Is Ozone a Valid Adjuvant Therapy for Periodontitis and Peri-Implantitis? A Systematic Review

Introduction: Ozone is a naturally occurring unstable compound with three oxygen atoms that generally transforms into an oxygen molecule, releasing one oxygen atom. This feature has been exploited in dentistry for numerous applications, including for periodontal diseases and peri-implantitis. Methods: This review was performed in relation to the PRISMA flow chart and was annotated in the PROSPERO register. PICO questions were used as research questions. The risk of bias in the non-randomized clinical trials was appraised using the ROBINS-I tool. Results: An electronic search found a total of 1073 records, in particular, 842 from MEDLINE/PubMed, 13 from Bio Med Central, 160 from Scopus, 1 from the Cochrane library databases, and 57 from the PROSPERO register. A total of 17 studies were included in the present systematic review. Information regarding the characteristics of the periodontal clinical and radiographic parameters for gaseous ozone, ozonate water, ozonate oil, and ozone gel, including clinical attachment loss (CAL) probing depth (PPD), bleeding on probing (BoP), plaque index (PI), gingival index (GI), and marginal bone levels (MBL), were obtained. Conclusions: The studies included in this systematic review show different results regarding the ozone in periodontal treatment in association with or without SRP.


Introduction
Ozone is a natural compound and an allotropic form of oxygen as it is composed of three oxygen atoms. It is also called triatomic oxygen or trioxygen [1].
In nature, it is generated when ultraviolet rays strike oxygen atoms, causing them to temporarily recombine into groups of three. Ozone is found in the form of gas in the stratosphere where it performs the important function of absorbing harmful ultraviolet radiation from the sun from water [1]. Therefore, ozone in the stratosphere plays an important role, protecting life on the earth's surface [2].
Ozone is an unstable gas that rapidly transforms into oxygen molecules, thus releasing an oxygen atom. This feature has been used to eliminate bacteria, fungi, to inactivate viruses and control bleeding and has therefore been exploited in medicine [3].
Ozone was already used at the end of the 1800s in the USA and then later also among German soldiers during the First World War for the treatment of post-traumatic gas gangrene, infected wounds, mustard gas burns and fistulas [4,5].
Today, ozone therapy is used in medicine and in dentistry where it is used for different applications and through various media such as gas, water, oil and gel. It is not used intravenously as an air embolism may form [6,7].
However, ozone therapy also has contraindications, which have been described by Nogales et al., such as pregnancy, ozone allergy, acute alcohol intoxication, severe anemia, autoimmune disease, active bleeding, hyperthyroidism, myasthenia gravis and myocardial infarction [8].

Study Protocol
This review was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) flow chart [25] and was annotated in the PROSPERO (International Prospective Register of Systematic Reviews) systematic review register (code ID: CRD42023397763), as recommended by Booth et al. [26].
PICO (Population, Intervention, Comparison, and Outcome) questions were used for the research questions [27].
The clinical question in the "PICO" format was: Is there a significant difference in clinical, radiographic and inflammatory parameters of periodontal and peri-implant tissues treated by ozone gaseous, oil or water with or without other interventions compared with placebo or other interventions? P (Population): subjects with periodontal or peri-implant diseases. I (Intervention): gaseous, oil or water ozone treatment alone or with other interventions. C (Comparison): subjects treated with placebo or periodontal or peri-implant treatment without ozone. O (Outcome): Clinical, radiographic and inflammatory periodontal and peri-implant tissue parameters.

Search Strategy
Studies published in the English language concerning ozone treatment using water and oil in periodontitis and in peri-implantitis were electronically searched without date restrictions until 1 February 2023 across major scientific online databases, including PROSPERO register, BioMed central databases, Scopus, MEDLINE/PubMed and the Cochrane Library databases by two authors (A.A. and F.D.A.) using the following keywords combined with Boolean operators, as show below: (periodontal disease OR periodontitis OR peri-implantitis OR dental OR peri-implant disease OR mucositis OR oral lesions) AND (ozone water OR ozone treatment OR ozone oil OR ozone OR gaseous ozone OR ozonide).
The following filters were applied: -English language on the Scopus library and on the MEDLINE/PubMed database; -Reviews, systematic reviews and metanalyses were excluded from the MEDLINE/ PubMed database.
No filters were applied to the BioMed Central database, to the PROSPERO register or to the Cochrane library.

