1. Introduction
Periodontitis is a multifactorial inflammatory disease associated with biofilm dysbiosis [
1]. However, although the disease remains stable after periodontal therapy, its progression occurs in sites related to poor oral hygiene [
2].
Chlorhexidine (CHX) rinses minimize biofilm formation and gingival inflammation following periodontal surgery. However, the impact of reducing the periodontal probing depth (PD) is unclear [
3]. Furthermore, CHX reduces bacterial recolonization after periodontal surgery, which favors healing and avoids the recurrence of the periodontal lesion [
4]. Although CHX is safe, stable, and effective in minimizing periodontal pathogen recolonization and preventing biofilm formation, several side effects, such as dental surface pigmentation, taste modification, scaly lesions on the mucosa, dryness of the tissues, and periodontal healing delay, among others, have limited its clinical use [
5].
Neutrophils and macrophages synthesize hypochlorous acid (HOCl) during phagocytosis of antigens as the final product of H
2O
2 by the action of the myeloperoxidase and Cl
2, and this is synthesized and stabilized to use in clinical medicine for skin infections, burn wound healing, and chronic leg ulcers [
6,
7]. HOCl is effective against many Gram-negative microorganisms recognized as periodontal pathogens using concentrations between 180 and 500 ppm [
8,
9] or low concentrations combined with stabilized acetic acid to reduce bacterial viability on oral biofilm [
10]. Moreover, HOCl is an oxidizing agent with an excellent viricidal effect, including SARS-CoV-2 [
11]. HOCl displays low toxicity and anti-inflammatory and proliferative cell effects. It is a promising molecule for post-surgical periodontal use [
6].
The primary objective of this study was to compare the clinical and microbiological efficacy of postsurgical protocols with HOCl at 0.05%/0.025% and CHX at 0.2%/0.12% as antimicrobial agents in patients with chronic periodontitis following 7, 21, and 90 days of surgical periodontal therapy. Adverse effects were also evaluated.
3. Discussion
According to the findings of this study, the 0.2%/0.12% CHX antiplaque treatment resulted in significant reductions in PI of roughly 65% at 7 and 21 days post-surgical periods. Comparable results were reported by previous post-surgical studies [
12]. HOCl also showed a reduction above 50% over time. CHX is more effective by 10% in reducing PI than HOCl at seven days, although after 21 days, individuals with non-regular hygiene are similar. Nevertheless, the HOCl protocol is not inferior in reducing PI because the limit of non-inferiority in this study was demonstrated. The efficacy of GI, BoP, and PD at 21 days post-surgery was similar between protocols. However, at 90 days, HOCl performed better than CHX in CAL gain > 3 mm. These results could be explained by the significant effect of HOCl on the gram-negative microorganisms and the anti-inflammatory and proliferative cell effects during the healing [
7,
8,
9,
10,
11].
Bacterial colonization of tooth surfaces is a relevant cause of periodontitis recurrence. Bacterial antimicrobial agents as control adjuvants have been proposed to reduce the bacterial colonization of tooth surfaces in the post-surgical period [
13].
P. gingivalis,
T. forsythia,
T. denticola, and
A. actinomycetemcomitans are essential microorganisms in periodontitis. Other microorganisms, such as
E. nodatum, are also related to periodontal destruction [
14]. Many of these species not only colonize periodontal pockets but are also present in oral mucosa, tongue, and tonsils and are commonly detected in saliva [
15,
16]. Full-mouth disinfection (FMD) therapy includes CHX adjunct to periodontal instrumentation in one or two appointments. However, no evidence exists to establish the FMD approach to provide additional clinical benefits [
17]. Otherwise, CHX in the post-surgical period of no regular hygiene has an evident impact on clinical parameters compared with prophylaxis [
18] or placebo [
19]. Other studies demonstrate the benefit of using CHX post-surgery to reduce the recolonization of periodontal pathogens, demonstrating the establishment of a less mature flora with a predominance of streptococci [
4].
