and oxygen, this index for erythrocytes was lower- than the control group (around 32%), which indicates the tendency to normalization of LPO.-

**Figure 6** depicts the DB index of lipids extracted from tumor and liver tissues. These were measured in our previous study for the first time in mice with tumors [20]. The DB index of tumors reduces by 83% and of the liver by 70% in the group of mice with smaller doses of ozone compared to the control group and the other groups treated with ozone and oxygen. This phenomenon was observed in both tumor and liver tissues and it seems to be a consequence of the modification in metabolism promoted by ozone. According to the preliminary studies, the cancer cells repressed the TIGAR enzymes. The lower concentrations of these enzymes keep the ROS concentration at an abnormal higher level [8] that caused the apoptosis of cancer cells [34]. The mice treated with ozone every second day and oxygen had higher values for DB index of tumor tissues, which is related with the cell activity observed microscopically. In fact, there was no significant statistic difference of the DB index measured in tumor tissue treated with ozone every second day and the control group. It seems that this dose had no effects over the tumor cell metabolism, contrary to the lower dose (every fifth day), where the mice's system was able to better regulate the oxidative stress induced in the treatment, and this was reflected in both the lower cell activity and the tissue DB index. In the group of mice treated with oxygen, the DB index increased 33% compared with the control group. This may be caused by the overexpression of some enzymes, such as FAS, which improved the cell proliferation by oxygen presence [9, 20].

However, the DB index of liver tissue increased by 50% compared to the control group. This variation points out to the sensibility of this index on the ozone dose, that is, this value decreased by 70% in mice dosed every fifth day and increased by 50% in subjects dosed every

**Figure 6.** DB-index values observed in tumor and liver tissues (n = 6).-

second day. In the group treated with oxygen, the DB-index in liver decreased 40% compared with the control, due to the decrease of the consumption of the energy caused by the decrease of tumor volume. The DB index of the liver suggests a modification in the energy consumption associated with the tumor activity because of the treatment. The accumulation and production of lipids appeared to be inhibited in mice treated with ozone (every fifth day) and oxygen [25]. This effect could be explained by two possible metabolic pathways of the fatty acids synthesis in the organism. The first considers that lipids in plasma come from energetic reserves of the liver (glucagon), which is regulated by the lipid reduction in blood indeed. The second assumesthat the fatty-acid cycle regulates the lipid concentration in plasma, which is also regulated by the liver, but there is no energetic transformation. Under the higher dose of ozone, the liver regulates the lipid imbalance to compensate the oxidized fatty acids. This additional lipid source may explain the increase of the DB index.-

On the other hand, under the smaller ozone dose, the lipid-accumulation effect was not observed. The last can be a result of the regulatory process conducted outside of the liver tissue that seems to justify the DB index reduction. The tumor cell activity correlated with the DB index of the lipids is obtained from the tumor tissue. When their activity was the smallest or almost zero, the DB index value was smaller also. This confirms that the DB index determination can be a reliable method to control the medical treatment efficiency for regulating the tumor growth and its activity as well.

### **6.2. Indirect applications: vegetable oils**

 The ozonated vegetable oils (OVO) have shown interesting applications in diverse fields, such as food, pharmaceutical, and cosmetic industries, since their applications have resulted in several positive invitro, invivo, and clinical effects. In addition to their therapeutic potential, the OVOs have some advantages over other ozone applications, since they are composed of stable reaction products [35]; thus, it is not necessary to produce them in situ. This is an additional advantage from a commercial point of view.

Among the most reported therapeutic effects of the OVO, one may list bactericidal, fungicidal, as well as inflammation and wound-healing mediators [35–38]. These effects are highly related with the oil type, as well as the ozonation degree.

The determination of the TU and DB index has been useful in the application of vegetable oils. By using these parameters, it is possible to control the ozonation conditions to achieve a certain ozonation degree, as well as observe the treatment's evolution and evidence of the biochemical changes derived from the treatment.

#### *6.2.1. Ozonation degree of vegetable oils-*

The therapeutic action of the OVO depends on the accumulated ozonation products. A lot of- techniques have been employed to characterize these compounds, such as the spectroscopic- methods (Fourier transform infrared (FT-IR) and hydrogen-1 nuclear magnetic resonance (<sup>1</sup> H NMR). The identified productsby these techniques corresponding to those described by the- Criegee's mechanism (iso-ozonides, poly-peroxydes, hydroxyperoxides) [12, 13, 17, 18, 39, 40].

Different studies have justified that the observed effects of the applied ozonated oils depend on the oil's ozonation degree. For example, the invitro tests showed that the bactericidal and fungicidal effects increase when the ozonation degree increases [41–43]. Complementary, the invivo evaluations showed that the adequate ozonation level depends on the treated illness and the vegetable source of oil [44–46].

For example, the inflammation process induced in the mice's skin by 2,4-dinitrofluorobenzene was inhibited after the application of olive oil ozonated 100% (iodine value = 0).-However, the repeated applications produced hair losses, hypervisibility and swelling reactions [44]. Another work demonstrated that the ozonated sesame oil showed diverse effectiveness for mice's wound healing, depending on the peroxide index of the applied oil [45]. The authors found that the better peroxide value was 1631±64 mEq/kg. The higher and lower values of oil's peroxide value were less effective.-

 In our previous work, we studied the ozonation of two oils: sunflower (SFO) and grape seed- (GSO) [16]. The different ozonation products (mainly ozonides) were identified by the spectroscopic techniques (FT-IR, <sup>1</sup> H NMR). Also, the changes in OVO's viscosity were associated- with the formation of poly-peroxides. We also determined the dynamics of >C=C-<decomposition and product accumulation. We found that the TU decrease was similar in both oils,- but the distribution of their ozonation products was different. It was established that the- maximum amount of ozonides were formed faster in GSO.-This oil accumulated a higher- proportion of poly-peroxides related with its viscosity, when compared with the SFO [16].

