Effects of 0.2% Hyaluronic Acid Gel‐Impregnated Dental Floss on Clinical Gingival Parameters: A Randomised Clinical Trial

ABSTRACT

Objectives

Gingivitis is a common inflammatory lesion caused by the build‐up of oral biofilm and is an essential precursor to periodontitis. For its treatment, oral hygiene habits, such as dental flossing, must be improved, and adjunctive materials, such as hyaluronic acid, may be used to reduce plaque formation and gingival inflammation. This study aimed to assess the effects of 0.2% hyaluronic acid gel‐impregnated dental floss on the clinical periodontal markers of patients with gingivitis.

Material and Methods

This clinical study adopted a split‐mouth, randomised controlled trial design. After clinical data were assessed at baseline, and supragingival scaling was performed, bilateral gingivitis regions were randomly allocated to either the hyaluronic acid gel‐impregnated floss group or just the floss group using a computer‐generated randomisation table. Clinical parameters were recorded at 1, 2 and 4 weeks after treatment.

Results

Over the 4‐week trial period, all patients showed significant improvements in all clinical periodontal markers. The 1‐week plaque index, 4‐week gingival index and 4‐week papillary bleeding index were significantly lower in the test group than in the control group (p < 0.05).

Conclusions

The use of hyaluronic acid‐impregnated dental floss has resulted in more significant improvements in clinical periodontal parameters compared to dental floss alone. For ease of use, ready‐made products with this innovative formulation may be produced.

Trial Registration: ClinicalTrials.gov identifier: NCT06307041

1. Introduction

Incomplete, inadequate or improper daily oral self‐care results in the formation of dental biofilm [1]. Dental biofilm with its complex structure can accumulate on teeth and cause gingivitis if it remains uninterrupted or unremoved for days or weeks [2]. This happens as a result of the loss of the symbiotic relationship between the biofilm and the host's immune‐inflammatory response [2]. Toothbrushing can remove supragingival plaque from the lingual/palatal and facial surfaces, but it is often suggested to use special tools to reach the interdental space [3]. There are various products for interdental cleaning such as dental floss, interdental brushes and plastic and wooden toothpicks [4].

Historically, flossing has been the most often recommended self‐care strategy for interdental cleaning, likely because it is the most widely applicable technique [5]. It is known that flossing in addition to toothbrushing significantly improves gingival parameters [6]. Adults who have limited interdental space between the teeth and papillae that either partially or completely fill it are advised to use dental floss [7]. In addition to eliminating food particles from the teeth, proper flossing can remove dental plaque from the surrounding surface, reaching up to 3 mm below the apex of the papilla; this promotes healthy gingiva without posing a risk to the periodontium [8].

Apart from mechanical cleaning, using regenerative biological materials is a strategy that aims to improve periodontal parameters [9]. Hyaluronic acid (HA) is a disaccharide polymer, the predominant glycosaminoglycan synthesised by connective tissue cells and located in the extracellular matrix (ECM) [10]. In both the hard tissues, such as cementum and alveolar bone, and the soft tissues, such as gingiva and periodontal ligament, HA is an essential component, and it performs a variety of physiological and structural tasks inside these tissues [11, 12]. By controlling osmotic pressure and tissue lubrication, it preserves the structural and homeostatic integrity [13].

Studies on its chemical and physicochemical properties and physiological role in humans have shown that HA is an ideal biomaterial for cosmetic, medical and pharmacological purposes [11].

Hyaluronic acid contributes to the treatment of periodontal diseases by yielding anti‐inflammatory, anti‐edematous and antibacterial effects [14]. It is routinely used as an adjunct to surgical and non‐surgical periodontal treatments [10, 11].

Hyaluronic acid is commonly used as a beneficial supplement in the treatment of gingivitis. Numerous studies have demonstrated a significant reduction in the gingival bleeding index, plaque index (PI) and gingival crevicular fluid (GCF) volume with the topical use of 0.2% HA gels or HA sprays [12]. The application of 0.2% HA gel to periodontal pockets following scaling and root planing has been reported to significantly reduce inflammatory infiltration [15]. No adverse effects of HA administration have been demonstrated [16].

Our study aimed to evaluate the effects of 0.2% HA‐impregnated dental floss on the clinical periodontal parameters of patients with gingivitis. The null hypothesis was that there would be no difference in the clinical periodontal parameters between groups.

2. Materials and Methods

2.1. Study Design and Ethical Considerations

The study was approved by the Gülhane Educational Research Hospital Clinical Research Ethics Committee (2023/55) and, was conducted under GCP and ICH, and, all volunteer patients signed an informed consent form. This study was a single‐centre, single‐blinded, split‐mouth, randomised clinical trial, which adhered to the Consolidated Standards of Reporting Trials Statement [17]. (Data).