HOCl has shown an antimicrobial effect for oral bacteria in preclinic microbiological studies using high concentrations, such as 180 ppm (332.8 uμM), 250 ppm (474 μM) to 500 ppm (948 μM) [
7,
8], and lower concentrations as 50 ppm (90 μM). [
20] However, the higher concentration of HOCl, such as 220 or 330 ppm, did not significantly decrease the minimum inhibitory volume ratio against the microorganisms [
20]. HOCl is associated with cellular alterations and stopping the cell cycle due to its oxidizing effect at low concentrations and the formation of chloramines [
21]. At low concentrations below 20 μM, HOCl stimulates increased free radical activity against tissues and activates preforms of collagenase-2 and gelatinase-B proteases through the oxidation of thiol groups. [
21]. High concentrations can inactivate proteases and the transport of glucose and amino acids, lipopolysaccharides, endo, and bacterial exotoxins; HOCl oxidizes specific cysteine residues in the active site of gingipains such as Rgp and Kgp (
P. gingivalis cysteine proteases), reducing their damaging potential on tissues [
6].
This study evaluated a non-inferiority hypothesis based on similar behavior between protocols. However, an equivalence study required a considerable sample, so an attempt was made to verify at least one hypothesis of no inferiority using specific statistical tests. When the difference is significant with a p < 0.05, it is verified that HOCl is non-inferior to CHX.
Although HOCl has low substantivity compared to CHX [
22], HOCl demonstrated a significant effect on gram-negative microorganisms associated with periodontitis, as previously reported [
4,
7]. The HOCl protocol was not inferior to the CHX protocol to avoid recolonization of
T. denticola at 21 days.
P. gingivalis recolonization was also similar in the groups. These results are relevant because CHX is the antiplaque substance considered the gold standard.
Neither CHX nor HOCl affected the recolonization of
A. actinomycetemcomitans. HOCl was not inferior in reducing and recolonizing
A. actinomycetemcomitans on days 7 and 21. The recolonization of
A. actinomycetemcomitans is frequent due to the adhesion capacity to epithelial cells employing a specific adhesive protein that subsequently binds to other species of bacteria through coaggregation phenomena [
23,
24]. Once this microorganism colonizes the supragingival plaque, it moves to the subgingival biofilm, invades the epithelium of the periodontal pocket, and penetrates the underlying connective tissue [
25].
Previous research has shown that HOCl has wide-spectrum antimicrobial properties, low or null systemic toxicity, and possible positive effects on cell proliferation [
6,
25]. Likewise, in vitro studies have shown a potent antiviral effect against SARS-CoV-2 that may favor its use as an antimicrobial agent [
26].
This study showed a similar reduction of the mean of PD and CAL in CHX and HOCl protocols. However, in gain >3 mm of CAL, HOCl tended to show a significant reduction compared to CHX. We could hypothesize that reducing the recolonization of periodontal microorganisms may favor tissue healing. However, HOCl has been shown to have an anti-inflammatory effect in atopic dermatitis, and it is favored when combined with taurine [
27,
28]. The anti-inflammatory effect of HOCl could also be related to the improvement of healing of periodontal tissues, unlike the CHX, which has been associated with a delay in the proliferation of gingival fibroblasts and the production of collagenous and non-collagenous proteins [
29,
30].
This study introduced a postsurgical brush after seven days in both groups to avoid the deterioration of the experimental substance. The introduction of soft surgical toothbrushes on days 3 to 14 twice daily, adjunct to CHX, is similar to days 14 to 28. An ultrasoft brush may be desirable even early in the postoperative period [
31]. Rinsing with CHX causes extrinsic tooth staining and other adverse effects such as calculus build-up, transient taste disturbance, effects on the oral mucosa, and dry mouth [
5]. HOCl at a concentration of 0.05% has a significant sensation of dryness at 7 days, 43.7% in the mucous membranes. However, upon lowering the HOCl concentration, it was reduced on day 21, like CHX.
