Effect of Ozone Therapy on the Healing Process of Tooth Extractions in Smokers: A Randomized Split-Mouth Study

ABSTRACT

Objective:  To evaluate the effects of ozone therapy on pain control and wound healing following dental extractions in smokers.

Material and Methods:  This split-mouth clinical study included 42 hospitalized male smokers requiring bilateral tooth extraction. The sample was divided into a test and a control group (n = 14 per group). Test treatments included suturing with ozonated oil (OZO), application of ozone gas (OG), and irrigation with ozonated water (OW). The control group received irrigation with 0.9% saline solution. Analyzed variables included post-operative pain, healing progression, biofilm formation, surgical technique, and procedure duration. Pain was assessed using the Visual Analog Scale (VAS) on post-operative days 1, 2, and 3. Healing was clinically and photographically evaluated on days 0, 2, and 7. Biofilm presence, soft tissue inflammation, epithelial proliferation, and granulation tissue formation were analyzed through blinded image assessments. Statistical tests were applied to compare pain and healing outcomes.

Results:  All groups exhibited a significant reduction in pain by post-operative day 3 compared to day 1. On day 2, the OZO group showed significantly reduced pain and enhanced healing compared to the control. The OG group also demonstrated pain reduction. No significant differences were found in surgical technique or duration across groups.

Conclusion:  Ozonated oil and ozone gas effectively reduced post-operative pain in smokers, with ozonated oil also promoting superior wound healing by post-operative day 2.

Keywords:
Ozone; Smoking; Tooth Extraction; Wound Healing.

Introduction

Pathophysiological factors, such as edema, ischemia, and infection, as well as metabolic factors including nutritional status and systemic diseases, can cause post-operative discomfort and delay wound healing in soft and hard tissues [¹]. Furthermore, in the post-operative period, the success of surgery can be influenced by clinical factors related to the technique and duration of the procedure, iatrogenic factors such as the surgeon's experience and operative skill [²], and emotional factors such as stress, anxiety, and previous experiences of pain [³]. The healing process of the dental socket is clinically characterized by the closure of the socket entrance through epithelialization and/or bone fill, as observed in radiographic examinations [²,⁴,⁵]. The application of ozone during the initial phases of the repair process in dental extractions may reduce inflammatory and infectious events [⁶,⁷]. Smokers experience microvascular alterations and delayed wound and bone healing, as well as greater bone loss during the healing process [⁸,⁹].

Ozone therapy has several biological effects, including increasing oxygen availability in the body, improving blood circulation, stimulating the immune system, and preventing or treating infectious conditions. It is also used to prevent inflammatory complications following surgery, such as edema, pain, and trismus following impacted third molar extractions [¹⁰-¹²]. In dentistry, ozone can be used in either gaseous or liquid form. It can be incorporated into water or ozonized oil [¹³-¹⁵]. Ozone gas is considered a potent oxidant, and when applied to human gingival keratinocytes (oral epithelium), it has demonstrable oxidative potential [¹⁶]. However, its half-life is only 40 minutes at 20°C, meaning it cannot be stored and must be produced in situ for each application [¹⁷].

Topical application of ozonized oil produces a more stable compound that promotes greater collagen proliferation and synthesis in wounds, thereby facilitating healing. It also increases the release of growth factors during tissue remodeling [¹⁵]. The aqueous form of ozone has antiseptic and biocompatible properties, making it ideal for oral application, as it is less cytotoxic to oral epithelial cells and fibroblasts than gaseous ozone and other antimicrobials such as 2% and 0.2% chlorhexidine digluconate, 5.25% and 2.25% sodium hypochlorite, and 3% hydrogen peroxide. Due to its antimicrobial potential, ozone might be useful in various dental applications, such as endodontic treatment, periodontal therapy, and caries prevention using mouth rinses [¹⁸,¹⁹]. However, there is a lack of clinical studies evaluating the potential of ozone as an adjuvant after exodontia, particularly in smokers.