2.2. Patient Selection

Patients who were referred to the Department of Periodontology of the Gülhane Faculty of Dentistry at the Health Sciences University from April to July 2023 were selected. According to the most recent classification of periodontal diseases, gingivitis is referred to as dental biofilm‐induced gingivitis [18]. Intraoral examination was conducted to screen for and diagnose gingivitis among patients aged 18–55 years, and each patient was then given a classification according to the most recent categorisation. Patients with no radiographic bone loss, pocket depths ≤ 3 mm at each site and more than 30% of bleeding sites after probing were diagnosed as gingivitis and were eligible for inclusion in the study [19]. Williams 14 W (Hu‐Friedy Mfg. Co. LLC, UK) probe was used for periodontal examination. Patients who freely consented to participate in the study and received routine dental scaling after being diagnosed with gingivitis were enrolled. Conversely, patients who had recently used antibiotics (within the last 3 months), had undergone periodontal therapy within the previous 6 months and had systemic diseases, such as hypertension, diabetes, autoimmune diseases, cardiovascular diseases, cancer, Crohn's disease, etc. were excluded from the study. Patients who have orthodontic appliances, removable dentures, tooth pain and interproximal caries as well as smokers, pregnant and lactating patients were excluded from the study. The main hypothesis of the research was to investigate the differences between and within groups. Similar studies that can be used in sample size calculation were examined and the highest sample size calculation was considered according to the statistical methods to be applied in line with the main hypothesis. In this study, the sample size was calculated at a 95% confidence level using the ‘G. Power‐3.1.9.2’ program. As a result of the analysis, α = 0.05, the standardised effect size was taken as 0.6434 (Sahayata [20] table 4: PBI) from a previous similar study and the minimum sample size for each group was calculated as 13 with a theoretical power of 0.95. Taking into account the possibility of loss of observations over time, 10% of the calculated sample size (4 observations) was added to the study. The minimum sample size for each group was calculated as 17. Since the study was conducted on gingivitis patients, the sample size was increased due to possible losses in follow‐up sessions. The flow chart of the study is shown in Figure 1.

FIGURE 1.

Flow chart of the study.

2.3. Experimental Approach

The gingival index (GI) [21], PI [22] and papillary bleeding index (PBI) [23] were recorded at baseline and 1, 2 and 4 weeks after treatment. During the trial, patients were asked not to use any mouthwash or whitening agents. Supragingival scaling was performed to ensure that no patient had extrinsic stains, dental calculus or dental plaque at the beginning of the study (AIRFLOW Prophylaxis Master; Electro‐Medical Systems, Switzerland). The same regular size, medium toothbrush with end‐rounded filaments (TePe Select; Malmö, Sweden), 65 mL tube of toothpaste (IPana Pro‐Expert; Gross‐Gerau, Germany) (Data S1) and instruction for brushing style (modified Stillman technique) were provided to all patient (Data S2) (https://www.mouthhealthy.org/all‐topics‐a‐z/brushing‐your‐teeth).

Patients were instructed on how to utilise dental floss during oral hygiene promotion according to the American Dental Association's recommendations (Data S3).

(https://www.mouthhealthy.org/all‐topics‐a‐z/flossing/) Thereafter, dental floss (30 m) (Sensodyne expanding dental floss; GSK Consumer Healthcare, UK) and 0.2% hyaluronic acid gel (Gengigel; Ricerfarma, Milan, Italy) (20 mL at 0.2% concentration) were given to patients. In addition to hyaluronan, the gel contains excipients and xylitol. Patients were asked to tear two equal pieces of floss of an appropriate size for cleaning and soak one of them in HA gel for 5 min (Data S4–, S9).

In a completely randomised fashion, some patients were asked to floss with the HA‐impregnated floss on their right side and regular floss on their left side and vice versa. The site (right/left) was selected using a computer‐generated randomisation table. In addition, written instructions and a checklist were provided to promote patient cooperation and ensure that each device was used properly on the correct side. While all clinical measurements and initial periodontal treatments were performed by the same investigator (O.S.A.), a different investigator conducted the randomisation (S.C.S.). The investigator who performed the measurements was blinded to which side of the patients was the test group. Intra‐examiner calibrations were conducted in five patients with gingivitis who were not included in the study. The Cohen's kappa value of the GI, PI and PBI was 0.80, 0.82 and 0.920, respectively.