Study Selection and Eligibility Criteria
The inclusion and exclusion criteria that were established before the start of the search and adhered to when selecting studies are shown below: Titles and abstracts obtained from the search as described were screened independently by two reviewers (F.D.A. and A.A.).
The Zotero (Version: 5.0.96.3.)reference manager tool was used to eliminate duplicates, and the obtained titles were analyzed by two Authors (A.A. and F.D.A) who independently read the abstracts of relevant studies.
From the abstracts that were judged to be the most relevant, the complete texts were obtained and independently screened by the authors. A third reviewer (F.G.) was consulted in cases involving doubts or disagreements.
Two authors also screened the bibliographies of the included reviews for any titles relevant to this umbrella review.
No restrictions were applied regarding the number or kind of studies included in this systematic review.
All studies analyzing treatments with gaseous, aqueous and oil ozone with periodontal parameters were considered in this review.

Data Extraction and Collection
Data were extracted by two reviewers (M.C. and A.A.) independently, and a third author was involved in case of disagreement (F.D.A.).
From each study included in this systematic review, the following data criteria were recorded:

•
First author, year of article, reference and study design; • Population, sample size and mean age of the study population of each study; • Type and regimen of gas, water or oil application used in periodontal treatment in each study; • Time points; • Clinical outcomes recorded, including clinical attachment loss (CAL), probing depth (PD), bleeding on probing (BOP), plaque index (PI) and gingival index (GI); • Radiographic outcomes recorded, including marginal bone levels (MBL) and cytokine profiles; • Conclusions.

Data Synthesis
The characteristics are presented in tabular form and summarized through a narrative synthesis.
Data were synthesized using Microsoft Excel software 2019 (Microsoft Corporation, Redmond, WA, USA).
In particular, with this review, we wanted to accomplish the following tasks: -Characterize the type and regimen of treatment performed with water or oil with ozone and make possible comparisons; -Compare the clinical radiographic results and cytokine profiles and bacteria amounts after ozone treatment (gaseous, water or oil) alone or with other treatments vs. placebo or other treatments.

Quality Assessment
The risk of bias in the non-randomized clinical trials was assessed using the ROBINS-I ("Risk Of Bias In Non-randomized Studies-of Interventions") tool [28].
In this tool, biases are classified as biases due to confounding, biases due to selection of participants, biases due to classification of interventions, biases due to deviations from intended interventions, biases due to missing data, biases in measurement of outcomes or biases due to selection of the reported result [28].
Low risk of bias: the study is judged to be at low risk of bias for all domains; Moderate risk of bias: the study is judged to be at low or moderate risk of bias for all domains; Serious risk of bias: the study is judged to be at serious risk of bias in at least one domain, but not at critical risk of bias in any domain; Critical risk of bias: the study is judged to be at critical risk of bias in at least one domain. The response options regarding bias are: Yes(Y), Probably yes(PY), Probably no(PN), No(N) and No information(NI). "Y" indicates a low risk of bias, PY indicates a moderate risk of bias; PN indicates a serious risk of bias, N indicates a critical risk of bias and NI indicates that there is no information related to bias.

Study Selection
An electronic search identified a total of 1073 records. In particular, 13 records were found using BioMed Central, 160 records were found using Scopus, 842 records were found using MEDLINE/PubMed, 1 record was found using the Cochrane library databases and 57 records were found using the PROSPERO register.
In total, 96 duplicates were eliminated, and 977 title abstracts were screened. Of these 977 title abstracts, only 29 abstracts were useful for the present systematic review, and of these 29 records, their full texts were obtained and screened, and 12 articles were excluded, as shown in Table 1.
The table above includes author names, years of publication of their studies and motivation for exclusion of their studies from the present review.
A total of 17 studies were included in the present systematic review ( Figure 1).

