Effectiveness of ozone therapy in the treatment of periodontal diseases: a systematic review

ABSTRACT The aim of this study was to evaluate the effectiveness of ozone as an adjuvant factor in the treatment of periodontal diseases. A systematic review was carried out using the PubMed, LILACS/BIREME, CoChrane, and Scielo databases and manual searches. Clinical studies published in English, Spanish, Portuguese, and French, between 1950 and 2020 that evaluated the effects of ozone in the treatment of periodontal diseases were included. The search resulted in 178 articles, of which 24 met the proposed eligibility criteria and were selected for this review. The risk of bias was assessed for each selected study in accordance with the Cochrane risk-of-bias tool - version 5.1.0. The selected studies evaluated the effect of ozone therapy on periodontitis, gingivitis, and peri-implant diseases. It was observed that the ozone in its oily form presented the best clinical results. In cases of periodontitis, it was concluded that ozone therapy in the gaseous form did not bring any additional benefit. With respect to gingivitis, ozone provided faster remission compared to the control group. And in peri-implant disease cases, ozone had the capacity to reduce the development of mucositis. Regarding microbiology, it was observed that ozone can continuously and significantly reduce periodontal microorganisms; however, with no difference from control groups. Further studies with adequate control of biases are suggested, using ozone in the oily or aqueous form, which are the most promising forms, also evaluating the possible effectiveness of ozone by-products.


INTRODUCTION
The development of periodontal diseases has been associated with anaerobic bacteria that initially affect the protective periodontium and can later reach the attachment periodontium, leading to bone destruction.As a result of this process, tooth mobility and even total tooth loss can be observed.Periodontitis is an inflammatory, chronic, multifactorial disease that begins with the accumulation of bacteria and their metabolic products.Thus, there is migration of the junctional epithelium in the apical direction, which promotes the deepening of the gingival sulcus and the formation of periodontal pockets associated with attachment loss [1].
Tobacco smoking, diabetes, medication, age, hereditary, and obesity have been related to increased risk of periodontal diseases.Similarly, other studies have suggested association between periodontal diseases and other diseases such as diabetes, hypertension, asthma, liver diseases, among others [2].Many studies have also suggested important association between periodontal disease and cardiovascular diseases, concluding that periodontal disease is a potential risk factor for cardiovascular events, such as stroke and coronary heart disease.Furthermore, it has been demonstrated that individuals with periodontitis have increased risk of developing cardiovascular diseases, including myocardial infarction, heart failure, peripheral artery disease, atherosclerosis and stroke [3].
In addition, there are a number of factors that favor the accumulation of dental biofilm, such as crowding, unsatisfactory restorations, poor contact point, mouth breathing and caries [4].For better control of the disease, periodontal treatment should be based on controlling the biofilm and the host response.The initial phase is the supraand subgingival mechanical debridement, which, although being the most important part of the treatment, cannot completely eliminate periodontal microorganisms, especially in very deep pockets (greater than 5-6mm) and furcation lesions [5].
Subgingival instrumentation failures reflected in periodontium with persistent inflammation can lead to indication of surgical procedure, which, in turn, can cause soft tissue recession and esthetic dissatisfaction [6].To enhance mechanical debridement, topical or systemic antimicrobial agents have been developed.Long-term use of systemic antibiotics is not recommended, as bacterial resistance may develop, in addition to their undesired adverse effects.The topical use of mouthwashes has some limitations as they do not seem to penetrate more than 3mm into pockets and may have poor substantivity [5].
In literature, there is controversy regarding the synergistic effect of subgingival irrigation combined with scaling and planing of root surfaces.Some authors believe that in this way, it is possible to release antimicrobial agents deep into pockets, thus being more effective in stopping the progression of the periodontal disease, reducing surgical interventions in the future [7,8].
Subgingival irrigation with antiseptics is described in literature mainly with the use of water, saline solution, povidone iodine, hydrogen peroxide, and chlorhexidine gluconate.The latter is the substance most used for this purpose, as it is an antiseptic with broad bactericidal spectrum, with bacteriostatic and antifungal function, and is also effective against gram-positive and -negative aerobic and anaerobic bacteria, [9,10].However, it also causes mucosal desquamation, damage to the healing of lesions, fixation of fibroblasts on root surfaces, changes in taste and tooth color [7].Allergic reactions have also been described, especially using povidone-iodine [8].
Recently, ozone has become an alternative antiseptic with good results in dentistry due to its strong action against bacteria, fungi, viruses, and protozoa, in addition to being a potent oxidant, acting as a metabolic and host response modulator [11].There are various forms of using ozone, whether gaseous or aqueous, such as in the treatment of early caries lesions; in tooth hypersensitivity, in the disinfection of cavities, root canals, and periodontal pockets; in mouthwashes and also for cleaning full dentures [7].Its use provides better healing of epithelial lesions such as canker sores and herpetic lesions, promotes hemostasis, and is also capable of sterilizing heat-sensitive medical and dental instruments [12].
Thus, the aim of this work is to carry out a systematic review of the effects of ozone as an adjuvant factor in the treatment of periodontal diseases.

