Encapsulated Chlorhexidine Gel Use in Adolescents Undergoing Orthodontic Treatment: A Randomised Trial

Abstract

Introduction

The majority of orthodontic patients present with plaquebiofilm and related manifestations in the early stages of active fixed orthodontic appliance use.

Aims

To evaluate the effects of a slow-delivery chlorhexidine:beta-cyclodextrin (Cx:β-Cd) gel compared with those of free Cx free formulations on clinical parameters in patients undergoing treatment with fixed orthodontic appliances.

Methods

Patients aged 12-18 years received prophylaxis and scaling and were randomised to treatment groups in a double-blinded manner. They were treated with Cx:β-Cd, Cx, or placebo gel for 15 or 30 days. Bleeding on probing and the visible plaque index were recorded, and gingival crevicular fluid samples were collected for the assessment of cytokine expression.

Results

At 15 days, the Cx:β-Cd group exhibited significantly less BOP than did the Cx and control groups (P < 0.001; n = 116). At 15 days, notable decreases in tumour necrosis factor-α and interferon-γ expression, as well as a substantial increase in interleukin-10 expression, were observed in the Cx:β-Cd and Cx groups relative to the control group (P < 0.05). The molecular encapsulation gel resulted in greater saliva Cx concentrations than did the free Cx gel released concentrations of 800 ng g⁻¹.

Conclusion

Cx:β-Cd gel is effective for the slow delivery of Cx, improving the gingival health of adolescents undergoing orthodontic treatment.

Clinical relevance

A single application of encapsulated Cx gel, was a more effective antiseptic than a free Cx application in fixed orthodontice appliance wearers.

Introduction

An estimated 60% of orthodontic patients present with considerable amounts of oral biofilm and related complications. In the early stages of fixed orthodontic appliance use, the plaque index and gingival bleeding increase notably.¹ The removal of dental plaque by brushing and the maintenance of good oral hygiene in the presence of orthodontic bands, wires, and ligatures are major challenges. Gingivitis development has been reported within 1-2 months after fixed orthodontic appliance placement.² Slight periodontal attachment loss may occur up to 2 years after the removal of such appliances, when patients are not completely and continually motivated to maintain oral hygiene.^3,4

The accumulation and permanence of large amounts of dental plaque trigger the imbalance of the host inflammatory response. Proinflammatory cytokines (interferon-γ [IFN-γ], tumour necrosis factor-α [TNF-α], and in turn interleukin-10 [IL-10]) play important roles in the initiation and progression of gingivitis and periodontal disease.⁵ Exposure to bacterial products leads to the activation of monocytes and macrophages, which promote the secretion of such cytokines and inflammatory mediators. This process triggers the release of matrix metalloproteinases,⁶ which are inflammatory cytokines and enzymes detectable in fluids such as saliva and used as cellular inflammation biomarkers.

Antimicrobial gels have been utilised to improve gingival health along with conventional tooth brushing in patients undergoing fixed orthodontic treatment.⁷ The slow-release application of antimicrobial agents in the periodontal pockets can eliminate pathogens and modulate the inflammatory response, thereby limiting tissue destruction. The efficacy and benefits of slow-release systems compared with systemic drug administration have been demonstrated.⁸

Products containing 2% chlorhexidine (Cx), including mouthwashes, toothpastes, lubricants, and moisturizers, are highly effective antimicrobial agents. Gels with lower Cx concentrations can be used to diminish the adverse effects of Cx,8, 9, 10 primarily (reversible) tooth discoloration. Orthodontists are advised to recommend the most effective antiplaque agent to their patients under treatment.

The encapsulation of Cx in Cd improves the effectiveness and long-term antimicrobial activity of oral gels.¹⁰ Gels containing Cx:β-Cd inclusion compounds exhibited more antimicrobial activity against oral pathogens than did free Cx in a long-term in-vitro study.¹¹ The complexation of Cd with other drugs has been shown to effectively improve macromolecular therapy by stabilising the guest molecules against aggregation and/or degradation.11, 12, 13, 14