In this study, 62.5% of the patients reported taste alteration for CHX 0.2% and 12.5% for HOCl, much lower for the HOCl protocol. Other studies reported inferior results to ours, with changes in taste in 23% with CHX at two weeks and 25% at four weeks [
32]. Previous studies report 47.1% to 80% dental pigmentation with CHX 0.2% between 7 days to six weeks [
12,
19]. The CHX protocol reducing the concentration of 0.2% to 0.12% evidenced similar dental pigmentation of 62.5% at 21 days. This pigmentation is associated with forming pigmented metal sulfides and dietary factors as modifiers [
33]. Surprisingly, the individuals of the HOCl group reported 43% of teeth whitening at 7 days using HOCl at 0.05%, which slightly decreased with the lowest concentration at 21 days at 10%. This effect could be due to an oxidation reaction of the HOCl, similar to that observed with hydrogen peroxide products [
34,
35]. Oxidizing substances destroy pigments by removing hydrogen while reducing substances’ activities by removing oxygen [
36,
37]. Future clinical studies should be directed to evaluate the effect of HOCl on dental enamel and lower doses of HOCl to evaluate the effectiveness and the reduction of adverse effects.
4. Materials and Methods
4.1. Study Design
A triple-blind, non-inferiority randomized controlled trial with two arms was conducted and registered at
ClinicalTrials.gov (accessed on 15 December 2019) under n° NCT05952921. This study follows the Consolidated Standards of Reporting Trials (CONSORT) checklist for reporting this clinical trial (CONSORT extension for non-inferiority trials). According to the Declaration of Helsinki on experimentation involving human subjects, the Ethics Committee of the School of Dentistry of Universidad El Bosque (Act #014-2015) approved the study design.
4.2. Participants
Thirty-two patients between 20 and 60 years old diagnosed with chronic periodontitis attending the periodontics Postgraduate Clinics of the School of Dentistry of the Universidad Antonio Nariño in Bucaramanga-Colombia between July and December 2019 participated in this study. All members signed informed consent and were instructed about the objectives and possible risks of the study. Participants had to have a minimum of 20 teeth with at least three sites with probing pocket depth (PD) > 5 mm and clinical attachment level (CAL) > 4 mm, radiographic evidence of bone loss, and good general health and required periodontal surgery. Exclusion criteria included smoking, antibiotic therapy, use of NSAIDs in the last four months, pregnancy or lactation, and systemic diseases.
4.3. Sample Size
The sample size was determined using a power and sample size calculator for a non-inferiority trial of continuous outcomes from
https://sealedenvelope.com/ (accessed on 14 may 2019), based on a significance level (alpha) of 5% and a power (1-beta) of 80%, assuming a non-inferiority margin of 20% of the observed effect size between HOCL and CHX and considering a hypothetical pre-recolonization of 25% in the CHX group and 50% in the HOCL group. The sample size estimates revealed a minimum sample size of 16 subjects per group.
4.4. Randomization
Thirty-two voluntary participants were randomly assigned to receive one of two post-surgical protocols after periodontal surgery: (1) a high-concentration rinse with 0.05% HOCl (7 days), followed by 0.025% HOCl (14 days) [
8,
22]; (2) a high concentration of 0.2% CHX (7 days), followed by 0.012% CHX (14 days) [
5]. The participants had no regular oral hygiene and incorporated a post-surgical brush only after day 14 until the end of the study. In this triple-blind study, opaque and sealed envelopes were used for the assignment of each subject; the investigators did not know what type of rinse was assigned to each patient, and the analysis of the results was performed blindly using a coding system that was not disclosed until the analysis was completed. Randomization was generated by computer using Minitab 18 statistical software. A balanced random permuted block method was assigned to the two treatments. A clinical epidemiologist (DDB) realized the randomization table in five blocks. The mouthwashes were masked and indistinguishable in consistency, packaging, and labeling, but the taste was variable.
4.5. Clinical Evaluation
The PI, GI, PD, and CAL and subgingival sampling for microbiological analysis were evaluated in the baseline by a calibrated periodontist. GI and PI were dichotomic (presence or absence of changes in gingiva color to clinical observation or the presence or absence of visible plaque evaluated with a periodontal probe). PD was assessed on days 0 after surgery and 90 days after with a North Carolina periodontal probe (Hu-Friedy, Chicago, IL, USA) at six sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiolingual), except for the third molar; these sites were also used to assess CAL and BoP (present or absent). On day 7, the suture was removed, and the PI, GI, saliva sample, and subgingival plaque were realized for microbiological analysis. These analyses were repeated at 21 and 90 days.