This controlled, split-mouth clinical study aimed to evaluate the effectiveness of ozonated oil, ozone gas, and ozonated water in alleviating pain and promoting healing following tooth extraction in smokers. The study hypothesized that ozone gas therapy may or may not influence the repair of exodontia in smoking patients.

Material and Methods

Ethical Considerations and Study Design

This study was approved by the Ethics Committee in Research of the Federal University of Paraná (Opinion no. 5.294.051). It adhered to the principles outlined in the Declaration of Helsinki and the CONSORT (Consolidated Standards of Reporting Trials) guidelines for split-mouth controlled clinical studies. The research was conducted between March 2022 and June 2023. All participants provided their written informed consent.

Study Population

The study population consisted of a convenience sample of adult male patients undergoing treatment for drug addiction at the San Julian Psychiatric Hospital, located in the city of Piraquara, PR, Brazil. This hospital exclusively admits male patients.

Anamnesis and intraoral physical examinations were performed on all study participants. Teeth indicated for extraction and their contralateral counterparts were examined and selected according to the research protocol. Panoramic radiographs were obtained from all patients prior to surgery for surgical planning purposes.

Sample Calculation

The sample calculation was based on the pain analogue scale (VAS) values reported in previous literature [¹⁰], considering mean ± SD values of 8.42 ± 1.40 pain scores for the control group and 4.62 ± 3.12 for the test group (ozone), a power of 90% and a type I error rate of 5%, the GPower software [²⁰] determined a required sample size of 14 patients per group.

Eligibility Criteria

Patients were included in the study if they were smokers for at least five years, consumed more than ten cigarettes a day, had good general health and cognitive and intellectual capacity, and required bilateral and contralateral maxillary or mandibular extractions. Patients with any systemic condition that contraindicated ozone therapy or who failed to complete the clinical, radiographic and photographic follow-up protocol during the study were excluded.

Allocation

Participants were recruited and allocated according to the flow chart into groups and received one of the following treatments: OZO Test: Exodontia + suture thread soaked in ozonized oil; OZO Control: Exodontia + suture thread soaked in 0.9% saline; OG Test: Exodontia + application of ozone gas to the alveolus; OG Control: Exodontia + irrigation with 0.9% saline solution; OW Test: Exodontia + irrigation of the alveolus with 15 mg/mL ozonated double distilled water and OW Control: Exodontia + irrigation with 0.9% sterile saline solution (Fresenius Kabi Brasil Ltda., Aquiraz, CE, Brazil).

Participants were recruited through posters and an explanation of the nature of the study. After recruitment, participants were randomly assigned to one of three treatment groups: ozonated oil/control, ozone gas/control, or ozonated water/control. Randomization was performed electronically using the Random.org until all groups reached the intended sample size. The side and the control side were randomly selected using a coin toss to determine which tooth would be extracted first, on the right or left. Two weeks were given before proceeding with the tooth extraction on the opposite side. None of the patients were aware of the details concerning the study and control groups.

Pilot Study

Two individuals underwent surgery to establish the parameters for obtaining ozonized water and ozone gas and to determine the methods for applying ozone and predicting the start and end times of the surgical procedure. Follow-ups relating to pain and healing were conducted at 0, 2 and 7 days post-operatively. Clinical photographs were taken to standardize the preand post-operative images. These individuals were not included in the study sample.

Surgical Procedure

All subjects were operated on by the same experienced oral surgeon. Prior to surgery, subjects received extraoral antisepsis with a 2% chlorhexidine digluconate aqueous solution and intraoral antisepsis with a 0.12% chlorhexidine digluconate mouthwash for one minute. Local anesthesia was administered using 2% Mepivacaine hydrochloride with 1:100.000 epinephrine, within the recommended maximum dose [²¹].

The study employed both closed and open extraction techniques [²²], categorized as follows: Technique I involved the use of forceps alone; Technique II combined forceps and levers; and Technique III included osteotomy with bone removal and/or odontosection. The selection of the technique was based on the specific tooth and the complexity of the extraction. Sutures were placed using 5-0 nylon thread (TechSuture Indústria e Comércio de Produtos Cirúrgicos, Bauru, SP, Brazil). All patients received post-operative guidance and dipyrone sodium 1g three times a day for three days. For each participant, the surgical techniques used and the time elapsed from the start to the end of the procedure were recorded.