In the present study, a new Cx gel formulation with a delayed delivery system was developed as proof of concept of its activity after a single application. The key compound included in the gel is β-cyclodextrin (β-Cd), which creates a hydrophobic capsule around the Cx. The hypothesis was that the inclusion of Cx in a Cd substrate would provide for the sustained release and increased permanence of Cx due to the mucoadhesiveness of the gel, enhancing the drug's local effects by reducing dental plaque and consequently gingivitis while avoiding the adverse effects of Cx-containing mouthwashes. This proof-of-concept study sought to demonstrate that a single application of molecularly encapsulated Cx would result in the persistence of the drug for some days, resulting in more efficient antiseptic effects than obtained with free Cx. Thereby, were evaluated the clinical outcomes (bleeding on probing [BOP] and the visible plaque index [VPI]) following the use of a 0.6% Cx gel with a slow delivery system in patients aged 12-18 years wearing fixed orthodontic appliances.²

Materials and methods

Materials and gel preparation

For this study, Cx (molecular weight, 578.37) and hydroxypropyl methylcellulose were obtained from Sigma-Aldrich® (São Paulo, Brazil), and β-Cd (molecular weight, 1134.98) was obtained from Cerestar® Co. (Milwaukee, WI, USA). Cx:β-Cd inclusion complexes (1:1 molar ratio) were prepared by freeze drying as described previously¹³ and used to prepare the gel formulation. The hydroxypropyl methylcellulose polymer (3% w/v) was dissolved in water and mixed by mechanical stirring at 25°C. The gels were fabricated for a pharmaceutical in a private company's facilities. Single batches were used exclusively for this study. Briefly, 0.6% Cx or 0.6% Cx:β-Cd solution was mixed with a solution of polysorbate 20 (0.15%) and propylene glycol (15%) until gelation occurred. The gels were maintained at 8°C in amber glass flasks. The polymers were colour coded and dispensed in syringes, and the colour assignments remained undisclosed until the end of the study for blinding.

Study design, ethical considerations, and patient selection

This randomised blinded clinical trial was designed to compare the effects of 0.6% Cx gel with those of 0.6% Cx gel encapsulated in β-CD as adjunctive topical agents for the treatment of gingivitis. The examiner and participants were blinded to the type of gel applied. This study was conducted in accordance with the 1975 Declaration of Helsinki, as revised in 2000, and approved by the Institutional Review Board of the Federal University of Minas Gerais (UFMG; protocol no. CAAE: 48127415.3.0000.5149, available at www.saude.gov.br/plataformabrasil). It was registered in the Brazilian Clinical Trials Registry (no. RBR-85kywk3).

Participants were selected at the Faculty of Dentistry, UFMG, and their parents or guardians signed a free informed consent form. Eligibility was determined by clinical evaluation and medical histories obtained at screening visits. In total, 146 patients aged 12-18 years who were undergoing fixed appliance orthodontic treatment at the Dental School´s Orthodontic Clinic were invited to participate between November 2015 and June 2017. The patients were accompanied by their parents or guardians during treatment, in accordance with the faculty's established protocols.

Eligible subjects were undergoing >1 year treatment, had been diagnosed with gingivitis, had not received medication or gingival treatment in the previous year, and had no systemic complicating factor. Other inclusion criteria were: treatment with fixed appliance without extraction for ≥6 months, no prior surgical periodontal treatment, absence of acute periodontal disorders (periodontal abscess and disease and/or acute necrotising periodontitis) at the time of examination, no smoking, no pregnancy, no use of 0.12% Cx mouthwash or other chemical agent, absence of fungal infection, no allergic reaction to the gel components, and no antibiotic therapy or prolonged medication use in the last 6 months.

Clinical procedures

Fixed appliance treatment was performed with 0.022” straight-wire system (Morelli®, São Paulo, Brazil), with brackets applied to the anterior teeth and bands applied to the permanent first molars. Every patient was given instructions to follow the orthodontic specialist's routine of doing oral hygiene procedures after meals at least twice a day.

Before antimicrobial gel application, initial plaque control, oral prophylaxis, and scaling were performed. For this double-blinded study, participants were randomised into three groups treated with different gel protocols: 0.6% Cx in Cd gel, free 0.6% Cx gel (active reference) and placebo gel (control). The interventions involved two 4-min applications of 4 ml gel in trays at the clinic, administered at 0, 15 or 30 days. The efficacy of the treatments was then compared. The patients remained under orthodontic treatment and were recalled at 0, 15 or 30 days after gel application, with the reinforcement of the oral hygiene instructions. After the trial ended, the participants had monthly check-ups and were never without archwires.