Since the therapeutic effects of ozonated oils are strongly related to the accumulation of the ozonation products, our previous investigations offer an alternative method for controlling the ozonation degree in the preparation of ozonated oils.

### *6.2.2. Inflammation and wound healing-*

The effectivity of TU and DB index in ozonated oils' applications was evidenced in our- previous work [22], where the anti-inflammatory and wound healing effect of the ozonated grape seed (GS) and sunflower (SF) oils in mice were tested (for wound healing, diabetic mice were tested). The ozonation degree of both oils (determined by TU) was related- with the invivo effect of oils. For comparison, the ozonated physiological solution was- applied (subcutaneous injection, only for inflammation test), as well as the commercial- drugs indomethacin and Furacin® for anti-inflammatory and wound-healing tests were- used, respectively.

Some differences on the biochemical effect of different treatments were found, depending on the oil type and their ozonation degree. These differences were revealed by the DB index values of the treated tissues [22].

In the case of the SF oil with the ozonation degree of 44%, the DB index of 2.32 × 10−4 mol g-1 corresponds to the inflammation inhibition (INI) which is about 32%. For the GS oil, the maximum INI was 25% under the ozonation degree of 24% and corresponds to the DB index of 2.26 × 10−4 mol g-1. In this last case, the increase of the ozonation degree of GS oil up to 41% decreases the INI down to 23%.-

The effect of the vegetable source of oils on both the INI and the DB index suggests an active participation of their minor compounds typically contained in oils, in the response of the immunological system (polyphenols, tocopherols, carotenoids, chlorophylls). **Figure 7a**–**d**  shows the chemical structures of some these compounds. As seen, they are susceptible to reacting with ozone, due to their unsaturated structures. So, they may participate in the therapeutic effects of ozonated oils. Even when the concentration of these compounds is low, in comparison with unsaturated fatty acids, they can consume considerable amounts of ozone. For example, the stoichiometry of the reaction of phenols with ozone is 1:3.5, while >C=C< with ozone is 1:1.

A lower INI and the DB index of the pavilion of the ear (15% and 1.57 × 10–4mol g-1, respectively) resulted from the application of ozonated physiological solution (PS), when compared with ozonated oils. This fact indicates that the action mechanism of ozone, when applied directly (dissolved in PS) or indirectly (ozonated oils), is different.-

In the case of wound-healing evaluation for diabetic mice, we also found promising results, considering that this sickness negatively affects the wound-healing process. We observed that the complete wound healing of diabetic mice treated with ozonated oils was obtained during 12days [22]. This time is comparable with the one reported for wound healing of nondiabetic mice (14days) treated with ozonated sesame oil [45]. It is worth mentioning that no infection signs were observed over the mice skin tissue. The glucose content was also monitored throughout the treatment. None of the agents showed a regulatory effect of glucose, as expected. Then, the cutaneous application of ozonated oil did not have a systemic effect which is considered a positive effect because these oils acted locally.-

Our results showed that minor compounds presented in oils may have biological activity, which contributes to the effect of the well-known therapeutic ozonation byproducts, namely Ozone Dosage is the Key Factor of Its Effect in Biological Systems 51 http://dx.doi.org/10.5772/intechopen.76843

**Figure 7.** (a) Structures of polyphenols. (b) Structures of chlorophylls. (c) Structures of carotenoids. (d) Structures of tocopherols.

ozonides, as a product of triglycerides' ozonation. This effect was more pronounced in the case of inflammation. For wound-healing tests, a slight improvement was observed in the implementation of the SF oil, compared to the GS oil (both ozonated up to the same ozonation degree). Based on these data, we may conclude that the positive clinical effect of ozonated oils depends on their ozonation degree and their nature and then the composition of their minor compounds.

### **7. Conclusion(s)**

The TUL determination was an adequate parameter to evaluate the effect of ozone on biological substrates. The versatility of this technique allowed the control of the ozonation degree- of oils, as well as the correlation of biochemical changes in tissues involved in ozone-based treatments for C6 tumor cells, inflammation, and wound healing, considering direct and- indirect applications. The effect of dissolved ozone dosage (direct application) on the tumor- evolution was observed, and the main result was that at low doses of ozone (every 5days),- there is a greater effect on the inactivity of C6 glioma cells, decreasing their reproduction- and therefore, reducing the DB index of tumor tissues, in comparison with other groups.- This effect depends on the type and stage of the disease. Since it has been reported that the- application of ozone reduces the size of certain tumors, in this context, it was observed that- although ozone had a positive effect with respect to the activity of the tumor quantified- by micro PET and the DB index determination, the volumetric growth of the tumor was- disproportionated. The results presented in this study demonstrated that the key factor for- controlling the tumor activity, inflammation, and wound healing through direct and indirect applications of ozone was precisely ozone's dose. Our results suggest that low doses of ozone may induce a micro-oxidative stress that stimulates the organism to perform a redox- regulation, which is reflected as a self-inhibition of the cancer tissue activity. The micro-dose- of ozone may have a systemic and prolonged effect on the organism. Therefore, in this study,- three injections for 15days were enough to get a decrease, > 80%, of tumor cell activity in- mice. In addition, the DB index values pointed at different reaction mechanisms of direct- treatment with ozone (dissolved in the physiological solution) and to ozonation byproducts- (ozonated oils). Ozone's administration route influenced the inflammation inhibition, ozonated oils being the best anti-inflammatory agents. We found that the ozone dosage, meaning,- the ozonation degree of oils, as well as the frequency of application, is a key factor in the- biological effect of ozone-based therapies.-