METHODS
To select studies to be included in this review, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA-P) protocol was obtained as reference, registered in the PROSPERO platform (number CRD42020165351), with evaluation of the study design, population under study, types of intervention, presence of control group, and analyzed outcomes (PICO) [13].
Clinical trials comparing the effectiveness of ozone as an auxiliary means of treatment for periodontal diseases in relation to other already established therapeutic modalities were selected.
The population under study was composed of patients with some type of periodontal disease, whether gingivitis, chronic or aggressive periodontitis, and peri-implant diseases.
Clinical trials evaluating the effects of ozone in the treatment of periodontal diseases were included.
Studies that evaluated in vitro ozone therapy related to periodontal diseases or other outcomes (endodontics, periodontal disease not induced by biofilm, gingival graft hypersensitivity), review articles, other types of studies (case studies, quasi-experimental), without description of exposure, those performed with specific populations (pregnant women, diabetic patients), those with missing abstract or with unavailability of the full article, were excluded.
The search strategy adopted was to include articles published in English, Spanish, Portuguese, and French between 1950 and 2020, in order to gather the largest number of scientific articles published in literature, within the inclusion and exclusion criteria.
The following databases were searched: PubMed, LILACS/BIREME, Cochrane, and Scielo.To search the grey literature, the Open Grey platform was used, and a manual search for authors of selected articles was performed.The following Boolean expressions were used: "periodontal diseases" AND "ozone"; "gingivitis" AND "ozone"; "periodontitis" AND "ozone".
To search the articles in literature, three researchers reviewed titles and abstracts.After the first search, titles repeated in different databases were excluded.Finally, studies that did not meet the inclusion criteria were excluded.In cases of doubt, in order to decide whether a study should be included or excluded from this research, full reading was performed and, thus, it was decided by mutual agreement.
The risk of bias was individually assessed for each study according to the Cochrane risk-of-bias tool (version 5.1.0)which determines low, high, or unclear risk of bias, according to the following possibilities: selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases not belonging to the aforementioned domains [14].