Gingivitis was defined according to the criteria proposed in the 2017 World Workshop¹⁵ as probing depth ≤ 3 mm, 10% or more of sites with BOP, no attachment loss, and no radiographic bone loss. BOP¹⁶ and VPI¹⁷ values were recorded at the first session (baseline), to ensure that patients had periodontal alterations. The experimental periods were 0, 15 and 30 days after gel application. Examiner alignment and evaluation were conducted under the supervision of a periodontics specialist.

BOP was measured with gingival sulcus softening using a periodontal probe (Williams 23; Trinity, São Paulo, Brazil) and recorded as present (1) or absent (0) at six sites (mesiobuccal, midbuccal, distobuccal, distolingual, midlingual and mesiolingual) per tooth, excluding the third molars, within 10 s after probing. The VPI was also recorded for six sites per tooth as 0 (absence of visible biofilm), 1 (visualization of biofilm after probing), 2 (clinically visible plaque) or 3 (abundant plaque).

Gingival crevicular fluid (GCF) samples were collected to assess cytokine/chemokine expression. The selected sites were isolated with dry cotton rolls, and the samples were cleaned immediately after collection. Four paper points were introduced into the GCF mesial and distal to the first (or second, when the first was absent) premolar. After withdrawal, the paper points were cut at 4 mm from the tips and dropped into microcentrifuge tubes, then stored at –70°C. RNA was extracted from the GCF.

To determine the cytokine levels, a convenience sample of 19% was chosen from each group (six individuals/group). A single calibrated examiner collected all gingival data at baseline and on days 15 and 30.

For the treatment, isolation from moisture was maintained with three cotton rolls and air drying prior to the application of two disposable trays of 5 ml gel over the teeth and gingiva. The patients used bite force to maintain the trays in position. The Cx concentration was normalised to 0.6% in the gels with and without Cd. The duration of application was arbitrarily set to 4 min, in alignment a previous report¹⁸ and to prevent muscle fatigue. After 4 min, excess gel was washed away. To assess the impact of Cd on the desorption of Cx from the molecular compound, the saliva Cx concentration was measured.

Determination of cytokine expression

Primer sequences were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA) based on nucleotide sequences available in the GenBank database of Biological Science Institute. Real-time PCR assays were performed using the Step One Real-time PCR System (Applied Biosystems). Complementary DNA was synthesised by reverse transcription reaction using 1 μg RNA. PCR was performed under the following standard conditions: a holding stage at 95°C (10 min); a cycling stage of 40 cycles at 95°C (15 s), followed by 60°C (1 min) and a melting curve stage at 95°C (15 s), 60°C (1 min) and 95°C (15 s).

The sequences were used for quantitative PCR analysis of IFN-γ, TNF-α and IL-10 expression. A SYBR-Green detection system (Applied Biosystems) was used according to the manufacturer's instructions for assay primer amplification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization, performed with each reaction. All samples were run in duplicate. Reactions were performed in a volume of 25 μL and contained 1 μg cDNA. Sequence Detection Software (version 2.0; Applied Biosystems) was used to analyse the data after amplification. The results were obtained as threshold cycle (Ct) values. Expression levels were calculated using the comparative 2^−ΔΔCT method. The means of duplicate runs for each patient were obtained, and the mRNA expression levels in all samples were defined as the ratios of the specific primers to GAPDH expression.

Determination of Cx release into saliva

The saliva Cx concentrations were measured at baseline and 2, 4, 8, 12, 16 and 24 h after gel application in eight patients (four each in the Cx and Cx:β-Cd groups). When the patients had appointments in the afternoon, they were told to spit unstimulated pooled saliva into collection cups for five minutes at each time point for 10 s. The samples were then analysed by ultra-pure liquid chromatography (UPLC) in relation to the calibration equation (r² = 0.992).

Chromatographic Cx quantification

Cx was analysed using an ACQUITY UPLC^TM H-Class system (Waters, Milford, MA, USA) equipped with a binary solvent delivery system. The chromatographic analysis was carried out by gradient elution using a BEH C18 column (1.7 μm, 2.1 × 50 mm; Waters). The mobile phase used was a mixture of water (eluent A) and acetonitrile (eluent B), both with 0.5% (v/v) formic acid, with the following gradient: 0-3.0 min, 80%-15% A; 3.0-3.01 min, 15%-80% A; and 3.01-4.5 min, 80% A. The column temperature was maintained at 45°C, the flow rate was maintained at 0.35 mL min⁻¹, and the injection volume was 2 μL. The gradient and UPLC-tandem mass spectrometry (MS/MS) conditions were based on and similar to those described in the literature.^19,20 The system was interfaced with a sequential (MS/MS) triple quadrupole mass spectrometer (Waters Inc., Bedford, MA, USA) equipped with an electrospray ionization source operated in positive mode. The conditions set for the MS/MS system are provided in Table 1.