Study Characteristics
The main features of the included studies are summarized in Table 2. Of note, all included studies were published between 2010 and 2022 and were RCTs.

Data Extraction and Synthesis
Detailed findings related to periodontal clinical, radiographic and inflammatory parameters are synthesized in Tables 3-6, respectively, for gaseous ozone, ozonate water, ozonate oil and ozone gel.
Due to the variability of the studies, the limited data on periodontal parameters, and the various types of ozone used, it was not possible to analyze the data at a statistical level and perform a meta-analysis.

Quality Assessment of the Included Studies
The risk of bias in the non-randomized clinical trials was assessed using the ROBINS-I ("Risk Of Bias In Non-randomized Studies-of Interventions") tool [28].
The quality assessment of the included studies are summarized in Table 7  Table 2. Characteristics of the studies included in the present systematic review: author(s) and year of publication; study design; population, sample size, mean age; type of ozone used; Main results and Conclusions. Periodontal and peri-implant clinical, radiographic and crevicular parameters, including clinical attachment loss (CAL), probing depth (PPD), bleeding on probing (BoP), plaque index (PI), gingival index (GI), marginal bone levels (MBL), cytokine profile including interlukine-1b (IL-1b); matrix metalloproteinase (MMP-8) and periodontal treatment with scaling and Root Planing (SRP); Full-Mouth mechanical debridement (FMMD) and Test Group (TG); Control Group (CG); Gaseous Ozone Therapy (GOT); Right Quadrants (RQ); Left Quadrants (LQ); mechanical debridement (MT). PI, GI, PD, BoP and CAL were similar for both groups. All inflammatory parameters (PTX-3, Hs-CRP and IL-1) were reduced at 3-month follow-up. Only the decrease in PTX-3 levels between baseline and 3-month follow-up was statistically significant. A higher percentage of plaque index (12%), gingival index (29%) and bleeding index (26%) reduction was observed using ozone irrigation compared to chlorhexidine. Treatment success was obtained in 50% of the implants in the ozone group and 36.6% of the implants in the control group. However, a statistically significant difference was not observed between the groups regarding treatment success.

PD Isler, 2018 [52] RCT
A significant difference was only detected in the PD values between the groups at the 3-month follow-up period in favor of the ozone group (p < 0.05).
At 12 months postoperatively, significant differences were observed in the PD values for both groups compared to baseline (p < 0.001). Table 4. Characteristics of the periodontal clinical, radiographic and inflammatory parameters for ozonate water, including clinical attachment loss (CAL), probing depth (PPD), bleeding on probing (BoP), plaque index (PI), gingival index (GI), marginal bone levels (MBL), Interlukine-1b (IL-1b) and periodontal treatment with Scaling and Root Planing (SRP).

CAL Ranjith, 2022 [42] RCT triple blind
The mean clinical attachment gain in moderate and deep pockets was significantly greater in the test group (p < 0.01).
Significant clinical attachment gain may be indicative of better tissue regeneration with the use of ozone therapy compared to saline.

BOP Kshitish, 2010 [45] RCT double blind
A higher percentage of bleeding index (26%) reduction was observed using ozone irrigation compared to chlorhexidine.
Ozone may be considered an alternative management strategy due to its powerful ability to inactivate microorganisms.

PPD Ranjith, 2022 [42] RCT triple blind
Ozone water irrigation resulted in significant reduction in pocket depth in deep pockets (p = 0.01) and the number of sites with a pocket depth ≥ 4 mm with bleeding on probing.
PPD reduction was statistically significant for deep pockets (>7 mm) and for pockets with depth ≥ 4 mm with bleeding on probing.