### **Acknowledgements**

The authors thank the Department of Graduate Study and Investigation of the Instituto Politécnico Nacional of Mexico (Projects 20170481, 20170590) and the Consejo Nacional de Ciencia y Tecnología of Mexico—CONACyT (Projects 83593, 83275, 153356, 156150). P. Guerra-Blanco thanks CONACyT for the scholarship support. Some of this study was carried out as part of the activities of the "Laboratorio Nacional de Investigación y Desarrollo de Radiofármacos LANIDER-CONACyT.

### **Conflict of interest-**

Authors declare no conflict of interest.-

### **Author details**

Tatyana-Poznyak<sup>1</sup> \*, Pamela Guerra-Blanco<sup>1</sup> , Arizbeth Pérez-Martínez<sup>2</sup> , Isaac Chairez-Oria<sup>3</sup> and Clara-L.-Santos Cuevas4-

\*Address all correspondence to: tpoznyak@ipn.mx-

1-Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Chihuahua, Mexico-

2-Instituto Politécnico Nacional-ESIQIE, Mexico City, Mexico-

3-Instituto Politécnico Nacional-UPIBI, Mexico City, Mexico-

4-Instituto Nacional de Investigaciones Nucleares, Mexico City, Mexico-

### **References**


### **Chapter 4**

## **Ozone in Dentistry**

Aysan Lektemur Alpan and Olcay Bakar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75829

### **Abstract**

Ozone (triatomic oxygen or trioxygen) is the combination of three naturally occurring oxygen atoms. Ozone therapy is an alternative to traditional approaches in dentistry. The main feature suggests that ozone can be used in dentistry as a strong antimicrobial agent. In addition, ozone has antimicrobial, immune system regulatory, metabolic rate, and biosynthesis-enhancing effects. Ozone affects cellular and humoral immunity. It has positive effects on oxygen transport in the body; production of adenosine triphosphate (ATP); and production of enzymes such as glutathione peroxidase,- catalase, and superoxide dismutase. Ozone use in dentistry can be made possible via ozone gas, ozonated water, ozonized olive, or sunflower oil. Ozone is used in periodontology (gingivitis, periodontitis, periimplantitis, surgical injuries, prophylaxis), oral pathologies (stomatitis, aphthous ulceration, candidiasis, herpes infections), endodontics (root canal treatment, the fistula, abscesses), oral surgery (hemostasis, wound- healing, implantation, reimplantation, tooth extraction), prosthodontics (disinfection of crowns, disinfection of the alloy part of partial dentures), orthodontics (TME function disorders, trismus, myoarthropathies), and restorative dentistry (caries, dentine hypersensitivity, cracked tooth syndrome, bleaching, disinfection of cavity). As a result of the studies performed, ozone therapy in dentistry should be considered as an aid to conventional treatments.

**Keywords:** antioxidant, antimicrobial, dentistry, immunostimulating, ozone

### **1. Introduction**

Ozone (triatomic oxygen or trioxygen) is the combination of three naturally occurring oxygen atoms. Ozone is present in the gas form in the concentration of 1–10 ppm in the stratosphere in nature. Molecular weight is 47.98 g/mol, and it is highly endothermic and also thermodynamically unstable as an oxygen compound. Depending on environmental

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conditions, such as short half-life, pressure, and heat, molecular oxygen in ozone can be converted to atomic oxygen in a short time [1]. Ozone is the third most powerful oxidant known that does not possess radical properties due to its chemical structure [2]. Ozone has a higher energy than atmospheric oxygen, 1.6 times more dense, and 10 times more soluble in water than oxygen [1]. In 1785, Van Marum noticed that when the spark occurred in the electrostatic machine, there was a peculiar smell of air around him. In 1801, Cruickshank heard the same smell on the anode side during electrolysis of water. Sconbein described this substance in 1840 as "Ozein," meaning to smell it in Greek. In 1856, Werner Von Siemens designed an ozone generator in 1857, which was used in the disinfection of operating theaters. Since these types of generators are the forerunners of later generations, these types of generators in the market are called "Siemens type" ozone generators. In 1860, the first ozone generator- in Monaco was used for plant treatment. In 1870, for the first time in medical treatment, it- was used by Lender [3].

In the nineteenth century, Dr. Fisch used ozonated water in his practice for the first time in dentistry and introduced it to Dr. Erwin Payr who was a German surgeon. Dr. Erwin Payr used ozone in surgery and he reported a publication (of 290 pages) entitled "Ozone Treatment in Surgery" (Über Ozonbehandlung in der Chirurgie) at the 59th Meeting of the German Surgical Society in 1935 [4].

As modern science is used in the practice of dentistry, it is also changing and developing with time. Ozone therapy is an alternative to traditional approaches and can be considered as a model to help in healing. Emerging technology has led us to less invasive and more conservative work.