Table 1.

Conditions set to the MS/MS systems during chlorhexidine analysis.

Common MS/MS conditions
Capillary voltage of 3.0 kV; desolvation temperature at 450 °C;
cone and desolvation gas flows of 10 and 650 Lh−1, respectively
MS/MS conditions in multiple reaction monitoring mode (MRM)
Analyte	Dwell time (s)	Cone energy (V)	m/z of the precursor ion	Collision energy (V)	m/z of the product ion
Chlorhexidine		44	505.2	44	170.0**
	0.52	40		30	184.0*
		40		20	335.9**

^⁎Significant difference (p < 0.05).

^⁎⁎Significant difference (p < 0.01).

To quantify Cx in the saliva samples, a calibration curve was prepared with blank saliva samples fortified at concentrations of 9, 26, 35, 65 and 296 ng g⁻¹ via the addition of adequate masses of working solution. To match the dilutions of the calibration and patient samples, adequate amounts of methanol:water (2:8, v/v) containing 0.1% formic acid solution (v/v) were added. Patient saliva samples were considered to be diluted properly when the Cx concentrations exceeded the highest concentration on the calibration curve. The curves were prepared in duplicate for each concentration level. Linearity was expressed as the linear correlation coefficient (r) for the calibration curve, calculated using the ordinary least-squares method.

Statistical analysis

The sample size was calculated using the Statistical Package for the Social Sciences (version 19.0 for Windows; IBM SPSS, Armonk, NY, USA), which indicated that a minimum of 24 individuals per group was required for analysis with a significance level of 5%. The minimal sample size for each group was chosen at least 26, assuming that 20% of participants would not finish the treatment and that 95% detection power and a significant alpha level of 0.05 would be attained.

Randomization and data analysis were double blinded. BOP and VPI values were analysed using one-way nonparametric analyses of variance. A boxplot was used to descriptively evaluate the empirical distribution of the Cx data. The normality of numerical data on cytokine levels in GCF was tested using the Shapiro–Wilk test. As the data were not distributed normally, the Kruskal–Wallis test was used to identify significant differences among samples and the Dunn post-hoc test was performed with Bonferroni adjustment for the number of comparisons (P < .05).

Results

Clinical characteristics

Of the 146 patients evaluated for inclusion in this randomised controlled clinical trial, 14 individuals did not meet the inclusion criteria, 2 refused to participate before study initiation, and 1 did not participate for another personal reason. The remaining 129 individuals were allocated by coin flip to the placebo (n = 43), 0.6% Cx:β-Cd (n = 44) and 0.6% Cx (n = 42) gel groups. Thirteen individuals (n = 6, 3 and 4, respectively) withdrew from the study (Figure 1).

Fig. 1.

Diagram of study flow.

Thus, data from 116 individuals were included in the final analysis. Sixty-one participants were female, with a mean age of 13.07 years, and 45 participants were male, with a mean age of 14.31 years (Table 2). No side effect or allergic manifestation caused by the drug tested was observed.

Table 2.

Demographic characteristics distribution on the groups after application of gels with Cx.

Demographic characteristics	Control: Plain gel		Cx:β-cd gel		Cx gel
N subject	37		41		38
Age (year) media	14.0		12.0		13.21
Women/men	20	17	22	19	27	11

At baseline, the mean VPIs in the three groups were similar (Table 2). At 15 days, the median reduction in the VPI was lesser in the control group than in the experimental groups (P < .05), with no difference between the latter. At 30 days, the values were similar to those obtained at baseline in all groups, although the difference between 0 and 30 days in the Cx and Cx:β-Cd groups differed from that in the control group (P < .001). The degree of increment in median VPI scores was more pronounced in the control group than in the Cx and Cx:β-Cd groups. While the differences between Cx and Cx:β-Cd were not statistically significant, there were significant differences in the increment in median VPI scores at periods of 0 (initial) and after 15 days for Cx and Cx:β-Cd. The VPI declined between baseline and 15 days in all groups (Table 2), but this effect was no significant at 30 days.