Obtaining Ozonated Double-Distilled Water and Ozone Gas

Medical ozone in gaseous form is a mixture of 5% O₃ and 95% O₂, which was prepared using a MedPlus ozone generator (Philozon-Indústria e Comércio de Geradores de Ozônio, Balneário Camboriú, SC, Brazil). The gas was applied using a 10 ml disposable silicone syringe (Terumo Medical do Brasil Ltda., São Paulo, SP, Brazil) coupled with a 26 G1/2 BD needle, which was inserted into the bottom of the alveoli of the wounds. The contents were then applied for two minutes. In the ozone gas group, the gas was applied at the end of the extraction.

The ozonized double-distilled water (Laboratório Sanobiol, Pouso Alegre, MG, Brazil) was prepared five minutes before the surgeries by an external collaborator. The same ozone generator was connected to a glass column with a catalyst and a microbubble diffuser in a stainless-steel tube. The double-distilled water absorbed 20-25% of the available ozone concentration [²³]. The ozone generator was set to deliver 60 μg/mL of ozone for five minutes into one liter of double-distilled water (Laboratório Sanobiol, Pouso Alegre, MG, Brazil), resulting in a final concentration of 15 μg/mL. This solution was applied to the socket using a silicone syringe (Terumo Medical do Brasil Ltda., São Paulo, SP, Brazil) at a volume of 10 mL after the exodontia suturing procedure.

Ozonized Oil

Ozoncare sunflower seed oil (Philozon-Indústria e Comércio de Geradores de Ozônio, Balneário Camboriú, SC, Brazil) was soaked into a 5.0 nylon thread (Techsuture Indústria e Comércio de Produtos Cirúrgicos, Bauru, SP, Brazil) and a gauze pad with ozonized oil was kept in place for 15 minutes following suturing.

Calibration

A single examiner (M.R.A.), who was experienced in oral and maxillofacial surgery, assessed 40 images of post-operative healing and biofilm in a blind study. These images corresponded to 23.8% of the total sample. The images were evaluated twice, with a 2-week interval between evaluations. The Kappa values were 1.00 for post-operative healing on the right side, 0.85 for the left side, and 0.80 for the biofilm variable.

Evaluation

The variables evaluated post-extraction were pain, wound healing, presence of biofilm, time and surgical technique.

Pain

Pain intensity was measured using the Visual Analogue Scale (VAS), ranging from 0 (no pain) to 10 (maximum possible pain). Participants completed the scale at 1, 2, and 3 days after surgery, following prior instruction on how to use the VAS [²⁴]. After completion, the scales were placed into folders labeled according to group.

Healing

Clinical and photographic monitoring of mucosal and socket healing was performed at 0, 2, and 7 days post-operatively. The clinical photographs were archived, coded, and inserted into a PowerPoint presentation for later blinded evaluation, without knowledge of the treatment groups. The evaluated criteria included: inflammation of the soft tissue, epithelial proliferation around the socket, and the formation of granulation tissue filling the socket. Healing was classified as excellent, good, moderate, or poor [²⁵].

Healing was considered excellent (score 3) when there was minimal or no soft tissue inflammation, a good amount of proliferating epithelium, and the socket was completely filled with granulation tissue; Good healing (score 2) was defined as minimal or no inflammation, a moderate amount of proliferating epithelium, and the socket fully filled with granulation tissue; Moderate healing (score 1) was characterized by moderate soft tissue inflammation, a small amount of epithelial proliferation, and the socket being half to two-thirds filled with granulation tissue; Poor healing (score 0) was assigned when the socket showed moderate to severe inflammation of the surrounding soft tissue, exposed bone surface, and insufficient granulation tissue. A dry socket was also considered poor healing [²⁵].