PPD Hayakumo, 2013 [46] RCT
There were statistically significant improvements in the full-mouth mean PPD in both groups from baseline to 4 weeks and 8 weeks. The differences in the full-mouth mean PPD were not statistically significant at each follow-up visit.
The reductions in mean PPD from baseline to 4 and 8 weeks in the NBW3 group were significantly greater than those in the water group. No statistically significant differences among the groups.

GI Al Habashneh, 2015 [49] RCT
The decrease sin gingival indexes were comparable in the two treatment groups.
Ozonated water irrigation as an adjunctive therapy to SRP produced no statistically significant benefit compared to SRP with distilled water irrigation.

GI Kshitish, 2010 [45] RCT double blind
A higher percentage of gingival index (29%) reduction was observed using ozone irrigation compared to chlorhexidine.
Ozone may be considered an alternative management strategy due to its powerful ability to inactivate microorganisms.
Salivary interleukin 1 beta reduced significantly in the test group after therapy.
The mean difference in scores between the groups was not statistically significant.
The mean difference in scores between the groups was statistically significant.
The mean difference in scores between the groups was statistically significant.  [53] RCT The adjunctive use of the OZO with SRP resulted in a significant improvement (p < 0.001) in BOP. A significant improvement in BoP was observed in both groups.
There was a non-statistically significant reduction in both groups, but not between the two groups.
Ozonate olive oil + SRP improved BOP. The ozonated olive oil mouthwash slowed the decrease in BoP index at the beginning of treatment. No differences between ozone olive oil + SRP and clorexidine group + SRP. Groups showed significantly different variances regarding PPD (p < 0.001 in Levene's test). The adjunctive use of the OZO with SRP resulted in a significant improvement (p < 0.001) in PPD. There was a non-statistically significant reduction in both groups, but not between the two groups. The adjunctive use of the OZO with SRP resulted in a significant improvement (p < 0.001) in PI.
PPD decreased both in the study and control group with no statistical significance. Ozonate olive oil + SRP improved PPD. No differences between ozone olive oil + SRP and clorexidine group + SRP. Ozonate olive oil + SRP improved PI.

PI
Nardi, 2020 [48] RCT A significant improvement in PI was observed in both groups. Ozonated olive oil slowed the decrease in PI index at the beginning of treatment (at T1).
SRP + ozonated oil led to a significant and faster reduction in saliva MMP-8 concentrations in patients with periodontitis.

PPD GI
Gandhi, 2019 [56] RCT double blind Patel, 2012 [50] RCT double blind No significant difference was found in the efficacy of ozonated olive oil and CHX in improving the PD. The adjunctive use of the OZO with SRP resulted in a significant improvement (p < 0.001) in GI.
No statistically significant differences were found between the CHX and ozonated olive oil groups regarding any of the clinical parameters at the follow-up visit. Ozonate olive oil + SRP improved GI.

CAL CAL CAL
Gandhi, 2019 [56] RCT double blind Nambiar, 2022 [53] RCT Patel, 2012 [50] RCT double blind No significant difference was found in the efficacy of ozonated olive oil and CHX in improving clinical attachment levels.
There was a non-statistically significant reduction in both groups, but not between the two groups. The adjunctive use of the OZO with SRP resulted in a significant improvement (p < 0.001) in CAL.
No statistically significant differences were found between the CHX and ozonated olive oil groups regarding any of the clinical and microbiological parameters at the follow-up visit. No differences between ozone olive oil + SRP and clorexidine group + SRP. Ozonate olive oil + SRP improved CAL. Table 6. Characteristics of periodontal clinical, radiographic parameters and inflammatory parameters for ozone gel, including clinical attachment loss (CAL), probing depth (PPD), bleeding on probing (BoP) and periodontal treatment with Scaling and Root Planing (SRP).

CAL Colombo, 2021 [51]
Prospective single-group and single-center RCT Significant intragroup differences were found between each time point for the sites treated with SRP plus chlorhexidine (p < 0.05), whereas for ozone, a significant improvement was found at T1 but not at T2. A significant intergroup difference was found between the sites at T2 (p < 0.05).

