Single application of 0.8% hyaluronic acid as a coadjuvant of nonsurgical treatment in nonsmoking patients with periodontitis: A split-mouth, randomized, controlled pilot clinical trial.

BACKGROUND: Topical coadjuvants might be indicated to increase the results of nonsurgical periodontal treatment and overcome some of its limitations. The aim of this study was to evaluate the clinical benefits of a single topical application of 0.8% hyaluronic acid (HA) as a coadjuvant to scaling and root planing (SRP) in periodontal patients.
MATERIALS AND METHODS: Sixteen patients diagnosed with periodontitis were recruited to participate in this split-mouth randomized controlled trial. At baseline, oral hygiene instructions, prophylaxis, and SRP were performed in both sides; additionally, a subgingival application of HA at 0.8% was made in the test side. Several clinical parameters were assessed at baseline, 6, and 12 weeks later: full mouth plaque score, gingival score, bleeding on probing (BoP), probing depth (PD), and clinical attachment level (CAL).
RESULTS: At the end of 12 weeks, there was a overall improvement in all periodontal parameters in both sides (P < 0.05). Test sides showed less BoP when compared to the control sides (9.4 ± 4.0 vs. 14.9 ± 8.9, P < 0.05). Other periodontal parameters such as PD and CAL showed a slight improvement in comparison with the control sides, but the difference was not statistically significant (P > 0.05).
CONCLUSION: Results from this study indicate that a single subgingival application of 0.8% HA seems to reduce gingival inflammation and improve clinical parameters, particularly BoP. Further studies are needed to evaluate the effect of repeated application of HA and long-term results. Copyright:

INTRODUCTION

Periodontitis is an inflammatory disease with multifactorial background that causes progressive destruction of teeth supporting tissues that ultimately can lead to their loss. The main goal of periodontal treatment is to turn affected surfaces into biocompatible areas where both connective tissue and epithelial cells are able to adhere and proliferate.[1]

Since scaling and root planing (SRP) may not be completely effective in some situations due to anatomical constraints or increased pocket depth, systemic and topical coadjuvants might be indicated in some cases.[2] Topical therapies guarantee a better delivery of high concentrations of pharmacologic agents to the periodontal tissues. A diverse array of agents can be used as adjuncts, and these can be antimicrobials such as tetracyclines or macrolides, antiseptics such as chlorhexidine or iodopovidone, photodynamic therapy, or topical application of other agents including hyaluronic acid (HA).[234] In recent years, HA has gained growing attention since promising results have been demonstrated in the treatment of inflammatory processes in medical areas such as dermatology, ophthalmology, and orthopedics.[4567] HA is a nonsulfated glycosaminoglycan of high molecular weight, highly present in the extracellular matrix of nonmineralized and mineralized tissues.[7] Most of the cells in the human body such as fibroblasts, osteoblasts, and cementoblasts are capable of synthesizing HA in their cell membrane and secreting it to the exterior.[8] Due to this, HA can be found in both hard and soft periodontal tissues.[79] Regarding HA's turnover, it can take place either by lymphatic drainage or local metabolism.[9]

HA regulates the inflammatory and immune response acting as an antioxidant. Out of all important properties related to HA, it is important to highlight its regenerative capability due to its osteoinductive effect.[10] HA promotes the migration of endothelial cells which form a network for the deposition of bone tissue, and this mucopolysaccharide also transports proteins which act as growth factors – essentially bone morphogenetic protein-2 and osteopontin. HA also reveals an anti-inflammatory and immunosuppressive effect, which acts by forming a complex that inhibits proteases.[71011] Because of its properties, HA has been studied as a possible coadjuvant in nonsurgical treatment of gingivitis and periodontitis.[121314] Its application has been tested in a variety of periodontal conditions and has even been used in the treatment of infrabony defects, reducing PDs and increasing the attachment level.[15] Although the coadjuvant application of HA in nonsurgical periodontal treatment has been evaluated in some clinical trials, they exhibit some methodological flaws and current evidence is insufficient to support the routine clinical use.[1213141516]

The aim of this study was to evaluate the therapeutic effect of a single topical application of 0.8% HA as a coadjuvant in the nonsurgical treatment of periodontitis, through the evaluation of several periodontal parameters.

