A Randomized Clinical Trial on the Clinical and Immunological Efficacy of a Mouth Rinse Containing Hydrogen Peroxide and Hyaluronic Acid in Patients with Gingivitis.

OBJECTIVE: The present study aimed to inves?gate the impact of HA/H 2 O 2 (Hyaluronic Acid/Hydrogen Peroxide) mouthwash on pro-inﬂammatory cytokines, speciﬁcally IL-1 b and IL-6, in individuals diagnosed with gingivi?s. Furthermore, a comparison was made between the eﬀects of HA/H 2 O 2 and those of CHX (Chlorhexidine) and placebo. MATERIALS AND METHODS : This clinical trial was randomized, double-blinded, and parallel. FiUy-four students with bioﬁlm-induced gingivi?s (18 to 23 years old) were randomly assigned to three mouthwash groups: HA/H 2 O 2 , CHX, and placebo. Uniden?ﬁed bo[les were delivered to par?cipants. For 14 days, 10 millilitres of HA/H 2 O 2 mouthwash formula?on, CHX (0.12%), and placebo were used twice a day. All subjects were assessed twice, once at baseline and once aUer 14 days. Before and aUer each par?cipant’s mouthwash usage, bleeding on probing was measured as a clinical parameter for inﬂamma?on, Salivary IL-1 b and IL-6 were also assessed by ELISA. Changes in salivary interleukins and bleeding between baseline and aUer treatment were determined and compared by the t test with an alpha of 0.05. RESULTS: There was a sta?s?cally signiﬁcant decrease in salivary interleukin-1β and IL-6 in all three groups. However, intergroup comparisons at second visits showed no signiﬁcant diﬀerence between HA/H 2 O 2 and CHX groups (P>0.05) for the interleukins tested. Furthermore, all treatments considerably reduced bleeding on probing, although mouthwash with hyaluronic acid (HA) and hydrogen peroxide (H 2 O 2 ) had a greater impact than the other two treatments. CONCLUSION: Mouthwash containing HA/H 2 O 2 demonstrated an an?- inﬂammatory eﬀect, sugges?ng they are immunomodulatory agents. These ﬁndings may be beneﬁcial and encouraging for the use of HA/H 2 O 2 in the treatment of gingivi?s; thus, HA/H 2 O 2 rinse has the poten?al to serve as an appropriate replacement for chlorhexidine (CHX).


Introduction
Gingivitis is an inflammatory reaction caused by the accumulation of bacteria-in the plaque around the margin of the gingival tissue [1,2].The prevalence of biofilm-induced gingivitis is widespread among human populations and represents a primary periodontal ailment.If left untreated during its initial stages, this condition has the capacity to escalate into more severe forms of periodontal disease [3].Cytokines are a category of soluble proteins with small molecular weight that are synthesized in response to an antigenic stimulus.They serve as chemical mediators that regulate the immune system that is innate as well as adaptive [4].Interleukin-1 beta (IL-1) is a potent h#p://den*stry3000.pi#.edupro-inflammatory mediator that binds to IL-1 receptor and is essential for host defense responses to infection and injury [5].It has been reported that IL-1 in the inflammatory site is responsible for increased local blood flow, leucocyte recruitment, and neutrophil infiltration [6].Interleukin-6 (IL-6) is a cytokine that exhibits diverse physiological effects, such as the differentiation and activation of macrophages and T cells, the growth and differentiation of B cells, the stimulation of haematopoiesis, the differentiation of osteoclasts, and the resorption of bone [7].
The mouthwash containing chlorhexidine (CHX) is considered the preferred option for complementing oral hygiene due to its exceptional effectiveness in preventing the development of dental biofilm [8,9].Nevertheless, the utilisation of this substance may result in various adverse effects, such as discoloration, modified gustatory perception, and infrequently enlargement of the parotid gland [10].The development of alternative mouthwashes with comparable efficacy, but without these adverse effects is desirable.The present study aimed to examine the efficacy of a mouth rinse solution comprising of hydrogen peroxide (H2O2) and hyaluronic acid (HA).H2O2 possesses oxygenating properties and exhibits antimicrobial activity by releasing oxygen.It has been demonstrated to be efficacious against both Gram-positive and Gram-negative bacterial strains [11,12].HA is a linear polysaccharide that occurs naturally and serves as a crucial constituent of the extracellular matrix in various human tissues [13].Additionally, HA is present in the periodontal ligament, as well as gingivae, while the cementum and alveolar bone contain minimal amounts of this substance [14].HA is involved in phagocytosis, cell migration, adhesion, and wound healing due to its anti-inflammatory and anti-bacterial characteristics [15].The objective of this investigation was to assess the impact of a rinse containing (HA+ H2O2) on proinflammatory cytokines, specifically IL-1B and IL-6, among individuals diagnosed with gingivitis, and to juxtapose these results with those obtained from CHX and a placebo solution.