Biofilm

The monitoring of digital images for the presence or absence of biofilm on the suture thread and in the central area of the surgical wound was conducted at 0, 2, and 7 days post-operatively, using the same images collected for wound healing evaluation.

Surgical Techniques and Time

For each participant, the surgical technique employed and the duration of the procedure from anesthesia administration to completion were recorded.

Statistical Analysis

The data were organized in Microsoft Office Excel spreadsheets (Microsoft Corp., Redmond, WA, USA) and analyzed using GraphPad Prism software, version 8 (GraphPad Software, San Diego, CA, USA). To compare VAS and healing scores across the different groups (oil, gas and ozonized water) on each experimental day, a Kruskal - Wallis test was performed, followed by a Dunn's multiple comparisons test. To assess healing scores across different post-operative days (0, 2, and 7) within the same group, the Friedman test was applied, followed by Dunn’s multiple comparisons. The Wilcoxon matched-pairs test was used to compare test and control scores within each group.

Biofilm presence was recorded using numerical values and frequency distributions for both test and control groups. The normality of the quantitative variables was checked using the Shapiro-Wilk test. The duration of the surgical procedure was analyzed by one-way ANOVA. A significance level of p < 0.05 was adopted for all statistical analyses.

Results

Of the 47 participants who took part in the study, 5 were excluded for not completing the research protocol (Figure 1). In the end, 42 male participants remained in the study, with a mean age of 40.71 ± 9.76 years, ranging from 21 to 59 years old.

Figure 1
Flowchart of the study.

The average daily cigarette consumption and duration of smoking were 22.48 ± 10.60 cigarettes and 17.48 ± 8.11 years, respectively. The corresponding value for tobacco exposure, calculated as years of smoking multiplied by the number of cigarettes smoked per day, was 17.57 ± 21.46.

The three experimental groups, as well as their respective controls, showed a significant reduction in Visual Analogue Scale (VAS) pain scores by the third post-operative day compared to the first day (p < 0.05) (Table 1).

Table 1
Pain scores (VAS) median of the experimental groups (ozonized oil, gas, or water) during the post-operative period.

Period	Oil	Gas	Water	p-value
Day 1
Test	2.00 (1.75-3.00)A	4.00 (2.75-5.00)B	4.00 (2.75-5.00)B	oil-water 0.01* oil-gas: 0.02*
Control	3.00 (02.00-4.00)	4.50 (2.75-6.25)	4.00 (2.75-5.25)	0.10
p-value	0.08	0.19	0.60
Day 2
Test	1.50 (1.00-3.00)	2.00 (2.00-3.25)	3.00 (1.75-4.00)	0.12
Control	3.50 (2.00-5.00)	3.50 (2.00-5.25)	2.50 (1.75-4.25)	0.69
p-value	0.03*	0.02*	0.87
Day 3
Teste	1.00 (0.75-1.25)	1.50 (1.00-2.25)	1.00 (1.00-2.00)	0.21
Control	1.00 (1.00-3.00)	2.50 (1.00-4.25)	2.00 (1.00-4.00)	0.48
p-value	0.14	0.10	0.20

When comparing each post-operative day individually, it was found that on the first day, the group treated with ozonized oil had lower pain scores compared to the gas or water groups (p < 0.05), although there was no statistically significant difference when compared to its own control (p > 0.05) (Table 1).

On the second post-operative day, the groups treated with ozonized oil and gas presented lower VAS scores than their respective controls (p < 0.05), but no significant statistical difference was observed when compared to the other experimental groups (gas or water) (Table 1). By the third post-operative day, no differences in VAS pain scores were observed among the experimental groups or their respective controls (p > 0.05) (Table 1).

Images from the experimental groups during the preoperative period and on post-operative days 0, 2, and 7 following ozone therapy are shown in Figure 2.

Figure 2
Images of the experimental groups using ozonated oil (A), ozone gas (B), and ozonated water (C) treatments. Preoperative images are shown in panels A1, B1, and C1; immediate post-operative images in A2, B2, and C2; images at 2 days in A3, B3, and C3; and images at 7 days in A4, B4, and C4.