At baseline, 26% of the sites tested bled, and the average BOP values for the three groups were similar (Table 3). At 15 days, the BOP value was significantly lower, and the median BOP reduction was significantly larger, in the Cx:β-Cd group than in the control and Cx groups (P < .05).

Table 3.

Comparison intra-groups and inter-groups the groups of Visible Plaque Index (VPI) and bleeding on probing (BOP) positive response after application of Cx:β-Cd, Cx and control plain gels in patients with orthodontic appliances.

Visible Plaque Index (VPI)
	Control plain gel		Cx:β-cd gel		Cx gel		P -value
	Median ± SD	CI	Median ± SD	CI	Median ± SD	CI
T0	0.62 ± 0.06	0.50; 1.37	0.79 ± 0.10	0.38; 1.21	0.64 ± 0.15	0.35; 0.93	NS
T15 days	0.35 ± 0.14*	0.42; 0.81	0.07 ± 0.07*,§	0.06; 0.33	0.56 ± 0.0§	0.04; 0.27	< .05
T30 days	0.56 ± 0.02†	0.37; 1.56	0.35 ± 0.01†,‡	0.27; 0.47	0.36 ± 0.07†,‡	0.20; 0.50	NS
Δ0-15	0.27 - 0.18		0.62 ± 0.03*		0.49 ± 0.14		< .05
Δ0-30	0.26 ± 0.04		0.44 ± 0.09*,¶		0.28 ± 0.08		< .05
BOP positive (%)
	Control plain gel		Cx:β-cd gel		Cx gel		P -value
T0	27		25		27		NS
T15 days	30		15*		23§		P < .05
T30 days	32		23		25		NS
Δ0-15	-3		10*		4		P < .05
Δ0-30	-5		2		2		NS

CI = Confidence interval, NS = Not significant.

^⁎Intra-groups comparison between T0 and T15.

^†Intra-groups comparison between T0 and T30.

^‡Intra-groups comparison between T15 and T30.

Δ_0-15—mean difference from T0 to T15_.

Δ_15-30—mean difference from T0 to T30_.

^§Inter-group mean differences for Δ_0-15.

^¶Inter-group mean differences for Δ_0-30.

Cytokine expression

Figure 2 shows cytokine expression in GCF in the three groups. The TNF-α and IFN-γ levels showed significant decreased the level after the gel application and the IL-10 level showed no significant differences between the experimental groups at 15 days, but was different when compare among they and control in the same time (P < .05). In the control group, the TNF-α and IFN-γ levels showed no significant differences.

Fig. 2.

Expression of TNF-α, IFN-γ and IL-10 genes in gingival crevicular fluid (GCF) of patients with human gingivitis after 30 days. Bars represent the mean values of samples recovered lines representing the standard error of the mean. The same letter indicates statistical difference P < .05 in T1 comparison with other times (T1 = initial, T2 = 15 days and T3 = 30 days) by the Mann–Whitney tests.

Cx release

Cx release from the Cx:β-Cd and Cx gels was evaluated using UPLC. The calibration curve obtained by the ordinary least-squares method demonstrated that the linear range evaluated was described by the curve equation. The Cx:β-Cd gel showed more concentrated and prolonged Cx release than did the Cx gel over 24 h (Figure 3).

Fig. 3.

The release profile of Cx from samples of saliva after application of gels of Cx and Cx: β-cd in orthodontic patients with gingivitis at time 2, 4, 8 12, 16 and 24 h.

Figure 3 shows the mean Cx concentrations (ng g-1) in saliva at 2, 4, 8, 12, 16 and 24 h after application. The 0.6% Cx:β-Cd gel released larger amounts of the active molecule over time (mean, 680 ng g-1). Major differences were observed between the Cx and Cx:β-Cd groups at 2, 4, 8, 12 and 16 h. At 24 h, only a small difference was observed between these groups. Thus, the new Cx:β-Cd formulation is advantageous in clinical situations in which individuals’ oral hygiene maintenance is deficient.

Discussion

In the present study, all patients were instructed to perform oral hygiene at least twice a day after meals, with no method specified. Patients wearing orthodontic braces should be oriented to use of interdental brushes to clean the braces’ sides. Given the possibility that some patients will neglect their oral hygiene, chemical control is necessary.