MATERIALS AND METHODS

The study protocol was approved by Egas Moniz Ethics Committee and was carried out in accordance with the Declaration of Helsinki (1975 revised in 2000). All patients signed a written informed consent after being thoroughly informed about the conditions of participation.

This split-mouth randomized controlled pilot clinical trial was undertaken between the period of March and September of 2016. Sixteen patients referred to the university clinic for periodontal treatment were enrolled in the study. To be included in the study, patients should be between 40 and 70 years of age and have a confirmed diagnosis of periodontitis. Patients which had received periodontal treatment in the last 6 months, pregnant women, smokers, patients with systemic diseases affecting periodontal disease progression or wound healing, and patients with recent history of antibiotic and nonsteroidal anti-inflammatory drugs therapy were excluded. In the definition of periodontitis cases, we have followed the criteria proposed by Eke et al.[17]

Before study initiation, the examiner that carried out all clinical evaluations (JL) was trained and calibrated by an experienced examiner. Six voluntary patients who did not participate in the study were used in this calibration process. The interexaminer agreement was 99.5% and the intraexaminer agreement was 99.6%, showing an excellent reproducibility.[18]

Two quadrants were randomly chosen by coin flip: in one of the quadrants, only SRP was conducted (control side), and on the test side, SRP was followed by a single subgingival application of 0.8% HA (Gengigel®, Ricerfarma, Italy) on all of the surfaces which presented periodontal pockets. The protocol for application of HA was as follows: the area was anesthetized with lidocaine 2% (Xylocaine, Dentsply), SRP of the affected surfaces was performed, hemostasis achieved applying pressure with a surgical dressing for 3 min, a needle was introduced inside the periodontal pocket, and the gel was deposited in an apicocoronal direction avoiding the incorporation of air bubbles. Oral hygiene instructions were given to the participants, and they were also instructed to refrain from using mouth rinses. After the application of HA, patients were instructed also to refrain from eating, drinking, and rinsing for at least 30 min.

The clinical outcomes measured in this trial were as follows: full mouth plaque score (FMPS), gingival index (GI), bleeding on probing (BoP), probing depth (PD), and clinical attachment level (CAL), all of which were determined using a CP-12 calibrated periodontal probe (Hu-Friedy, Chicago, IL, EUA). The variables FMPS and GI were assessed in 4 different surfaces (mesiobuccal, centrobuccal, distobuccal, and lingual) whereas the variables PD, BoP, and CAL were assessed in six surfaces (mesiobuccal, centrobuccal, distobuccal, distolingual, centrolingual, and mesiolingual). Three measures were taken, one before the treatment, one at 6 weeks, and the final one at 12 weeks after the end of nonsurgical treatment. The operator who performed the periodontal assessment was unaware of the treatment modality.

During the course of the study, no patients were lost. The statistical analysis comprised descriptive measures, such as absolute and relative frequencies, means and standard deviations and inferential statistics, where the intraclass correlation coefficients were calculated and T-student test for paired samples and Anova for repeated measures were applied. The Levene and Shapiro-Wilk were conducted to test for homoscedasticity and normality. When the assumptions were not satisfied, the Wilcoxon test was employed in alternative to T-student and Friedman test in replacement of Anova for repeated measures. The significance level was set to 5% (P< 0.05). Statistical Package for the Social Sciences (IBM, Armonk, NY, USA) version 22 was used for the statistical analysis.

RESULTS

This pilot study included 16 patients with ages between 41 and 68 years (mean of 55 years), with 38% of the participants belonging to the age group between 50 and 60 years of age, 37% between 40 and 50, and 25% between 60 and 70.