Experimental Design
This parallel three-arm doubleblinded clinical trial was conducted between March and April 2023 at University of Baghdad's Faculty of Dentistry.The Helsinki Declaration and CONSORT 2010 Statement for multi-arm studies were followed.Volunteers visited the hospital's dentistry clinics at the start and end of the 14-day study.The Research Ethics Committee of the University of Baghdad, Faculty of Dentistry approved the study (Project No. 748622, December 28-2022).In 2023, "Clinicaltrials.gov"registered this clinical trial under protocol number "NCT05787600".

Study Subjects
The trial's sample comprised of undergraduate students who were receiving treatment at the dental clinics of University of Baghdad's Faculty of Dentistry.A notice was displayed in the reception area of the clinics, soliciting the participation of students as volunteers in the research investigation.Individuals who satisfied the predetermined criteria for inclusion were extended an invitation to partake in the present investigation.
The study's inclusion criteria encompassed individuals who were in good systemic health, did not smoke, possessed over 20 natural teeth, and had been diagnosed with biofilminduced generalised gingivitis.This diagnosis was characterised by the presence of over 30% bleeding sites with no PPD greater than 3 mm, an intact periodontium, and no loss of periodontal attachment [16].Furthermore, participants were required to have no visible supra-or sub-gingival calculus and to regularly perform brushing their teeth.The study's exclusion criteria comprised individuals with periodontitis, current use of oral rinse, habitual smoking behaviour, extensive neglected dental caries, dental appliance wearers, individuals who underwent h#p://den*stry3000.pi#.eduperiodontal therapy within the past six months, those who received antibiotic therapy three months prior to the study, pregnant or breastfeeding women, and individuals with a previous record of allergic reactions to any of the items used in the investigation.
Following the process of participant selection, the objective of the inquiry was communicated to every potential participant, and written consent was procured.The decision was made to refrain from providing additional guidance on oral hygiene in order to preserve the participants' current dental hygiene routines.

Calibration
The reproducibility of the examiner's recording of clinical periodontal parameters (bleeding on probing) as determined by intra-examiner calibration.The examiner recorded periodontal parameters twice with a 2-hour interval for five participants.The acceptable limit for agreement for all clinical parameters was > 0.75.In this research, the Kappa value for parameter was 0.91.

Sample Size
To calculate sample size, IL-6 was used as a primary outcome of the study.The concentration of the biomarker in health is estimated to be equal to 6.69 ±0.89 and during gingivitis is equal to 7.85±0.82(20).The software G*power version 3.1.9.2 was utilised to determine the appropriate sample size with a 95 % power of study, an alpha error of probability of 0.05.The results indicated that each study arm should consist of 18 participants, which includes 16 calculated individuals and an additional 2 to account for any potential dropouts.Therefore, the total sample size for the study was 54 participants.

Study Interventions
The clinical trial employed a threearm design, utilising three different mouth rinses.The first is a positivecontrol rinse, consisting of 0.12% Chlorhexidine and marketed under the brand name KIN Gingival by KIN, Spain.The second rinse was a placebo, consisting of distilled water with added food colouring to ensure double-blind conditions.The third rinse, known as Perhyal, was used as the test intervention.Perhyal is manufactured by BMG Pharma in Milan, Italy and contains H2O2 (1.80%) and HA (0.10%) as active ingredients, with the remaining portion consisting of water (97.3%) and inert additives.The mouthwashes were stored in impermeable bottles that were uniform in appearance and were randomly labelled with letters (A, B, and C) by an individual who was not involved in the clinical trial.Therefore, maintaining volunteer and investigator blindness throughout the clinical investigation was crucial.The process of decoding was conducted subsequent to the conclusion of the inquiry.The randomization, blinding, and intervention allocation procedures were followed as reported in previous investigations [17].