Numerous antiseptic formulations containing chemical agents have been used in orthodontic clinics to facilitate dental plaque control. Despite being regarded as an effective anti-plaque agent for the reduction of gingival inflammation, bleeding and erythema, Cx has been found to have several adverse effects, including tooth discoloration and loss of taste. The dose-dependent nature of these effects has prompted research into reduction of gingivitis following orthodontic appliance placement. Moreover, the prolonged use of Cx is not advised due to its adverse effects. Thus, a well-designed home care regimen and shorter (e.g., 3-monthly) prophylactic intervals may prove to be equally advantageous.

With the widespread use of Cx gels, Cx has frequently been compared to other materials and used in a variety of formulations and dosages (most commonly 2% and 1% Cx gels), prohibiting the comparison of study findings.²¹ The Cx:β-Cd inclusion compound promotes the regulated release of the active molecule, which may enable the minimization of the Cx dosage.

The antimicrobial activity of low-concentration Cx:β-Cd systems has been shown to be more effective against periodontal biofilm than is pure Cx.^12,13 In this study, the use of a controlled Cx release system as an antibacterial agent co-adjuvant to mechanical treatment to alleviate gingivitis was examined.

Mechanical prophylaxis has been shown to effectively control plaque; however, the intensity and time expended determine the quality of the results.²² In the present study, the VPI and BOP were reduced at 15 days after Cx:β-Cd gel application and had returned to the pre-treatment baseline at 30 days. The lack of a difference between the Cx and Cx:β-Cd groups at 15 days could be explained by the gels’ promotion of drug retention and adhesion on the oral cavity surfaces, and slow drug release due to the polymeric (hydroxypropyl methylcellulose) network. The effects of 0.12% Cx mouthwash solutions are less enduring than those of gel formulations. At 96 h, no significant difference was noted in the efficacy of mouthwashes in managing supra- and sub-gingival biofilm.²³

Favourable outcomes of experimental treatment involving the insertion of 0.2% Cx gel into extraction sockets enabled the performance of less-traumatic surgeries, particularly in older patients.²⁴ The intra-alveolar placement of adhesive 0.2% Cx gel intraoperatively after surgical impacted third molar extraction was found to provide a more direct and prolonged therapeutic effect, helping to prevent infection.²⁵ A temperature-sensitive Cx hydrochloride gel was successfully developed in situ as a broad-spectrum anti-microbial agent used in the treatment of periodontal biofilm using a cold method and poloxamer 407 and carbopol 934P as gelling agents.²⁶ The physical characteristics, pH, and drug contents of Cx formulations were found to be satisfactory.²⁷

In this study, we used hydroxypropyl methylcellulose due to its greater fluidity and ability to penetrate the gingival sulci, and these properties were corroborated by the results. Hydrogels provide for the sustained local delivery of a variety of therapeutic agents. The use of the natural polymer hydroxypropyl methylcellulose as a release system material in hydrogels has been pursued strongly dues to its biocompatibility, low toxicity and biodegradability. Gels containing this polymer have also shown high-water swell ability, high oxygen permeability, ease of drug loading and release and structural diversity.^28,29

Other semi-solid systems have been used for Cx release; for example, the use of Cx chips in periodontal pockets reduced the number of subgingival microorganisms of the red complex in patients under treatment for chronic periodontitis.³⁰ Similarly, a study related reduction of papillary bleeding, mouthwashes should be performed twice to strengthen the effects of chlorhexidine, which supports the results of studies showing that the antimicrobial agents included in β-Cd presented best effects than the free drug.³¹

In the present study, patients’ clinical indices remained consistent with the initial values, with no significant difference between groups, at 30 days after mechanical treatment and Cx and Cx:β-Cd gel use. These results are comparable to those reported by Karkhanechi et al.³² and Kouraki et al.³³, who also established that extended orthodontic appliance use is associated with elevated incidences of periodontal pathogen biofilm and haemorrhage. Chemical therapy resulted in substantial decreases in clinical indices relative to mechanical therapy alone across all timepoints assessed in the current investigation. Haas et al.³⁴ also demonstrated the utility of chemical methods for the control of plaque and gingivitis, especially in patients receiving orthodontic treatment.