The main feature suggests that ozone can be used in dentistry as a strong antimicrobial agent. It is effective against Gram (+, −) bacteria, viruses, and fungi. Ozone, which is used in dental prosthesis, endodontics, restorative dentistry, periodontology, and oral and maxillofacial surgery, offers great advantages in addition to traditional treatments [5, 6].

Ozone shows the antimicrobial effect by creating cell membrane damage. It reacts with the double bonds of the hydrocarbons in the cell membrane and causes the modification of the cell content by the action of the secondary oxidant. Ozone is highly effective against antibiotic-resistant species. The antimicrobial activity of ozone is increased in liquid and acidic pH [6]. The basis of the ozone action mechanism in viral infections is inhibiting infected cell peroxide sensitivity and the synthesis of viral proteins by altering the activity of the reverse transcriptase enzyme [7].

Ozone affects cellular and humoral immunity. It stimulates the proliferation and immunoglobulin synthesis in immune cells, accelerates the sensitivity of macrophage phagocytosis, and activates other macrophage functions. This activation results in the production of specific molecules called cytokines. This suggests that ozone administration at low doses is beneficial to people with an impaired immune system. Ozone also promotes the synthesis of biologically active molecules such as interleukins, leukotrienes, and prostoglandins, thereby helping to reduce inflammation and improve wound healing [6, 8].

Ozone increases the partial oxygen pressure in the tissues and increases the oxygen transport in the body causing changes in cell metabolism. This change increases the use of oxygenated respiration and therefore energy sources (glycolysis, cycling of the Krebs, β-oxidation of fatty acids). It also prevents the erythrocytes from collapsing and increases the contact surface of erythrocytes for oxygen transport. It activates the Krebs cycle, which stimulates adenosine triphosphate (ATP) production, and causes a significant reduction in nicotinamide adenine dinucleotide (NADH) leading to oxidation of cytochrome C. Ozone therapy stimulates production of enzymes such as glutathione peroxidase, catalase, and superoxide dismutase which act as free radical scavengers [9].

It promotes intracellular protein synthesis by stimulating mitochondria and ribosomes. This alteration may lead to activation of cell functions and regeneration potential of tissues and organs. Ozone causes dilation in arterioles and venules by stimulating the release of vasodilators such as nitric oxide [6, 10].

### **1.1. Dental treatment modalities of ozone therapy**


Ozone use in dentistry can be possible via ozone gas, ozonated water, ozonized olive, or sunflower oil [2]. Ozone, a very unstable molecule in gas form, lasts a few minutes in the air, while the aquatic life lasts a few days. However, it has been reported that ozone can be measured for months and years when dissolved in an oil-based content such as 100% pure olive oil [11].

### **1.2. Ozone toxicity**

It should not be forgotten that ozone is a toxic gas if it is inhaled. Eyes and lungs are very susceptible to ozone. For this reason, long-term exposure to ozone results in some side effects, such- as epiphora, irritation of the upper airways, bronchoconstriction, rhinitis, cough, headache, and vomiting may occur, depending on the time of the ozone exposure. In such cases, it has been reported that administering supportive treatment such as oxygen, ascorbic acid, vitamin E, and N-acetylcysteine to the patient would be beneficial [12–14]. Pathological and anatomical studies showed that blood clotting is impaired in a typical table ozone poisoning and has been shown to- occur in lung hematomas. However, the ozone gas is not a chemical disinfectant, and after completing the disinfection task due to its unstable structure, it rapidly transforms into oxygen [2].

As ozone therapy is an atraumatic treatment method, the areas of use in dentistry are increasing with the protection of healthy and decayed dental caries, disinfection of dental unit water systems, antibacterial effect in avulse teeth, and healing properties of oral lesions.-

### **1.3. Contraindications of ozone therapy**


Ozone can be used for prophylaxis in dentistry due to its biological properties and for the treatment of various diseases. Ozone is used in periodontology (gingivitis, periodontitis, periimplantitis, surgical injuries, prophylaxis), oral pathologies (stomatitis, aphthous ulceration, candidiasis, herpes infections), endodontics (root canal treatment, the fistula, abscesses), surgical procedures (hemostasis, wound healing, implantation, reimplantation, tooth extraction), prosthodontics (disinfection of crowns, disinfection of the alloy part of partial dentures), orthodontics (TME function disorders, trismus, myoarthropathies), and restorative dentistry (caries, dentine hypersensitivity, cracked tooth syndrome, bleaching, disinfection of cavity) [15].

### **1.4. Ozone gas generating systems**

1. Corona discharge ozone generators: with the corona discharge method, ozone gas (O3 ) is formed by breaking the double bond of the oxygen molecule (O2 ) by passing electric current and combining the other free oxygen atom [6].

**Figure 1.** An ozone-generating system used in dental practice [17].


Thanks to innovative technology, ozone has become painless, safe, effective, and easy to apply in many areas of medicine. New-generation medical ozone generators can produce ozone at very narrow therapeutic ranges (0.1–2.1 µg /s) from oxygen molecules present in the atmosphere or in liquids. The applied microcurrent (max 100 µA) is completely harmless to both the patient and the implementer [16] (**Figure 1**).

### **2. Role of ozone on restorative dentistry**

In recent years, ozone treatment has begun to be used as a new method in the treatment of caries. It has been suggested that application of ozone to caries stops or hardens these lesions. Application of ozone to caries will provide an alternative to conventional treatment modalities. It was demonstrated in studies that ozone can be used to eradicate bacteria in carious lesions, painlessly.