In the experimental groups (ozonized oil, ozone gas, and ozonized water) at the three evaluated time points, healing scores for the surgical wounds on the test side were similar to those on the control side on post-operative days 0, 2, and 7, with no statistically significant differences observed, except for the ozonized oil group on the second post-operative day (p = 0.031) (Table 2).

Table 2
Healing scores of the experimental groups (ozonized oil, gas, or water) on post-operative days 0, 2, and 7.

Group					Days								p-value
	0				2				7
Oil
Scores	0	1	2	3	0	1	2	3	0	1	2	3
Test (n)	14	0	0	0	12	1	1	0	12	1	1	0	0.263
Control (n)	10	3	1	0	7	5	1	1	8	5	1	0	0.07
p-value	0.125				0.031*				0.437
Gas
Scores	0	1	2	3	0	1	2	3	0	1	2	3
Test (n)	13	1	0	0	12	1	1	0	12	1	0	1	0.272
Control (n)	8	6	0	0	7	5	1	1	11	1	2	0	0.249
p-value	0.062				0.062				>0.99
Water
Scores	0	1	2	3	0	1	2	3	0	1	2	3
Test (n)	13	1	0	0	11	2	1	0	12	2	0	0	0.367
Control (n)	13	1	0	0	6	6	2	0	11	3	0	0	0.001*
p-value	0.062				0.062				>0.99

• Healing Scores: 0 (Poor), 1 (Moderate), 2 and 3 (Excellent); Data across rows were analyzed using the Friedman test followed by Dunn’s multiple comparisons; Data within columns were analyzed using the Wilcoxon matched-pairs test;
• ^*
  No significant difference in Dunn’s post-test.

Biofilm was observed in less than 10% of the cases evaluated across the three experimental groups and their respective controls. In the group treated with ozonated oil, the number of participants with biofilm was higher in the control group compared to the test group, remaining stable between days 2 and 7 in both subgroups. In the ozone gas group, no participants in the test group presented biofilm at any of the evaluated time points, while one participant in the control group exhibited biofilm, with no variation between days 2 and 7. In the group treated with ozonated water, the number of participants with biofilm was the same in both the test and control groups on post-operative day 2; however, by day 7, a reduction was observed in the test group, whereas the control group showed an increase in the number of cases with biofilm (Figure 3).

Figure 3
Number of participants presenting biofilm in the experimental groups on post-operative days 0, 2, and 7.

The frequencies of the surgical techniques in the experimental and control groups are presented in Table 3. No statistically significant differences were found in surgical time among the experimental groups (ozonized oil, gas, or water) or their respective control groups (p > 0.05) (Table 3).

Table 3
Surgical techniques and surgical time in groups test and control.

Groups	Oil	Gas	Water	p-value
	N (%)	N (%)	N (%)
Test
Technique I	8 (57.0)	6 (43.0)	3 (21.0)	-
Technique II	1 (7.0)	4 (28.0)	6 (43.0)	-
Technique III	5 (36.0)	4 (28.0)	5 (36.0)	-
Control
Technique I	7 (50.0)	7 (50.0)	0 (0.0)	-
Technique II	6 (43.0)	6 (43.0)	8 (57.0)	-
Technique III	1 (7.0)	1 (7.0)	6 (43.0)	-
Surgical Time
Test*	15.36±4.16	17.07±5.01	15.14±4.31	0.47
Control#	14.21±1.58	15.50±3.54	14.43±4.30	0.74

• Technique I: Forceps; Technique II: Forceps and levers; Technique III: Osteotomy with bone removal and/or odontosection;
• ^*
  Data with normal distribution (verified by the Shapiro-Wilk test) were analyzed using one-way ANOVA; #Data with non-normal distribution were analyzed using the Kruskal-Wallis test.