Clinical Research Design
One experienced, calibrated examiner selected, enrolled, and evaluated the individuals.54 students volunteered and agreed to the research criteria.A comprehensive intraoral examination followed.Bleeding on probing, gingival index and plaque index were assessed with sterile Williams' periodontal probes (MEDESY 546-1, Italy).Clinical examiners would clarify the study's aims and protocols if individuals met inclusion criteria.Participants who accepted the conditions and guidelines signed the informed consent form and started the investigation.
In the baseline visit at the beginning of the study period, saliva samples were taken from all 54 participants before starting with measures of the clinical parameters.The collection of unstimulated whole saliva was conducted utilizing a previously described technique [18].The study's participants were directed to abstain from engaging in oral hygiene practices, such as brushing, flossing, and using mouth rinses, as well as consuming food, beverages, or chewing gum for a period of one hour prior to the collection of saliva samples.Subsequently, all the subjects underwent a 30-second rinse h#p://den*stry3000.pi#.edu with 10 mL of tap water, followed by expectoration.
The participants expectorated at least 5 mL of unstimulated whole saliva in sterile containers containing lyophilized protease inhibitor solution.On the ice, saliva samples were collected.Before analysis, aliquots were prepared, and samples were frozen at -80°C .Whole saliva samples were tested for the presence of interleukin (IL)-6 and interleukin (IL)-1β by using an enzyme-linked immunoassay (ELISA) kit (USCN Business Co., Ltd., Wuhan, China), used for determining protein levels in salivary samples.The analysis was done following the manufacturer's instructions for each kit.The ELISA procedure for this study was the sandwich ELISA technique.
Then, measurement of bleeding of probing (BOP) for the six surfaces by gently introducing the periodontal probe to the level of the gingival sulcus removed coronally and waiting thirty seconds to see whether there is any bleeding (1: bleeding, 0: no bleeding) [19].14-days after baseline (the first visit) saliva collection and clinical periodontal parameter scoring (BOP) were repeated.
Examiners regulated mouthwash usage by giving one patient a 140mL bottle at the start of each week and asking for the returning the previous week's bottle.The mouth rinse bottles were recovered, and the remaining liquid was computed to determine usage compliance (280 ml/bottle for two weeks).

Randomization
The Simple Randomization method was used to allocate patients into three distinct groups for enrollment.
Each cohort of subjects was allocated a letter (A, B, or C) to match up with the intervention.A computer-based random number generator, implemented in Microsoft Excel 2016, was employed to generate a set of random numbers.These numbers were subsequently utilised to rearrange the order of participants (n=54) and groups, which were predetermined.Each group was assigned an equal number of participants (n=18) in a 1:1:1 allocation.Subsequently, bottles that were appropriately labelled with the corresponding interventions were dispensed to the patients, accompanied by detailed guidelines for their administration.The participants were directed to perform a mouth rinse for a duration of one minute, using 10 mL of undiluted mouthwash, after brushing their teeth.They were also advised to refrain from consuming any food or beverage for a period of 30 minutes following the mouth rinse.In order to mitigate potential interference with the effectiveness of the interventions, participants were instructed to perform oral hygiene procedures twice a day, with a 12-hour time interval between each session.Every individual employed a toothbrush featuring filaments of a soft hardness and toothpaste from the brand COLGATE® by Colgate-Palmolive in India.The tooth-brushing method of the subjects was not allowed to be modified.

Statistical Analysis
The Statistical Package for the Social Science (SPSS) version 21, developed in Chicago, Illinois, United States of America, was utilised for the purposes of data description, analysis, and presentation.The agreement between investigators was assessed by means of Cohen's kappa coefficient.The Shapiro-Wilk test was employed to assess the normality of the distributions.To compare interventions, statistical measures such as mean and standard deviation are utilised to describe the scores of bleeding on probing %.The study utilised a parametric analysis, specifically a paired t-test, to compare the aforementioned variables within the same group.
The researchers employed a one-way analysis of variance (ANOVA) to detect potential variations among the interventions.
Subsequently, the Tukey HSD posthoc test was employed to ascertain the presence of a statistically significant distinction between every intervention pair.The Tukey HSD posthoc test was employed to determine the proportion of participants who achieved a state of good health.The effect size of the interventions in comparison to the placebo was calculated.P < 0.05 was h#p://den*stry3000.pi#.educonsidered to indicate statistical significance.