Baysan et al. [18], found a significant decrease in *Streptococcus mutans* and *Streptococcus sobrinus* numbers on primary root carious lesions which were applied ozone gas for a period of 10 s. Then, the in vitro study was adapted to a randomized clinical trial and the results of the controls were measured by using DIAGNOdent and ECM.-A significant increase in remineralization was observed in ozone groups. In a randomized trial, Holmes evaluated the effect of- ozone on surface hardness (soft, brittle, stiff) on root caries. At the end of 12months, 100% of- the teeth treated with ozone treatment had hardened caries surfaces, and 37% of caries in the control group without treatment reported that the lesions were getting worse [19]. Samuel et al [20] evaluated the effect of ozonated water in remineralizing artificially created initial enamel- caries using laser fluorescence and polarized light microscopy. According the results, reduced- DIAGNOdent scores and greater depth of remineralization were gained following application of ozonated water and the ozone-treated group exhibited maximum remineralization under the polarized light microscopy. Polydorou et al. [21] used two different bonding systems, 40- and 80s, in ozone-applied cavities in an invitro study. Two different bonding systems wereperformed without any application of ozone to the control group. The teeth were then restored with composite resin. *Streptococcus mutans* ratio in the ozone group for 80 s values was statistically decreased in comparison to the other groups. These results are promising for applications directed at *Streptococcus mutans*, which is the most important pathogen responsible for caries. On the other hand, in a Cochrane review, authors concluded that application of ozone on caries provides no evidence in terms of arresting or reversing the decay process [22].

Tooth structure can lessen via attrition, abrasion, erosion, trauma from occlusion, and it may cause wearing away of enamel and dentin, thereby causing hypersensitivity. Ozone application has been found to reduce sensitivity in exposed enamel and dentin and also in cases of root sensitivity. It was found that 40–60s application of ozone offers pain reduction in sensitive teeth. Ozone initiates removal of the smear layer, opens the dentinal tubules, and widens them so that the remineralizing agents—calcium and fluoride ions—can enter the dentinal tubules easily, readily, and completely, preventing the fluid exchange from dentine tubules. Depending on this, termination of sensitivity occurs following ozone application in a short time and also lasts longer than conventional treatment modalities [9].

Delay and Holmes [23] reported that the ozone application provides a reduction in the symptoms of patients with cracked tooth syndrome. Medozon, an ozone-generating device, claims that ozone application of 60–120 s to the cracked area in the fractured tooth syndrome provides long-lived restorative material [24].

Ozone can be used on root canal-treated discolored teeth by irradiating the root canal for 3 min. This treatment provides good esthetic result by bleaching the tooth. Tessier et al. [25] evaluated the ozone efficacy in an experimental rat model used to lighten tetracycline-stained incisors. At the end of the study, it was found that ozone application could be successfully used for lightening the yellowish tinge of tetracycline-stains incisors.

### **3. Role of ozone on periodontology**

The main ozone application area in periodontology is relayed on its antimicrobial properties. It seems to be effective against both Gram (+) and Gram (−) bacteria, viruses, and fungi. It can be applied into the periodontal pocket with the different tips of generators (**Figure 2**), ozonated water, or ozonated oil.

 Nagayoshi et al. [27] investigated the effect of ozonated water on cell permeability and viability of microorganisms. Gram-negative bacteria (*Porphyromonas gingivalis*, *Porphyromonas endotalis*) was found to be more sensitive to ozone than streptococci and *Candida albicans*. In addition, the ozonated water has strong bactericidal action against *Streptococcus mutans* bacteria in the plaque biofilm. Also it was reported that ozonated water inhibited the experimental bacterial plaques in vitro. In another study, it was concluded that high concentrations of ozone water (20 µg ml−1) had an antibacterial effect equal to 0.2% concentration of chlorhexidine, whereas highly concentrated ozone gas (≥4gm−3) had an antibacterial effect of as much as 2% chlorhexidine and more effective than 0.2% chlorhexidine [28]. Ramzy et al. [29] used 150 ml of ozonized water for periodontal pocket irrigation (5–10 min once weekly, 4 weeks)

**Figure 2.** Ozone application into periodontal pocket [26].

in patients suffering from aggressive periodontitis. Statistically significant decreases in terms of pocket depth, plaque index, gingival index, and bacterial count were observed. In a study, authors compared the effect of oral irrigation with ozone water, 0.2% chlorhexidine and 10% povidone iodine, in chronic periodontitis patients and concluded that local ozone application could be used as a powerful atraumatic and antimicrobial agent in the nonsurgical treatment of periodontal disease for both home care and professional practice [30].