2.4. Statistical Analysis

Descriptive statistics (numbers, percentages, means, standard deviations, medians, minimum values and maximum values) were used to present the data. As the first step of the statistical analysis, the normality assumption was checked using the Shapiro–Wilk test and the homogeneity of variance using the Levene test. An independent sample t‐test was conducted for comparing two independent groups with a normal distribution and the Mann–Whitney U test for comparing those with a non‐normal distribution. A dependent sample t‐test was performed for comparing two dependent groups with a normal distribution and the Wilcoxon signed rank test for comparing those with a non‐normal distribution. All analyses were performed using IBM SPSS 25.

3. Results

A total of 30 patients, 15 women and 15 men, aged between 18 and 47 years, were included in the study. The distribution and comparison of clinical measurements obtained at different times in both groups are shown in Table 1. PI, GI and PBI values at baseline and the 1st, 2nd and 4th weeks were compared in HA‐impregnated floss and floss groups. The tests revealed significant differences between the baseline and 1st‐, 2nd‐ and 4th‐week PI, GI and PBI in both groups (p < 0.05).

TABLE 1.

Comparison of clinical measurements obtained at different times according to patient groups and time.

	Time	Test group	Control group	t/U	p
		Mean ± SD	Mean ± SD
PI	Baseline	1.41 ± 0.49	1.42 ± 0.49	‐0.114 a	0.910
	1 week	0.49 ± 0.26	0.63 ± 0.28	−2128 a	0.038
	2 week	0.36 ± 0.25	0.38 ± 0.26	427.0	0.734
	4 week	0.23 ± 0.18	0.29 ± 0.23	391.0	0.383
GI	Baseline	1.31 ± 0.50	1.24 ± 0.40	435.0	0.824
	1 week	0.55 ± 0.29	0.68 ± 0.21	−1974 a	0.053
	2 week	0.39 ± 0.24	0.48 ± 0.28	350.0	0.139
	4 week	0.26 ± 0.20	0.40 ± 0.25	293.0	0.020
PBI	Baseline	1.00 ± 0.40	1.02 ± 0.58	410.0	0.554
	1 week	0.31 ± 0.21	0.42 ± 0.24	−2005 a	0.050
	2 week	0.28 ± 0.28	0.35 ± 0.31	370.5	0.240
	4 week	0.16 ± 0.14	0.24 ± 0.15	280.0	0.012

^a Independent sample t‐test.

Clinical measurements obtained at different times were compared between floss and HA‐impregnated floss groups (Table 2). The tests showed significant differences in the 1st‐week PI, 4th‐week GI and 4th‐week PBI between the groups (p < 0.05); accordingly, there were significant clinical improvements in these parameters in the HA‐impregnated floss group compared with the floss group.

TABLE 2.

Comparison of clinical measurements for patient groups over time.

	Baseline to 1 week			Baseline to 2 week			Baseline to 4 week
	Dif. (%)	t/Z	p	Dif. (%)	t/Z	p	Dif. (%)	Z	p
Test group
PI	64.88	12.786 a	< 0.001	73.49	12.337 a	< 0.001	82.37	−4762	< 0.001
GI	58.25	−4960	< 0.001	71.72	−4679	< 0.001	77.58	−4762	< 0.001
PBI	66.75	8688 a	< 0.001	74.70	−4535	< 0.001	82.47	−4782	< 0.001
Control group
PI	51.90	9600 a	< 0.001	69.89	−4782	< 0.001	77.72	−4782	< 0.001
GI	47.86	6818 a	< 0.001	67.61	7856 a	< 0.001	63.61	−4762	< 0.001
PBI	52.90	−4355	< 0.001	60.79	−4474	< 0.001	68.93	−4782	< 0.001

^a Dependent sample t‐test.

4. Discussion

Gingivitis is an early and reversible form of periodontal disease. It is crucial to treat gingivitis to prevent periodontitis. Although not all cases of gingivitis progress to periodontitis, it is recognised that both periodontal lesions have the same aetiology and follow the same inflammatory process [24]. The interproximal surfaces of teeth are inefficiently reached by toothbrushes. As a result, in addition to brushing, other methods of controlling interdental plaque should be used [25]. This study aimed to evaluate the effects of HA‐impregnated dental floss usage in addition to tooth brushing on clinical periodontal parameters of gingivitis patients.

Some studies have reported that dental flossing in addition to brushing increases the success of the treatment of gingivitis [6]. Muniz et al. [26] reported that using dental floss with chlorhexidine caused a greater reduction in supragingival biofilm than using conventional dental floss but had no additional effect on marginal gingival bleeding.

HA has been successfully used in the treatment of numerous inflammatory diseases and conditions [10]. Especially in recent years, many studies in the field of periodontology recommend the use of HA [27, 28]. In some of these studies, HA‐containing sprays and mouthwashes were used by the patients [29, 30]. On the other hand, HA‐containing gels were applied to the gingiva by the physician in many studies [31, 32], while in some studies, patients applied them at home [20, 33, 34]. In our study, we examined for the first time whether using HA‐impregnated dental floss has a greater impact on clinical periodontal markers than using regular dental floss.