Discussion

To date, no studies have evaluated the effect of ozonized oil, ozonized water, and ozone gas on pain and healing following simple tooth extractions in smokers. A split-mouth study design was selected for this research because it involves symmetrical, bilateral surgical intervention in the hemiarches, an approach that has been adopted by other researchers [¹²,²⁶,²⁷]. This design allows for a reduced sample size, minimizes interindividual variability, and increases the statistical power of the study [²⁸]. Because this was a split-mouth study, a two-week interval was maintained between extractions in the different groups to prevent symptom overlap and potential residual effects from the previous ozone treatment. This same interval has been used in previous studies to assess short-term outcomes such as swelling, trismus, and pain following third molar extractions [¹²,²⁷,²⁹].

Ozone gas is not considered a medication and does not produce systemic side effects when used in appropriate doses and for the correct indications [⁸], which makes it suitable as an adjunct in oral surgeries. Ozone has beneficial effects on cellular redox balance modulation (allowing the cell to counteract oxidative effects in the extracellular environment), as well as on inflammation and ischemia modulation [³⁰]. It also has analgesic and antimicrobial properties [²³]. Therefore, ozone therapy supports the healing process and reduces post-treatment discomfort, justifying its use as an adjunctive treatment in tooth extraction procedures [¹¹,¹²].

In the present study, pain reduction was observed both with and without ozone application in the three days following extractions. The highest pain scores were recorded on the first day compared to the third, regardless of the treatment applied. Post-operative pain typically peaks between 3 to 5 hours after the surgical procedure, coinciding with the end of the anesthesia’s effects and persists for 2 to 3 days before gradually decreasing by the seventh day. These findings are consistent with previous studies [¹²,²⁶,²⁷].

Another significant finding of our study was that, on the second post-operative day, the groups that received ozonized oil and ozone gas showed a significant reduction in pain. Therefore, ozonized oil and ozone gas demonstrated pain-reducing effects during the initial inflammatory phase of wound healing [³¹], an effect that was likely enhanced by the prescribed post-operative analgesic. The control group also experienced a reduction in pain due to the analgesic effect.

Topical applications of ozone, whether through ozonized water or oil, have antimicrobial properties and enhance the healing process. When injected into the tissue, ozone exhibits analgesic properties. The proposed mechanism involves the oxidation of algogenic mediators, which inhibits pain signaling and activates the body's antinociceptive system [³²]. The analgesic action of ozone begins when it mixes with blood, leading to the formation of hydrogen peroxide, an important messenger in the activation of the immune system. This initiates a cascade of chemical reactions and the production of interleukins, interferons, and other cytokines, which help modulate and regulate immune function and restore normal thalamic processing in the pain cycle [³³]. These proteins play a crucial role in cell growth and differentiation, reducing inflammation, and promoting wound healing [³⁴].

Oil serves as a medium that preserves the active form of ozone gas (O₃) for a longer period, up to two years when stored under refrigeration at 4°C. It also allows ozone to remain in contact with the wound for a longer duration compared to ozonized water or ozone gas [³¹]. This may explain the prolonged effect of ozonized oil observed in the present study, as compared to o ozonized water.

Ozone gas, on the other hand, has a more rapid effect compared to other forms of application. This can be attributed to the higher concentration of ozone delivered during gaseous application [³⁵]. In the present study, ozone gas was shown to reduce pain on the second post-operative day. A clinical study involving 1521 individuals investigated various forms of ozone application (ozonized oil, ozone gas, and ozonized water) and their effectiveness in controlling pain from alveolitis following tooth extraction. The gaseous form proved to be the most effective in reducing more intense pain and acted more rapidly [³⁵].

Smokers are prone to early inflammatory effects inherent to tobacco use, which include cellular alterations in chemotaxis and in the activation of neutrophils and macrophages. They also exhibit suppressed eosinophil function and reduced production of inflammatory cytokines [³⁶]. These inflammatory responses influence both pain and wound healing, particularly in moderate to heavy smokers, those with more than five years of tobacco use and a daily consumption of 10 or more cigarettes [³⁶]. In the present study, we observed that both the average daily consumption and the duration of tobacco exposure among participants were high. However, healing outcomes did not differ significantly, except in the ozonated oil group on the second post-operative day.