In contrast, Eltaş and Yavuzer applied ozone gas in addition to scaling and root planning in- acute gingivitis patients. Changes in plaque index, pocket depth, and clinical attachment levels did not differ significantly between groups at 4weeks after treatment [31]. Yılmaz etal. [32] investigated the changes in clinical and microbiological parameters of mechanical treatment, mechanical treatment + erbium: yttrium-aluminum-garnet laser, and mechanical treatment +- gas ozone application in chronic periodontitis. Attachment gain and pocket depth reduction- were found to be greater in the laser group than in the other groups. Although not statistically significant, the decrease in anaerobic flora was observed in both laser and ozone groups.-

 Karapetian et al. compared ozone therapy with surgical procedures and conventional methods in patients with periimplantitis and stated that the most effective method to eliminate bacteria was ozone therapy [33]. In an in vitro study, gaseous ozone (140 ppm, 33 mL/s) for 6 and 24 s was applied to saliva-coated titanium (SLA and polished) and zirconia (acid etched and polished) disks to determine the antibacterial effect on periimplantitis caused by bacteria such as *Streptococcus sanguinis* and *Porphyromonas gingivalis*. Gaseous ozone showed selective efficacy to reduce adherent bacteria on titanium and zirconia without affecting adhesion and proliferation of osteoblastic cells [34].

### **4. Role in bone regeneration**

Besides the antiseptic and disinfectant properties of ozone, it was also investigated for its effects on bone regeneration in recent years. One of the first study about this subject belonged- to Özdemir et al. [35]. According this study results, ozone application combined with autograft provided an increase the amount of total bone area and osteoblast count.

Kazancioglu et al. [36] compared the effects of low-dose laser therapy and ozone treatment on- bone regeneration in 5-mm critical-size defects in rats, and all defects were restored with biphasic calcium phosphate grafts. According to the histomorphometric measurements, the new bone area in the ozone group was statistically higher than the control and low-dose laser group.

In another study comparing the effects of hyperbaric oxygen and systemic ozone administration, the rats were sacrificed on days 5, 15, and 30, postoperatively. There was no difference in bone formation between hyperbaric oxygen and ozone [37].

Lektemur Alpan et al. [38] used diabetic rat calvarial defects with xenograft, and they concluded that the ozone accelerated bone morphogenetic protein-2 and osteocalcin positivity followed by accelerated xenograft resorption and enhanced bone regeneration.

### **5. Role of ozone in oral surgery**

Ozone therapy has a vast range of applications in oral surgery because of its biological properties such as enhancing wound healing, improving several properties of erythrocytes, and facilitating oxygen release in the tissues. All these biological events cause and hence improve the blood supply to the ischemic zones leading to use of ozone in cases of wound-healing impairments, following surgical interventions like tooth extractions or implant dentistry.

Ozone treatment can be applied in cases such as disinfection of wound area, treatment of soft tissue lesions (aphthous ulcers, herpes simplex, herpes zoster, etc.), healing disorders in bone and soft tissue, alveolitis, periimplantitis, bisphosphonate-related osteonecrosis, tooth transplantation, and decontamination of root surfaces of avulsed teeth planned to be reimplanted [39]. It is possible to apply ozonized water to infections that may occur after osteotomy in oral surgery. In some prospective studies, it has been shown clinically and histologically that ozonized water has a positive effect on soft tissue healing. In a prospective study involving 250 patients, application of ozone water as a cooling and flushing agent during third molar osteotomies has been shown to reduce infectious complications after surgery [39]. Kazancioglu etal. evaluated the effect of ozone therapy on pain, swelling, and trismus following third molar surgery, and they concluded that ozone application effectively reduced postoperative pain; however, it had no effect on swelling and trismus [40].

Ahmedi et al. [41] evaluated the efficacy of ozone gas on the reduction of dry socket, which occurred after surgical extraction of lower jaw third molars. Two groups were evaluated: in the control group, saline solution was used for irrigation of extraction sockets and, intraalveolar ozone was applied at 12 s (Prozone, W&H, UK) in the experimental group. They concluded that the ozone gas has a positive effect on reducing the development of dry socket and pain following third molar surgery depending on metabolic capabilities of ozone for promoting hemostasis, increasing the supply of oxygen, and inhibiting bacterial proliferation.

In a study, ozone therapy was compared with the photo-biomodulation therapy in mental nerve injury by counting Schwann cells and fasciculated nerve branches and measuring fascicular nerve areas. At the end of the study, a better healing pattern was observed in thetreatment groups. The number of Schwann cells was markedly larger in the ozone treatment and photo-biomodulation groups than in the control group [42].

The effects of ozone therapy stimulating cell proliferation and soft tissue healing must be taken- into account in the treatment of bone necrosis in patients using bisphosphonates [43]. Many studies have been carried out on the use of ozone in osteonecrosis cases occurring in jaws due to the use of bisphosphonates. Similar results were obtained in these clinical trials with different routes of administration of ozone (gas, ozonated water, and oil) [44–46]. After radiotherapy in maxilla or mandible, the amount of oxygen in the affected area is considerably reduced.- Radiotherapy leads to obliteration of intrabony vessels and inadequate vascular support in spongiosal medullary spaces resulting in xerostomia, mucositis, or loss of taste sensation. As a result, fibrosis and aseptic osteonecrosis may occur. Recovery after surgical procedures is- impaired after tooth extraction from this kind of bone in comparison to healthy bones which have adequate blood supply. Such cases are always at risk of persistent osteoradionecrosis [47].

Akdeniz et al. [48] performed a study on human primary gingival fibroblasts exposed to cytotoxic concentrations of bisphosphonates. They concluded that ozone gas plasma therapy significantly decreased the genotoxic damage and this application provided 25%, 29%, and 27% less genotoxic damage, respectively, in bisphosphonate groups and improved the wound closure rate on human gingival fibroblasts.-

Doğan etal. [49] investigated the effects of ozone on cancer progression and survival with radiotherapy. Experimental tongue cancer was formed in rats and were separated into four groups. Ozone groups were received 1 ml at a concentration of 15 mcg/ml ozone (rectal 4 sessions, for 5 days after 22 week). Groups that received ozone showed more histopathologic improvements in comparison to other groups. Radiotherapy combined with ozone therapy has provided more survival rate and tumoral reduction than the other groups.