A clinical study reported that the use of HA in addition to a coronally positioned flap in the treatment of furcation defects showed superiority in hard tissue measurements [35]. In another study, Sahayata et al. [20] found that HA therapy reduced gingival bleeding, decreased gingival fluid flow and improved gingival health among patients with gingivitis. Jentsch et al. [33] demonstrated that topical therapy with 0.2% HA twice daily for 3 weeks improved the PI, PBI and GCF volume in individuals with gingivitis. In the study by Pistorius et al. [29] topical administration of an HA spray (five times daily for 1 week) reduced the sulcus bleeding index, PBI and GCF volume. Similarly, our study showed that the use of HA‐impregnated dental floss resulted in greater improvements in the GI, PI and PBI than did the use of regular dental floss. This is a result of the antibacterial and anti‐inflammatory activity of HA, and the method we used effectively achieved the access of HA to the interproximal region.

In the study by Liu et al. [36] only concentrations of HA between a particular range (0.01%–0.1%) encouraged cell adhesion, migrationand proliferation; at concentrations above 0.1%, these activities were either reduced or even inhibited. In a previous study in the field of periodontology, 0.2% HA was found to be suitable for use by patients at home, while 0.8% HA was noted to be more suitable for application by clinicians in the office [37]. In our study, we preferred to use 0.2% HA, in accordance with the literature.

It is well known that HA, a glycosaminoglycan of the ECM, has different biological functions depending on its molecular weight [38]. Low‐molecular‐weight HA (LMW‐HA) exhibits pro‐inflammatory and immunostimulatory behaviours, whereas high‐molecular‐weight HA (HMW‐HA) has anti‐inflammatory and immunosuppressive properties [39]. The patented medical product in this study, like most HA‐based agents used in periodontal therapy, contains HMW‐HA and is highly pure [40]. In this study, we used HMW‐HA and observed its positive effect on the healing of gingivitis.

During our study, a female patient developed allergic dermatitis on the side of the HA application and presented to our clinic on the third day. The allergic symptoms improved immediately after the patient stopped using HA. Although it has been reported in the literature that there are allergies encountered in the dermo‐cosmetic use of HA [41], we could not observe such data in the field of periodontology. This possibility must be considered in future studies.

To eliminate individual differences in host response, our study used a split‐mouth model in which two treatments were randomly assigned to either the right or the left dentition. The so‐called carry‐across effect, which causes a leaking effect from one site to the other [42], is a limitation of our study model. Comparison of half‐mouth scores rather than comparing the interproximal data separately scores is also a limitation of our study. Another important limitation of our study is that only clinical periodontal parameters were used. Our findings can be supported by further studies in which biochemical and microbiological examinations are performed. Patient compliance and ability are factors that cannot be ignored in the effective use of dental floss. Therefore, another important limitation of this study is the interindividual differences.

5. Conclusion

Within the limits of the present study, the use of 0.2% HA gel‐impregnated floss was found to significantly improve clinical periodontal parameters such as GI and PBI of patients with gingivitis more than standard flossing. It can be concluded that the use of HA‐impregnated floss increases the accessibility and applicability of HA to the interproximal region, thus adding chemical activity to the mechanical properties of the floss. Ready‐made products with this innovative formulation should be produced for ease of use.

6. Clinical Relevance

6.1. Scientific Rationale for the Study

The use of dental floss for interdental cleaning is a part of oral hygiene, and reinforcement with a chemical agent may be useful in increasing its clinical efficacy.

6.2. Principal Findings

Using HA‐impregnated dental floss yields more favourable improvements in clinical parameters than does using regular dental floss.

6.3. Practical Implications

The addition of chemical activity to the mechanical properties of dental floss using a tissue‐friendly agent with antibacterial and anti‐inflammatory effects such as HA is substantially effective in treating gingivitis.

Author Contributions

All authors designed the study. O.S.A. and G.U. conceived the idea for the study. O.S.A., S.C.S. and G.U. identified suitable patients. O.S.A. measured the clinical periodontal parameters. S.C.S. performed the randomisation. G.U. collected the data. E.O. analysed the data. O.S.A., S.C.S. and G.U. led the writing of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1. IPana Pro‐Expert toothpaste ingredients.

Data S2. Instruction for brushing style.

Data S3. Instruction for dental floss usage.

Data S4. HA‐impregnated and regular dental floss.

Data S5. HA and dental floss.

Data S6. HA and dental floss prepared for dipping.

Data S7. Dipping the dental floss in HA.

Data S8. Soaking the floss in HA for 5 min.

Data S9. HA‐impregnated dental floss.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.