Since data did not follow a normal distribution (verified by the Shapiro–Wilk test), Friedman's test was employed. Considering the evolution of periodontal parameters in the control sides, from the first appointment (baseline) to the third appointment (12 weeks), there were no statistically significant alterations in the FMPS (P > 0.05) [Table 1], although statistically significant differences were found in the GI (17.1 ± 12.3 vs. 7.6 ± 9.6) [Table 2], BoP (33.2 ± 19.5 vs. 14.9 ± 8.9) [Table 3] with a mean decrease of 18.3%, PD (3.9 ± 0.6 vs. 3.3 ± 0.6) [Table 4] with a mean decrease of 0.6 mm, and the CAL (4.8 ± 1.2 vs. 4.3 ± 1.0) revealed a gain of 0.5 mm [Table 5].

Table 1

Changes in Plaque index after 6 and 12 weeks compared to baseline

	M1(SD)	M2(SD)	M3(SD)	P**
Control Side	25.7ab (17.5)	20.2bc (16.1)	16 . 8c (13.3)	0.034+
Test Side	20.6 (17.7)	23.1 (17.7)	16.5 (13.6)	0.193
P*	0.256#	0.371	0.915

* Control side vs. Test side comparison (paired samples t-Student test, except (#), where Wilcoxon test was used). ** Time comparison within sides (repeated measures ANOVA, except (+), where Friedman test was used) different lower case letters indicate significant differences between mean M1/M2/M3 (SD): Mean and standard deviation at baseline, 6 weeks and 12 weeks respectively

Table 2

Changes in Gingival index after 6 and 12 weeks compared to baseline

	M1(SD) (%)	M2(SD) (%)	M3(SD) (%)	P**
Control Side	17.1a (12.3)	6.2b (6.3)	7.6b (9.6)	0.005+
Test Side	8.9 (6.2)	10.7 (13.5)	4.0 (5.2)	0.130
P*	0.011	0.172#	0.209#

Control side vs. Test side comparison (paired samples t-Student test, except (#), where Wilcoxon test was used). Time comparison within sides (repeated measures ANOVA, except (+), where Friedman test was used) different lower case letters indicate significant differences between mean M1/M2/M3 (SD): Mean and standard deviation at baseline, 6 weeks and 12 weeks respectively

Table 3

Changes in Bleeding on Probing after 6 and 12 weeks compared to baseline

	M1(SD) (%)	M2(SD) (%)	M3(SD) (%)	P**
Control Group	33.2a (19.5)	21.1b (15.4)	14.9b (8.9)	0.004+
Test Group	32.1a (17.3)	15.8b (11.9)	9.4c (4.0)	< 0.001
P*	0.811	0.101	0.009

* Control side vs. Test side comparison (paired samples t-Student test). ** Time comparison within sides (repeated measures ANOVA, except (+), where Friedman test was used) different lower case letters indicate significant differences between mean M1/M2/M3 (SD): Mean and standard deviation at baseline, 6 weeks and 12 weeks respectively

Table 4

Changes in Pocket Depth after 6 and 12 weeks compared to baseline

	M1(SD) (mm)	M2(SD) (mm)	M3(SD) (mm)	P**
Control Group	3.9a (0.6)	3.4b (0.7)	3.3b (0.6)	<0.001
Test Group	3.8a (0.6)	3.3b (0.6)	3.2b (0.5)	< 0.001
P*	0.379	0.506	0.303

* Control side vs. Test side comparison (paired samples t-Student test). ** Time comparison within sides (repeated measures ANOVA) different lower case letters indicate significant differences between mean M1/M2/M3 (SD): Mean and standard deviation at baseline, 6 weeks and 12 weeks respectively mm: milimeters

Table 5

Changes in Clinical attachment loss after 6 and 12 weeks compared to baseline

	M1(SD) (mm)	M2(SD) (mm)	M3(SD) (mm)
Control Group	4.8a (1.2)	4.4b (1.1)	4.3b (1.0)	0.005
Test Group	4.9a (1.1)	4.3b (0.9)	4.2b (1.0)	< 0.001
	0.702	0.667	0.445

* Control side vs. Test side comparison (paired samples t-Student test) ** Time comparison within sides (repeated measures ANOVA) different lower case letters indicate significant differences between mean M1/M2/M3 (SD): Mean and standard deviation at baseline, 6 weeks and 12 weeks respectively mm: milimeters

In the test side, no statistically significant difference was found from the baseline to the third appointment for the FMPS (P > 0.05). Regarding the GI (8.9 ± 6.2 vs. 3.79 ± 5.17), BoP (32.1 ± 17.3 vs. 9.4 ± 4.0) with a mean reduction of 22.7%, PD (3.8 ± 0.6 vs. 3.2 ± 0.5) with a mean reduction of 0.6 mm, and CAL (4.9 ± 1.1 vs. 4.2 ± 1.0) with a mean gain of 0.7 mm, the differences between baseline and final evaluation were all statistically significant.