RESULTS
The database search strategy resulted in 168 articles.Of these, 81 were duplicates and 57 were excluded after analysis of titles and abstracts.Thus, 30 articles were read in full and, based on the proposed eligibility criteria, 24 articles were selected for this review (figure 1).Interventions were performed with gaseous ozone in eight studies, topical in the oily form in seven studies, and liquid (ozonated water) in nine studies.In all cases, the manufacturer's instructions regarding time, potency, pressure, and quantity were followed.
In most studies, interventions performed were accompanied by hygiene instructions to participants, being well described in the articles, with the exception of seven studies [4,7,[15][16][17][18][19], which did not provide information in this respect.
In relation to the control group, in 33% of studies, only scaling and root planing were performed, without the use of any irrigation substance.In another 33%, isolated chlorhexidine gluconate was used.In addition, the use of common water, distilled water, oxygen, indocyanine green (photosensitizing agent), combined with laser, hydrogen peroxide, and saline solution was also observed.
The general characteristics of studies in relation to intervention and control groups are described in table 1, which also show the main characteristics of the population of selected studies, the outcomes analyzed, and the results obtained.No statistically significant difference was observed between intervention and control groups in terms of plaque index, gingival index, and probing depth (p>0.05).Statistically significant difference was observed only in the gingival bleeding time index between groups (p=0.0436).
The reduction of signs and symptoms was faster in the group treated with ozone.The treatment efficacy at 180 days was considered good in 98% of patients in the experimental group and in 78% in the control group (p=0.00239).
Higher reduction percentage was observed in the plaque index (12%) (p=0.23),gingival index (29%) (p=0.042), and bleeding index (26%) (p=0.03) using ozone irrigation.The percentage reduction of Actinobacillus actinomycetemcomitans using ozone was 25% compared to no change using chlorhexidine.No antibacterial effect was observed on Porphyromonas gingivalis and Tannerella forsythensis with ozone or chlorhexidine.In addition, ozone had antifungal effect at baseline (37%) and on the 7th day (12.5%),unlike chlorhexidine, which showed no antifungal effect.All clinical parameters evaluated in the study groups were reduced after treatment.Scaling with ozone therapy provided increase in MMP levels in patients with chronic periodontitis and reduction in MMP levels in patients with aggressive periodontitis.Scaling followed by ozone therapy does not lead to further improvement in clinical periodontal parameters in patients with chronic and aggressive periodontitis.Chlorhexidine 0.2% is more effective than ozonated water and can be used as adjunct to mechanical therapy to achieve significant reduction in inflammatory periodontal changes and also in the reduction of periodontopathogenic microflora.The present study showed significant results in both groups regarding the improvement of clinical parameters.When comparison was made between the two groups, the ozonated water group showed slightly higher improvement than the chlorhexidine group.However, statistically significant difference was observed only for the plaque index.Ozone therapy had no additional effect on periodontal parameters.All inflammatory parameters, PTX-3, Hs-CRP, and IL-1β, were reduced at 3 months of follow-up.However, only PTX-3 levels were significantly lower at ozone sites compared to those at control sites at three-months.Verma  Statistically significant difference in clinical and microbiological parameters was observed in the study group and in the control group from baseline to 2 months.However, no statistically significant difference was observed in clinical and microbiological parameters between groups.The trends for significant benefits were in increasing order of effectiveness: oxygen + NaCl; oxygen + hydrogen peroxide; ozone + NaCl, and ozone + hydrogen peroxide.Ozone with or without hydrogen peroxide can reduce the development of peri-implant mucositis and should be considered in clinical trials to assess its effect in the treatment of peri-implantitis.Table 2 shows the basic form of treatment for all subjects described in the studies, the clinical markers used to assess the disease, and the complementary exams performed.