BOP Colombo, 2021 [51]
Prospective single-group and single-center RCT Significant intragroup differences were found between each time point for both groups.
Significant intergroup differences were found between the sites (p > 0.05).

PPD Colombo, 2021 [51]
Prospective single-group and single-center RCT Significant intragroup differences were found between each time point for both groups.
Significant intergroup differences were found between the sites (p > 0.05). An important risk related to confounding bias is smoking status and smoking history, as reported by several studies [57,58].
In particular, McKenna, Ranjith, Kshitish, Yilmaz, Nicolini, Nardi and Ghandi [42,44,45,47,48,55,56] excluded smokers of any kind, specifying this in their exclusion criteria; in contrast, other authors such as Rapone et al. excluded subjects who smoked more than 10 cigarettes per day [40]. Isler included smokers, but it is not clear whether their study only included those who smoke fewer than 10 cigarettes per day [52].
Additionally, some authors such as Al Abashneh, Hayakumo and Tasdemir did not specify anything about smoking generating a significant bias in their results [43,46,49].
Nardi also included the inclusion criterion of participants who have a plaque index (PI) ≥ 35% and Gingival index (GI) ≥ 35%, which could be another confounding factor [48].

Discussion
The gold standard for periodontal treatment of periodontitis is scaling and root planing (SRP); however, many additional remedies have been described including the use of ozone [21][22][23]59,60].
Ozone is used in various branches of medicine, in particular in dentistry, due to its antimicrobial properties as it has been observed to have an effect on bacteria, viruses and fungi [3,61].

Clinical Periodontal and Peri-Implant Parameters in Patients Treated with Gaseous Ozone
The topical administration of ozone in gaseous form can be performed with an open system or with an aspiration system to prevent patients from inhaling the gas and therefore causing adverse effects [3].
Dengizek and Tasdemir did not find significant differences in CAL values between the groups treated without ozone and the group treated with gaseous ozone [41,43]. However, they considered short follow-up periods of 1 month and 3 months, respectively. In contrast, Rapone and Isler found statistically significant differences among the groups, and Isler considered a follow-up of 12 months [40,52].
Several results were discovered regarding BOP and PPD. In particular, Tasdemir did not find significant differences between SRP alone and SRP combined with ozone therapy at 3 months follow up [43], whereas Rapone described better results for SRP combined with ozone therapy in periodontitis compared to SRP alone at follow-up after 3 months [40]. However, the differences obtained could be caused by the different type of ozone application used. Tasdemir [43] considered the application of topical ozone in periodontal pockets twice a week for 2 weeks during active periodontal therapy. In contrast, Rapone proposed a protocol that envisaged both the use of ozonated water and cycles of gaseous ozone in pathological pockets [40].
McKenna instead compared treatment with O 3 + NaCl with treatment with O 3 + H 2 O 2 and found that they were equally the most effective treatments, whereas O 2 + NaCl was the least effective in controlling the bleeding around implants [55].
Yilmaz compared three different groups: an SRP only group, an SRP + ozone group and an SRP + laser group, and only the laser + SRP group showed statistically significant differences among the groups [44].
Tasdemir and Dengizek found no statistically significant differences regarding PI among the groups. McKenna found that treatment with O 3 + NaCl and O 3 + H 2 O 2 were equally the most effective treatments for GI and PI, whereas O 2 + NaCl was the least effective [41,43].