The reductions in the VPI and BOP were greater in the Cx:β-Cd group than in the other groups in the current investigation. Only in this group did the BOP remain considerably lower than baseline at 30 days. Isolated Cx treatment, especially with 0.12% Cx, has shown significant benefits in terms of plaque reduction and the improvement of oral health indices in similar clinical trials.³⁵ The efficacy of gingivitis control is contingent on multiple factors. Adolescents are susceptible to a wide range of gingival and periodontal diseases,^36,37 which may necessitate chemical or mechanical support, depending on the specific indication. Patients who have difficulty controlling plaque may benefit from the use of a gel containing Cx for additional local support, as well as the development of good hygiene habits, including compliance with daily dental cleansing. In this study, single gel application was performed; long-term studies evaluating patient acceptance, the emergence of side effects, and maintenance of effective gingivitis control are needed.

Pro-inflammatory cytokine activity is regulated inversely by anti-inflammatory cytokines, and an imbalance between them can trigger an inflammatory response. In the present study, the hypothesis that Cx and its derivatives would decrease pro-inflammatory cytokine activity, reducing inflammation and stabilising gingivitis, in adolescents was tested. Pro-inflammatory and immunoregulatory cytokines are important for the pathogenesis of periodontitis. IFN-γ is produced by helper T1 cells, and the pro-inflammatory agent estradiol induces TNF-α production by macrophages and prostaglandins. IFN-γ has been demonstrated to inhibit osteoclastogenesis by interfering with the RANKL-RANK signalling pathway. IL-10 suppresses pro-inflammatory agents, inhibiting the formation of osteoclasts and bone resorption, as it can reduce the mRNA to steady-state levels of TNF-α and IFN-γ.

In a similar study, Tavares et al.³⁸ showed that pro-inflammatory and immunoregulatory cytokine expression remained lower in the context of periapical inflammatory response at 15 days after 2% Cx gel dressing application. In the present study, Cx:β-Cd gel reduced IFN-γ and TNF-α expression and increased IL-10 expression relative to the control and Cx groups at 15 days after treatment.

The effect of Cx on inflammatory periodontal disease is corroborated by in-vitro data.³⁹.The release of Cx from the Cx:β-Cd inclusion compound could promote longer action against pro-inflammatory agents such as TNF-α, thereby reducing the possibility of gingivitis development. Self-hygiene is a critical strategy for the achievement of good oral health, and toothbrushing is the most popular mechanical method for the prevention of gingivitis. Randomised controlled trials examining the impact of ≥4 weeks Cx mouthwash use to supplement mechanical oral hygiene procedures on gingivitis in children and adults have provided substantial evidence for the reduction of dental plaque, notwithstanding extrinsic tooth discoloration and calculus.^40,41 The current investigation demonstrates reductions in gingivitis-related clinical parameters under extremely severe conditions (i.e., fixed orthodontic appliances use) with the use of Cx incorporated molecularly into Cd, which alters its chemical parameters and extends the antiseptic's 24-h persistence.

Conclusion

A gel containing 0.6% Cd was incorporated molecularly into β-Cd to develop a more efficient controlled antiseptic release method. This incorporation facilitated the release of the active molecule of Cx, thereby enhancing the drug's local effects and subsequently reducing gingivitis and oral plaque. It had a prolonged effect and reduced BOP and the VPI in adolescents under orthodontic treatment with fixed appliances. Thus, this formulation is a viable candidate for use as a co-adjuvant therapeutic agent alongside dental hygiene. Further long-term (≥6 months) investigation with microbiological analysis is warranted.

Clinical relevance

Scientific rationale for the study

Cx is the gold standard antiseptic treatment for the control of dental plaque and gingivitis. A new slow-release Cx gel system with encapsulation of the active molecule was developed and showed excellent bactericidal effects and less cytotoxicity in recent research. Based on its physicochemical characteristics and release profile, this clinical study was performed to examine the local application of this gel in individuals using orthodontic appliances. This application does not need to be repeated, is more comfortable for patients and can be performed at the same time as the dental control needed to improve oral hygiene habits.

Principal findings

One topical application of Cx:β-Cd gel successfully in controlled plaque and reduced gingivitis-related clinical parameters for up to 15 days, and improved oral hygiene. Its performance during treatment appointments for orthodontic patients may be sufficient to reduce plaque and prevent gingivitis without the need for daily product application.

Practical implications

The use of a Cx:β-Cd gel in patients with gingivitis may be an effective means of achieving good oral hygiene. It reduces the frequency of product application and has an immediate antimicrobial effect and long duration of action on the teeth and oral mucosa.

Funding

The funding organizations CNPq and Fapemig are acknowledged by the authors for their assistance.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.