PERI-IMPLANT DISEASES
Non-surgical supra-and subgingival scaling was performed in most studies, with mechanical and/or ultrasonic instrumentation without antibiotic prescription.Some studies reported the use of anesthesia for this procedure [8,[23][24][25].The duration and number of scaling sessions ranged from 1 to 7 sessions and from 30 min to 1 hour per patient.Irrigation Microbiological study with a sample of the gingival sulcus or periodontal pocket on the day of admission to the study and at 180 days of postoperative evolution.
Subgingival plaque was collected and analyzed by CRP.
Plaque index, bleeding on probing, sulcus bleeding index, probing depth, clinical attachment loss.
On the clinical exam days, saliva at rest was collected for laboratory analysis of MMP-1 enzyme levels.
Probing depth, clinical attachment level, probing percentage bleeding recorded at all six sites per tooth (mesial, distal, and middle on the buccal and lingual sides).
Microbiological exam performed immediately before and after treatment and 1 and 8 weeks after treatment.
Samples from representative periodontal areas were collected for the evaluation of microbiological parameters at time 0 and 90 days.
Probing pocket depth, bleeding on probing, clinical attachment loss, gingival recession (all measured at 6 sites on each toothmesial, distal, and middle on the buccal and lingual sides).Plaque index and gingival index (assessed at 4 sites -mesial, distal, buccal, and lingual) in the Ramfjord teeth.
After examining each patient, 5 mL venous blood sample were obtained after a 10hour fast period, at T0 and T1, to measure highly sensitive serum C-reactive protein concentrations.
Shoukheba & Ali (2014) [11] Scaling and root planing of all teeth using manual and ultrasonic instrumentation.
Plaque index, gingival index, bleeding on probing, probing depth, and clinical attachment level.
Before treatment, a subgingival plaque sample was collecetd from the deepest periodontal pocket (6 mm probe depth) for real-time CRP analysis.

Chaudhari et al. (2016) [7]
Scaling and root planing.Gingival bleeding index, probing depth, attachment level (all recorded at baseline and at 3 weeks post intervention).
Plaque samples were collected for microbiological study at baseline and immediately at the following day, but without identification of microbiological types.
Scaling and root planing of the entire mouth with irrigation.
Gingival index and probing depth at day 0 and 30 after treatment.
Plaque samples were collected for microbiological analysis from the mesial surface of the 1st molar of each quadrant 20 seconds after the end of the prescribed treatment.
Saeed et al. ( Scaling and root planing with manual and ultrasonic instrumentation, entire mouth polishing in 1 session. Probing depth, clinical attachment level, bleeding on probing, plaque index (all measured before treatment, and 1, 3, and 6 months after treatment).
Active salivary matrix metalloproteinase levels were analyzed by PerioSafe® (Dentagnostics, Jena, Germany), a diagnostic test for the prevention of periodontal and peri-implant disease.
Bacterial plaque samples were obtained for microbiological analysis in nine studies.Other complementary analyses carried out were blood samples, saliva samples, and gingival fluid samples.The collection frequency varied from before treatment (baseline=t0), the day after treatment, the 4th day, the 15th day, the 18th day, the 21st day, 1 month, 2 months, 3 months, up to 6 months.
After treatment, subjects were recruited within a period of 1 week, 14 days, 1 month, 2 months, 3 months, 4 months, or up to 6 months for the evaluation of clinical parameters.
Regarding methodological quality, the risk of bias results of selected studies are summarized in Figure 2.With regard to selection bias, low risk of bias was identified in 58% of studies in the random sequence domain and in 54% in the allocation concealment domain, as details about the randomization process were presented in these studies.As for performance bias, 62.5% of studies presented low risk, as blinding tools were well described in the methodology section.In 37.5% of studies; however, it was not possible to identify forms of blinding, being classified as undefined risk of bias.With regard to detection bias, 54% of studies had low risk of bias.Attrition bias was considered low in 62.5% of studies because, in these cases, when there was loss of follow-up, it was considered small and well justified.Regarding reporting bias, although some studies had not published a protocol, outcomes were reported as proposed in the methodology section and the treatment was well described and reproducible, and thus the probability of bias of this nature was considered low in 79% of studies.