In the experimental groups, at all three time points evaluated, wound healing progressed normally regardless of ozone application and despite participants being smokers, except for the ozonized oil group on the second post-operative day. This finding can be explained by the topical form of O₃ accelerating the repair of acute skin wounds, promoting collagen synthesis and fibroblast proliferation at the injury site, and increasing the expression of growth factors such as PDGF, TGF-β, and VEGF, all of which contribute to enhanced wound healing [³⁷].

Ozone induces increased expression of cytokines, particularly TGF-β, which plays a critical role in the early phase of wound healing, contributing to cell proliferation and the chemotaxis of monocytes and fibroblasts at the injury site. It also enhances the expression of other cytokines such as PDGF and VEGF [³⁸]. The application of ozonated water enhances local oxygen availability in the wound environment, thereby promoting tissue regeneration through increased proliferation of fibroblasts and epithelial cells [³⁷]. A recent systematic review and meta-analysis assessed the therapeutic efficacy of topical ozone-based solutions in wound healing, analyzing 22 studies, 13 conducted in animal models and 9 in human subjects. The results demonstrated that the topical application of ozonated water or oil was associated with a significantly greater reduction in wound size and a shorter initial healing time when compared to control groups [³⁹]. Similarly, consistent findings were reported in another systematic review and meta-analysis, which concluded that ozone therapy contributes to the reduction of both the size and pain associated with traumatic and autoimmune oral ulcers when compared to placebo [⁴⁰].

In the oral mucosa, bacterial colonies and food debris form a biofilm that contributes to wound infection [⁴¹,⁴²]. Once a biofilm establishes on a wound, the healing process is inhibited both by the bacteria themselves and by the physical barrier the biofilm creates. It is known that fewer than 10% of acute wounds contain biofilm, whereas in chronic wounds, this figure rises to approximately 60% [⁴³]. These findings are consistent with our study, which detected biofilm in eight wounds. This outcome is likely due to the use of nylon suture material, which impedes bacterial accumulation and is considered the first choice for surgical closure in tooth extractions [⁴⁴].

In the present study, most extractions were performed using surgical techniques I and II. Regardless of ozone treatment, there was no significant difference in surgical time between groups. Similar results were reported by [¹¹], who found no differences in surgical time in clinical studies of impacted third molar extractions using a split-mouth design with ozone therapy. These findings were expected, as simple extractions generally require less surgical time and cause less trauma to soft tissue and bone structures compared to third molar surgeries [¹¹]. Notably, no significant cases of edema or trismus were observed, complications that are common in third molar procedures.

This is the first study in smokers to evaluate the use of ozone as an adjunct in dental extractions, employing different application forms (oil, gas, and water).

Ozone therapy is a non-pharmacological, easily applicable, and minimally invasive alternative; however, its outcomes in minor oral surgery remain inconsistent. Therefore, additional studies are necessary to further evaluate early post-operative pain, alternative methods of ozone application, and the management of analgesic prescriptions to confirm its clinical efficacy.

The limitations of this study include methodological heterogeneity among different investigations and the lack of a standardized protocol for ozone application in dentistry. This research focused on post-operative pain and early wound healing outcomes in smokers. Therefore, it is recommended that other outcomes, such as qualitative comparisons and evaluations of pain and long-term wound healing, be the subject of future studies to further corroborate the efficacy of ozone therapy.

Conclusion

Both the gaseous form and ozonized oil demonstrated pain-reducing effects during the early post-operative period; however, only the ozonized oil significantly enhanced alveolar healing by the second post-operative day in smokers.

Financial Support

None.

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

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Correspondence

Corresponding author: Maria Ângela Naval MachadoE-mail:[e-mail]

Academic Editor:

Wilton Wilney Nascimento Padilha

Conflict of Interest

The authors declare no conflicts of interest.

Publication Dates

• Publication in this collection
  31 Aug 2026
• Date of issue
  2026

History

• Received
  01 Sept 2025
• Reviewed
  16 Oct 2025
• Accepted
  15 Dec 2026