Clinical Periodontal and Peri-Implant Parameters in Patients Treated with Ozonated Water
Ozonated water was a very effective oral disinfectant thanks to its marked action against bacteria, fungi and viruses. Furthermore, it is cheaper than other oral chemical products. The aqueous form of ozone has proved to be less effective than the gaseous form but is more applicable both for addressing minor side effects (indeed, ozone gas has toxic effects if inhaled) and for its ease of use [3].
Ranjith, in a triple-blind RCT, compared CAL, PPD and IL-1b values in a group treated with SRP + ozonized water irrigation with a group treated with SRP + saline irrigation and showed that SRP + ozonized water irrigation possessed better results compared to SRP+ saline irrigation after a follow-up period of 4 weeks [42].
Al Abashneh [49] showed that treatment with ozonated water irrigation and SRP compared to treatment with SRP with saline irrigation showed no statistically significant differences regarding GI after a follow-up period of 3 months, and the same results were described by Nicolini regarding PI [47].
Different results are probably obtained due to the fact that each author used different protocols to compare different groups.
For example, Kshitish considered one group treated with ozonated water and one with chlorhexidine gluconate for a period of 18 days divided into two time intervals with a 4-day washout period followed by a second 7-day time interval. He showed a BOP, PI and GI reduction using ozonated water and thus concluded that ozone could be a valid alternative for management of periodontitis [45]. Furthermore, Vasthavi showed statistically significant differences between groups regarding PI and GI [54].

Clinical Periodontal and Peri-Implant Parameters in Patients Treated with Ozonated Olive or Sunflower Oil
Ozonated sunflower or olive oil has been proven to be an excellent antimicrobial agent and has been proven to be particularly effective against Staphylococci, Streptococci, Enterococci, Pseudomonas, Escherichia coli and, above all, against Mycobacteria [61][62][63][64].
This type of treatment can be used after in-office treatment with ozone gas or ozonated water or even simply at home using a 25 gauge blunt needle [3].
The use of ozonated olive oil in the treatment of periodontitis has been evaluated by many studies.
Some authors have shown that the addition of ozonated olive oil with SRP has proven benefits, while others have not.
Notably, Patel and Nardi described that ozonated olive oil and SRP improve BOP and also PPD [48,50], whereas Nambiar found no difference in BOP and PPD between treatments using SRP with olive oil and chlorhexidine groups with treatment using SRP [53].
Nardi evaluated salivary MMP-8 metalloproteinase levels in the periodontium, demonstrating that adding ozonated olive oil mouthwash to non-surgical periodontal treatment produced better results on salivary MMP-8 reduction compared to non-surgical treatment alone [50].
In some studies, Gandhi [56] and Nambiar [53], found no significant difference in the addition of ozonated olive oil to SRP compared to the use of chlorhexidine for improved PI, GI, PD, CAL and reduced periodontopathogenic bacteria.
However, other authors such as Patel have described that the addition of ozonated olive oil to SRP led to significant improvements in CAL, GI and PI [48].

Clinical Periodontal and Peri-Implant Parameters in Patients Treated with Ozonated Gel
Ozonated gel is another available formulation of ozone, but it has only been analyzed by Colombo.
Colombo evaluated in an RCT the difference between ozonated gel + SRP, compared to SRP + chlorhexidine gel with follow-up periods of 1 month and 3 months regarding PPD, CAL, GI, PI and BoP values; the results showed that the use of the ozonated gel in addition to SRP had no greater efficacy than treatment involving SRP + chlorhexidine [51].
Better results were obtained with SRP + chlorhexidine regarding CAL and GI at follow up after 3 months [51].

Conclusions
The studies included in this systematic review show different results regarding the influence of ozone related to the improving periodontal treatment with or without SRP.
Furthermore, the heterogeneity of the included studies and their risk of bias should be considered. Ozone shows encouraging therapeutic effects with regard to periodontal and peri-implant disease. However, the gold standard for the treatment of periodontitis remains SRP, whereas studies with sufficiently large samples and with low risks of bias are necessary to aid in our understanding of the effectiveness of ozone in the different formulations proposed regarding periodontal parameters.
Another important factor to consider is that, among the various formulations in which ozone is available, it should be borne in mind that, although the gaseous formulation may seem to be the most effective, it is also the most dangerous as inhaling ozone can be toxic for the respiratory system and other organs.
The complications caused by ozone therapy are rare but possible and range from minor side effects such as rhinitis, cough, headache, nausea and vomiting to more serious effects such as circulation and respiratory problems, heart problems and even ischemia. Data Availability Statement: All data generated or analyzed during this study are included in this article.

Conflicts of Interest:
The authors declare no conflict of interest.