DISCUSSION
It is known that ozone has several physicochemical properties such as immunostimulant and analgesic, antihypoxic and detoxifying, antimicrobial, bioenergetic and biosynthetic, by activating the metabolism of carbohydrates, proteins, and lipids [30].
In periodontics, the therapeutic use of ozone has gained popularity due to the antimicrobial properties of ozone against gram-positive and -negative bacteria, viruses, and fungi [5,7,8,23,25], in addition to its ability to stimulate the circulatory system and modulate the immune response [28].Regarding gingivitis, only three studies were included, and in one of them, ozone provided faster remission compared to the control group [4].However, these studies were classified as having undefined risk of bias, indicating the absence of relevant information to judge their validity.
Based on articles with lower risk of bias in all six categories analyzed [22][23][24][25], it could be concluded that ozone therapy did not bring any additional benefit, nor any significant improvement in periodontal parameters in terms of periodontitis.On the other hand, in studies by Hayahumo et al. [8] and Yilmaz et al. [16], which were also classified as having lower risk of bias in five categories (lack of information on concordance for the assessment of clinical parameters), significant improvement was observed only in clinical parameters, for Hayahumo et al. [8], and in clinical and microbiological parameters for Yilmaz et al. [16].The lack of standardization in the way ozone is applied for the treatment of periodontal diseases, length of use, and frequency could explain differences between results of these studies.Specifically for peri-implant diseases, two articles were identified [28,29] with low risk of bias in all categories.In both studies, the conclusion was that ozone can reduce the development of mucositis, significantly improving clinical parameters in addition to radiographic evidence of significant improvements.It is noteworthy that the inclusion of periimplant conditions and diseases in the Classification of Periodontal Diseases and Conditions [31] is recent.Thus, further studies relating these diseases to ozone therapy are needed for a better understanding of its therapeutic effect.
Regarding the form of ozone use, it could be concluded with studies by Skurska et al. [6], Uraz et al. [23], Dengizek et al. [24], and Tasdemir et al. [25] that gaseous ozone therapy does not lead to additional improvement in clinical and microbiological parameters.Only the study by Yilmaz et al. [16] found improvement in clinical parameters such as attachment gain and reduction in pocket depth with this form of administering ozone.This can be explained by the instability of ozone in the gaseous medium, which has half-life from 30 to 40 minutes at 20 °C [32,33].
In the study by Kshitish & Laxman [9], good results using ozonated water under pressure from 350 to 500kPa were obtained.On the other hand, in the study by Pandya et al. [5], chlorhexidine was more effective than ozonated water produced at 0.595-350kPa, suggesting that the absence of pressure standardization interferes with the result obtained.Hayakumo et al. [8] used nanobubbles as alternative, with significant results for clinical and microbiological parameters.
The oily form of ozone was the form that presented the best clinical results in the evaluated studies [1,4,11,15,17,27].The advantage of this method is that the oil remains in contact with the surface for longer period of time, performing its functions.In addition, it can be stored under refrigeration for several months, eliminating the need for generator.This characteristic is opposed to that of gaseous ozone, which needs to be generated and used immediately, since when exposed to air, the gas decomposes quickly.
On the other hand, the chemical evaluation of the ozonation of some types of oils such as sunflower, castor, olive, almond, and propylene glycol was carried out.The absence of ozone percentages and the presence of formaldehyde were observed in all analyzed samples.It is known that the hydrolysis of ozonated oils can generate hydrogen peroxide, aldehyde, and ketones, which are antimicrobial.This suggests that the bactericidal and curative results of ozonated oils may be related to their by-products rather than to the ozone itself [34].
In the study by Uraz et al. [23], reduction of potent pathogens of the orange and red complexes such as Porphyromonas gingivalis (Pg) was observed in the first month and Tannerella forsythia (Tf) and Prevotella intermedia (Pi) in three months.Shoukheba & Ali [11] observed significant reduction in Pg and Actinobacillus actinomycetemcomitans (Aa) between 1 and 3 months in the ozone group; however between 3 and 6 months, no significant differences between groups were observed.In the study by Saeed et al. [1], reduction in Pg was observed for three months and Hayakumo et al. [8] also reported a more than 95% reduction in all sites under study.Verma & Indurkar [18] observed reduction in the four periodontal pathogens (Aa, Pg, Pi, and Tf) in both groups, which can be attributed to the fact that repeated subgingival irrigations were performed, interfering with the recolonization of the subgingival microflora.In the work by Kshitish & Laxman [9], percentage reduction was only observed in Actinobacillus actinomycetemcomitans, with no antibacterial effect on Porphyromonas gingivalis and Tannerella forsythensis, using ozone or chlorhexidine.
Two studies [6,27] evaluated the presence of high MMP-8 levels (metalloproteinases) in saliva and gingival fluid.In the study by Nardi et al. [27], it was observed that scaling and root planing, accompanied by subgingival irrigation with ozone in the oily form, was more effective in reducing this metalloproteinase in saliva than scaling and root planing alone, with significant difference between groups.MMP-8 levels are associated with the disease severity and help understanding the pathogenesis, diagnosis, and treatment of periodontal diseases.