When comparing both test and control sides, the only statistically significant differences can be found in the third appointment regarding the BoP parameter (14.9 ± 8.9 vs. 9.4 ± 4.0) (P < 0.05) and in the first appointment GI parameter (17.1 ± 12.3 vs. 8.9 ± 6.2) (P < 0.05). Regarding the PD and CAL, no statistically significant differences were found, although the values improved over time.

During the course of the study, no adverse effects attributable to the application of HA were detected.

DISCUSSION

The aim of this pilot study was to assess the clinical effects of a single application of 0.8% HA as a coadjuvant to SRP.

Previous studies have conflicting results with some of them showing a significant improvement in several periodontal parameters,[1213] whereas other studies showed improvements only in some of the parameters such as PD and BoP[141516] or no effect at all.[1019] Previous systematic reviews described only the results of the existing studies due to the impossibility of carrying out a meta-analysis.[720] Despite the existence of studies reporting the use of HA in the treatment of periodontal disease, the biological mechanisms involved are poorly understood.[21]

The different methodologies of previous studies, namely in the periodontitis case definition, inclusion criteria, treatment frequency, follow-up periods, product concentration, and outcome measures[13141516] make comparisons difficult and justify the execution of additional studies with a well-defined methodology before recommending routine clinical utilization. A clinical trial featuring split-mouth design allows a more precise estimation of the outcomes and effect of the treatment, even in smaller samples, since patients are used as their own control comparison.[22]

The results of the present study are in accordance with the first ones since reductions were found in all of the outcomes measures, even though some of them were not statistically significant. It is important to point out that the mean age of the participants was 55 years since it has been documented that an increase in age has an effect on the overall capacity of wound healing, making it slower, due to the delay in inflammatory and proliferative response.[23] Both in the control and test sides, all of the outcomes (FMPS, GI, BoP, CAL, and PD) improved from baseline up to the 12th week, which confirms the efficacy of the nonsurgical treatment of periodontitis.[161924] When a comparison of test and control sides was made, only BoP showed statistically significant differences. These findings suggest that the HA's anti-inflammatory and bacteriostatic properties might play a role in reduction of periodontal inflammation and bleeding.[920] These results are also corroborated by other studies.[24] By analyzing in detail the CAL and PD, it is interesting to note that the CAL was higher in the test sites at baseline and PD was lower, when compared to the control sites, allowing us to deduce that gingival recession was higher in the test sites. CAL reduction in the test sites was higher and PD did not show any significant improvement over time, and an improvement of the gingival recession was noticed. These results are probably due to the regenerative effect that HA can exhibit, by promoting adhesion, migration, proliferation, and cellular activation since it can store and deploy growth factors. By having these properties, HA's contribution in the formation of an extracellular matrix, tissue organization, and fixation of the gingival epithelium to the basal lamina is possible.[2526]

Regarding the limitations of this study, we highlight the short follow-up period and the fact that it was a single application of HA, which might be insufficient. Increasing the application frequency would theoretically increase HA's beneficial properties. The effect on smokers should also be evaluated since tobacco is one of the most important risk factors for periodontal disease and can counterbalance the beneficial effects of HA.

CONCLUSION

At the end of the observation period, an improvement of all of the clinical periodontal parameters was observed in both test and control sides. When both sides were compared, only BoP showed statistically significant differences, which might be attributed to the anti-inflammatory properties of HA.

Financial support and sponsorship

This study was financially supported by Gengigel® (Ricefarma).

Conflicts of interest

There are no conflicts of interest.