4.6. Microbiological Evaluation
Bacterial samples from the six sites with the greatest PD were collected with sterile paper points size 40 (Maillefer, Dentsply®) (Maillefer Instruments Holding SA; Ballaigues; Suiza. Dentsply Sirona; Pennsylvania, PA, USA) for 60 s and introduced into a sterile 1.5 mL tube labeled with the patient’s name. The samples were refrigerated at −20 °C until processing. Tris-EDTA (TE) buffer pH 7.4 buffer was added to the tubes containing the subgingival plaque tips and mixed by vortexing for 20 min. The supernatant was removed and transferred to a 1.5 mL tube for centrifugation at 14.000 rpm for 10 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 300 µL of sterile deionized distilled water molecular grade. Once homogenized in the vortex, it was frozen at −20 °C overnight. For DNA extraction and subsequent polymerase chain reaction (PCR), a protocol established in the Oral Microbiology Laboratory of the UIBO Institute was used, which consisted of DNA extraction by heat shock. Real-time PCR with absolute quantification allowed confirmation of the number of colony-forming units (CFU). All samples were amplified in a BioRad CFX 96 thermal cycler. The absolute quantification was carried out with the help of calibration curves made for each bacterium with DNA from reference strains with known amounts of bacteria in CFU; the data were transferred to Log10 for statistical analysis.
To identify
P. gingivalis, primers and the probe reported by Boutaga et al. in 2003 were used [
38], previously standardized in our laboratory [
39]. To identify
A. actinomycetemcomitans, the protocol reported by Boutaga et al. in 2005 [
40] was used and previously standardized in our laboratory [
41].
T. forsythia was identified according to the protocol reported by Morillo et al. in 2004 [
42]. and
T. denticola according to the protocol of Yoshida et al. in 2004 [
43]. The identification of
E. nodatum, the protocol previously standardized by our group, was used [
44].
4.7. Adverse Effects
A survey was carried out for each of the participants at 7 and 21 days of the study to identify clinical adverse effects such as burning sensation, burning or pain in the oral mucosa, sensation of dryness or dryness, and changes in the perception of taste or the color in the teeth. Microbiological adverse effects were performed through saliva samples taken at 0, 7, 21, and 90 days to verify the absence or presence of opportunistic flora associated with mouthwashes.
4.8. Statistical Analysis
A descriptive analysis was carried out to compare the groups according to the sociodemographic, clinical, and periodontal status variables. Obtained data were reported as the mean and standard deviation or expressed in median and interquartile range according to the type of distribution based on the Shapiro–Wilk test. PD, CAL, and BoP were compared at baseline and 90 days with paired t-student. Group comparison was performed with a t-student with a significance level of 5% (
p < 0.05). Repeated measures ANOVA adjusted for treatment–time and time–treatment interaction was used to assess plaque and gingival index at 0, 7, 21, and 90 days. A mixed linear model of repeated measures adjusted for treatment, time, and time–treatment interaction was used. The different bacterial species in times and frequencies of adverse events between groups were determined using a Chi-square/Fisher’s exact test with a significance level of 5% (
p < 0.05). For the non-inferiority analysis, it was predetermined that HOCL would be considered non-inferior to HOCL rinse administration if the upper boundary of the two-sided 95% confidence interval for the difference between the groups was less than the margin, Δ = −20% [
45]. Estimations were conducted using the Hodges–Lehmann hypothesis estimation for non-inferiority with Hodges–Lehmann confidence limits or the hypothesis test for difference in proportions for non-inferiority [
46], as appropriate. All analyses were performed using the statistical software programs STATA 14 ((StataCorp LLC; Texas, TX, USA) and Stat Graphics v.18
® (Statgraphics Technologies, Inc; Virginia, VA, USA).