### **6. Role in prosthodontics**

 Dentures are commonly inhabited by microbial plaque, especially *Candida albicans*. Denture stomatitis is routinely encountered in clinical practice, which can be prevented by effective denture plaque control. One successful method to do so is the use of ozone as disinfecting agent to clean denture. Arita et al. [50] concluded that exposure of dentures to flowing ozonated water (2 or 4 mg/l) for 1 min can reduce the number of *Candida albicans*.

Oizumi el al. [51] compared the microbicidal effect of gaseous ozone with that of ozonated water on oral microorganisms (*Streptococcus mutans, Staphylococcus aureus, Candida albicans*). They concluded that direct exposure to gaseous ozone seems to be a more effective microbicide than ozonated water for decreasing the microorganisms.

In another study, ozonized olive oil efficacy was evaluated in the treatment of oral lesions and conditions (aphthous ulcerations, herpes labialis, oral candidiasis, oral lichen planus, and angular cheilitis) (**Figure 3**). The ozonized olive oil was applied twice. All of the conditions showed improvement in the signs and symptoms at the end of 6 months [52].

**Figure 3.** Ozone application on herpes labialis.

Temporomandibular disorder (TMD) is a pathological condition involving both the muscular and skeletal system in the temporomandibular joint region (TMJ). This is characterized by pain in the preauricular region during jaw movements, limitation during mandibular movements, pain during chewing muscles and palpation in the TMJ region, and TMJ voices. Pain usually occurs during chewing or mandibular movements [53]. Temporomandibular disorder is a collective term embracing several problems that involve the temporomandibular joint, masticatory muscles, or both and treated usually with conservative and reversible therapy. Regular application of the ozone to the TMJ region with special probes developed for deep tissue stimulation allows for access to deep tissue under the skin. Ozone application increases the oxygenation of muscle and cartilage tissue and the creation of anti-inflammatory effect. This can be used as a noninvasive treatment method in patients with TMD. In addition, there are different current treatment modalities reported in the literature, including medication therapy, low-level laser therapy, vibratory stimulation, and, more recently, bio-oxidative ozone therapy which reduced pain in the TMJ region and improved in TMD-induced mouth opening problems after regular treatments [54, 55].

### **7. Role of ozone in endodontics**

Ozone is intensively used in root canal therapy due to its strong antimicrobial properties and absence of cytotoxicity. Ozone can be an effective agent when it is used in adequate concentration, time, and applied in a correct way into the root canals after other treatment steps have been performed. Most of the studies on effect of ozone in endodontics investigate its antimicrobial activities in the form of ozone gas, ozonated water, and ozonized oil.

Ozone is a powerful antibacterial agent. In a study, ozone was found to disinfect the bovine tooth dentin tubules effectively [56]. Nagayoshi et al. [27] demonstrated that in concentrations of 0.5–4 mg/L, ozonated water killed pure cultures of *Porphyromonas endodontalis* and *Porphyromonas gingivalis* effectively. These species were found more vulnerable to ozonated water than Gram-positive oral *Streptococci* and *Candida albicans*.

In a study, Hems et al. [57] evaluated antibacterial potential of gas form (produced by Pure zone device) and aqueous (optimal concentration 0.68 mg/L) ozone on the test species *Enterococcus faecalis.* They found that ozone in solution has antibacterial effect on planktonic *Enterococcus faecalis* after 240s treatment; however, it shows not much antibacterial- effect on *Enterococcus faecalis* within a biofilm.-

Estrela et al. [58] studied two forms of ozone—ozonated water and gaseous ozone—with 2.5% hypochlorite and 2% chloehexidine in infected dental root canals. All agents contacted 20 min and none of them had killed *Enterococcus faecalis* in human-infected root canals.

Use of gas delivery of ozone at a flow rate of 0.5–1 1/min with a net volume of 5 gm/ml for 2–3 min gave favorable result in eliminating pathogen species in the root canal [59].

As an intracanal irrigant, ozonated water can be used in infected necrotic canals, and as intracanal dressing, ozonized oils can be used to reduce target anaerobic biota. Ozone also enhances tissue regeneration and bone healing when used as a canal irrigant. Moreover, ozone water with sonification has antimicrobial effect in comparison to 2.5% NaOCl when used in the disinfection of the root canal [60].

Conflicting reports in term of antimicrobial effect of ozone on endodontic infections were presented in a review [61]. As a result, contradictory results regarding the efficacy of endodontic ozone administration have been reported in the literature.

### **8. Role of ozone in orthodontics**

In orthodontic treatments, diffuse opacity of enamel is commonly seen due to the effect of bonding material on enamel surface, as well as white spot lesions have been seen in first 4 weeks of the treatment. White spot lesion formation usually begins at bracket and toot interface and can reach beneath the bracket area. Hence, prophylactic therapy of enamel has an immense importance in orthodontic treatments.

Ghobashy et al. [62] studied on reducing demineralization of enamel bonded to the orthodontic bracket using ozonized olive oil. Patients who used ozonized olive oil gel with traditional oral hygiene instructions had significantly less decalcification areas during the orthodontic- treatment.