Results
The eligibility of 75 individuals was assessed, and 54 of them were selected following the application of inclusion and exclusion criteria.Thus, the final analysis included a total of 54 individuals.The study reports that the HA+H2O2 mouthwash group had a mean age of 21.22 ±0.87 years, and the Placebo group had a mean age of 21.16 ±0.92 years.The distribution of participants based on sex and treatment are shown in Table 1.

Groups
HA/ H2O2 0.12%CHX Placebo Overall, IL-1β is a significant contributor to the neutrophil migration capability as it prompts the dissemination of these cells via the bloodstream.Diminishing the concentration of the aforementioned cytokine could potentially lead to a decrease in migratory requirements, as the inflammatory process would be mitigated.Through the mechanical elimination of the biofilm as a component of periodontal therapy, the difficulty was diminished, resulting in a decrease in the production of these cytokines.A recent study found the resolution of gingivitis is associated with significant reductions in both gingival inflammation and plaque levels, as well as systemic inflammatory markers [25].In a comparison between HA/ H2O2 with CHX mouthwashes, the present findings revealed that CHX mouthwash had the highest effect in lowering IL-1B and IL-6 levels when compared to HA/ H2O2 mouthwash.Moreover, these results revealed that there was no statistically significant difference between HA/ H2O2 mouthwash and CHX in reducing of IL-1B levels at day 14.
The findings of our study indicated a statistically significant variance in the average bleeding on probing index percentage among all mouthwash treatments (HA/ H2O2, CHX, and Placebo) prior to and following a 14day period of use.Nevertheless, the intergroup comparisons regarding bleeding on probing (BOP) during second visits did not exhibit any significant disparity between the HA/ H2O2 and CHX groups (P>0.05).The results of our study indicated a significant reduction in BOP scores when using the HA+ H2O2 mouthwash in comparison to both the baseline data and the negative control.This finding can be explained by antiinflammatory properties of both constituent (HA, H2O2) and efficacy against both Gram-positive and Gram-negative bacterial strains of both ingredients [13,15] .The present data are compatible with Abdulkareem et al. [26], who found that a new formulation containing 0.025% HA with a 0.12% chlorhexidine mouth rinse compared to placebo resulted in a statistically significant reduction in gingival inflammation when compared to both the baseline and the placebo.Furthermore, the findings indicated that there was no significant statistical disparity between the effectiveness of HA/ H2O2 mouthwash and CHX in diminishing the BOP score on the 14th day.
On the other hand, the third (placebo) group in both biomarkers showed positive results.It was expected that the placebo group would also experience a decrease in gingival inflammation scores, as the patients were motivated to uphold optimal oral hygiene.The Hawthorne effect, a well-known phenomenon, could have played a role in the enhanced results observed in both cohorts.This may be attributed to an increased consciousness of oral health care [27] and the influence of the regular monitoring visits.
The trial's limitations included a short 14-day evaluation interval and the absence of instances of supra-sub calculus in the intervention group, as well as healthy controls.In addition, the trial was limited to students between the ages of 18 and 23.In addition, no effort was made to modify the subjects' diets, which h#p://den*stry3000.pi#.edu could have had a long-term impact on the results, particularly salivary cytokines; additional research is required to determine the extent of this influence.financing.

Conclusion
Mouthwash containing HA/H2O2 demonstrated an anti-inflammatory effect, suggesting an immunomodulatory effect.These findings may be beneficial and encouraging for the use of HA/ H2O2 in the treatment of gingivitis; thus, HA/H2O2 rinse has the potential to serve as an appropriate replacement for chlorhexidine (CHX).

Table I :
Demographic variables of the study groups.

Table 2 :
Bleeding on probing by study groups and intervals.Pa?ents with Gingivi?s.

Table 3 :
IL-6 levels by study groups and intervals.

Table 4 :
IL-1β levels by study groups and intervals.Pa?ents with Gingivi?s.