CONCLUSION
Works that discuss the use of ozone as a therapeutic alternative in periodontics do not reach any conclusion regarding a protocol to be used.It could be inferred that the antimicrobial effectiveness of ozone is related to the form of presentation, concentration (dose), exposure time, and microorganism evaluated.Thus, well-defined criteria must be implemented to provide the quality and reliability of the ozone produced in any of its forms.
Further studies with adequate control of biases are suggested, using ozone in the oily or aqueous form, as the most promising forms, also evaluating the possible effectiveness of ozone by-products.

Collaborators
Palma PV, substantially contributed in the conception and design, acquisition of data, analysis and interpretation of data; drafted the article and revised it critically for important intellectual content; and approved of the final version to be published.Cunha RO, substantially contributed in the conception and design, acquisition of data, analysis and interpretation of data; drafted the article and revised it critically for important intellectual content; and approved of the final version to be published.Leite ICG, substantially contributed in the conception and design, acquisition of data, analysis and interpretation of data; drafted the article and revised it critically for important intellectual content; and approved of the final version to be published.
consultation for each quadrant.Plaque index, gingival index, bleeding on probing, and probing depth.In the 3 pockets ≥ 5 mm in depth, gingival fluid and plaque were collected at time 0 and 1 and 3 months after treatment for CRP analysis by Elisa.Dengizek et al. (2019) [24] Scaling and root planing, supra-and subgingival, throughout the mouth with irrigation, with manual and ultrasonic instruments.Plaque index, gingival index, probing depth, clinical attachment level (performed one month after the second ozone application).Total antioxidant status and total nitric oxide oxidizing status, 8-hydroxy-2'-deoxygguanosine, myeloperoxidase, glutathione, malondialdehyde Transforming growth factors were evaluated from saliva samples collected before and one month after the second application of each gingival index, probing depth, bleeding on probing, percentage of sites with bleeding on probing, and clinical attachment level.Gingival fluid, interleukin-1β, and highsensitivity CRP protein were evaluated.supra-and subgingival scaling and root planing with irrigation, with manual and ultrasonic instruments.Plaque index, gingival index, probing depth, and clinical attachment level.Subgingival plaque samples were collected on the preliminary visit and after 6 weeks, and sent for analysis (CRP).The following periodontal pathogens were analyzed:

Figure 2 .
Figure 2. Classification of the risk of bias of selected studies based on the Cochrane risk-of-bias tool -version 5.1.0.
No significant differences were found between groups of patients treated with ozonated oil and chlorhexidine, with remission of the condition in both groups.
Statistically significant reduction was observed in the mean values of plaque index, gingival index, probing depth, clinical attachment level, and in the four periodontal pathogens analyzed in both groups.However, differences were not statistically significant when control and intervention groups were compared.
The term chronic edematous gingivitis has been replaced by gingivitis according to the new 2018 Classification of Periodontal and Peri-implant Diseases and Conditions.

Table 2 .
Type of standard treatment, clinical markers, and complementary exams performed in studies.

Table 2 .
Type of standard treatment, clinical markers, and complementary exams performed in studies.

Table 2 .
Type of standard treatment, clinical markers, and complementary exams performed in studies.