Ozone also has a strong oxidizing effect that might cause weak adhesions between tooth- and resin due to the negative effect of oxygen inhibition of polymerization. Cehreli etal. [63] evaluated the effect of prophylactic ozone pretreatment of enamel on shear bond strength- of orthodontic brackets bonded with total or self-etch adhesive systems. Study revealed that ozone pretreatment of enamel did not have an effect on the shear bond strength of- adhesive systems. Shear bond strength values of specimens in ozone group were even slightly higher.

### **9. Role of ozone in pedodontics**

Ozone treatment has become more and more popular in the dental clinic every day, and it has become effective in many treatment and application procedures in pedodontics.-

Applications of ozone in pediatric dentistry [4].


The use of ozone has positive effects on children, especially in terms of cooperation, such as not making noise, having very small hoods, not generating heat or bad smell, water spray or loud sounds of suctions, and not needing hand tools [4, 64]. Ozone prevents the caries formation via inhibiting the reproduction of pathogenic microorganisms, or destroying the cell wall by neutralizing or blocking [21, 65, 66] (**Figure 4**).

During this time, ozone attacks glycoproteins, glycolipids, and other amino acids and blocks enzymatic control systems of cells. Thus, the permeability of the cell membrane increases, extending to stop cell viability. After that, the ozone molecules can quickly enter the cell and cause the death of microorganisms [66].

In addition, ozone is an oxidative agent and can provide remineralization of demineralized dentin [21, 65]. The strongest acid naturally produced by acidogenic bacteria during caries formation is pyruvic acid. Pyruvic acid reacts with ozone and decarboxylates oxidatively to acetate and carbon dioxide. The remineralization of the initial caries lesions is supported by the buffered plaque fluid formed by the production of acetate [68]. Theoretically, ozone can be

**Figure 4.** Ozone can be used easily on children [67].

used to reduce the number of bacteria in active caries lesions and consequently can temporarily stop caries progression; caries restoration can be delayed or prevented [22, 69].

In particular, studies have shown that ozone efficacy on pit and fissures where bacterial elimination was very difficult and the most susceptible areas for development of caries [70].

In a randomized clinical trial conducted by Huth et al. [71], 41 children aged 3–7 years were evaluated. A total of 51 patients with pairs of teeth having cavitation-free initial decay were separated into two groups and 40 s ozone (HealOzone-Kavo Dental GmbH Germany) was administered to the study group. After 3 months of clinical observation and DIAGNOdent measurements, regression and remineralization of initial caries were observed in ozonetreated teeth, but the results were not statistically significant.-

In dental ozone applications, it is aimed to fix early lesions without altering the anatomical shape of the tooth, thanks to specially developed prophylactic tips. The first patient group to use this technique, which prevents unnecessary hard tissue loss in anterior and posterior initial caries lesions, is children.

A study was conducted with apprehensive children to determine ozone efficacy in open singlesurface caries lesions. A total of 82 patients with single-surface caries lesions were separated into two groups and ozone was not applied to the control group, while ozone (HealOzone-Cavo Dental GmbH Germany) was applied to the other group for 20 sec. In ozone-treated group, hardness values improved in comparison to the control group. At the same time, when cooperative evaluation and assessment of dental anxiety was conducted on children, it was stated that ozone application was less worrying in child patients and more acceptable due to short-time application, sound, and water withdrawal [72].

 In cavitated lesions, especially ozone gas application provides an antibacterial effect in the- cavity surface and satisfies intended to stop progression of the lesion. Although there is not enough clinical experience about the interval and dose of ozone gas to prevent the lesion becoming active after a certain period of cavitated caries lesions, it is thought that promising results can be obtained especially in children who have difficulties in cooperativeness, and this- technique may be developed widely and used in routine clinical practice.

### **10. Conclusion**

Scientific studies show that ozone can be a promising therapeutic agent in the practice of dentistry. Besides atraumatic application and antimicrobial effects of ozone, carrying toxic risk and can have deadly consequences of wrong actions also incomplete understanding of the mechanism of action many clinicians approach suspicious to ozone. Considering the studies done so far, it can be said that ozone can be used as an additional application besides applications such as antiseptics and local antibiotics, which are given in addition to dental treatments. As a result, more clinical studies on ozone therapy should be performed and welldefined parameters should be established. However, more studies on ozone are required in order to be used routinely in dental treatments.

### **Author details**

Aysan Lektemur Alpan1 \* and Olcay Bakar2

\*Address all correspondence to: ysnlpn@gmail.com

1 Faculty of Dentistry, Department of Periodontology, Pamukkale University, Denizli, Turkey

2 Faculty of Dentistry, Department of Periodontology, Erzincan University, Erzincan, Turkey

### **References**


## *Edited by Ján Derco and Marian Koman*

Ozone has an important and irreplaceable function in nature and human society. It preserves life on the Earth by stratospheric ozone layer. On the other hand, the formation of ground-level ozone by reactions of hydrocarbons with nitrogen dioxide in the presence of sunlight has adverse efects on humans and animals as well as on various materials. Tis book concentrates on the protection of stratospheric ozone and prevention of ground-level ozone formation; applications of its strong oxidizing properties in the treatment of water, wastewater and sludge; odor and color removal; uses in medicine as a disinfectant; and various other ozone therapies. It also deals with catalytic ozonation in water treatment, control methods for ozone applications on biological systems, various areas of ozone use in dental care, follow-up therapy and prevention.

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