Local delivery of hyaluronic acid as an adjunct to scaling and root planing in the treatment of chronic periodontitis in smokers and non-smokers: A clinical and microbiological study

Abstract

Objective:

The objective of the study is to assess the effects of adjunctive use of hyaluronic acid (HA), clinically and microbiologically in smokers and nonsmokers, in the management of chronic periodontitis.

Material and Methods:

48 sites from 24 chronic periodontitis patients, including smokers and nonsmokers with probing depth >5 mm, were selected for the study. A split-mouth design was followed. Scaling and root planing (SRP) was performed for all the patients. The test sites received 0.8% HA gel whereas the control site received placebo gel. The clinical parameters plaque, gingival and bleeding indices, Pocket Probing Depth (PPD), and clinical attachment level (CAL) were evaluated at baseline, 1 and 3 months. Microbiological parameters were evaluated at baseline and 1 month.

Results:

The results showed reduction in PPD and gain in CAL in both smokers and nonsmokers at the end of 3^rd month. The improvements in the test sites were statistically significant when compared with that of control sites. The microbiological analysis showed a significant reduction in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis at the test sites when compared to the controls in both the groups.

Conclusion:

The findings in both groups suggest that the use of locally delivered 0.8% HA gel may be valuable as an adjunct to SRP in the treatment of chronic periodontitis.

INTRODUCTION

Periodontal disease is considered as the outcome of an imbalance in the host-parasite interaction. Gram negative bacteria including Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis have been found consistently in periodontal lesions.[1]

Scaling and root planing (SRP) has been established to be effective in reducing gingival inflammation and probing depths. During recent years, additional improvements in clinical parameters have been noted when SRP is combined with nonmechanical therapies.[2] Local drug deliveries are used as an adjunct to SRP, maintenance therapy and for whom surgery is not indicated or those who refuse surgical treatment.[3]

Hyaluronan, presents in the extracellular matrix, plays a role in tissue hydrodynamics, cell migration, proliferation, and bacteriostatic action.[2] It also has an anti-inflammatory action by inhibiting tissue destruction and facilitating healing. Hence, it has gained popularity as a local chemotherapeutic agent in periodontal treatment.[4]

The development of periodontitis is influenced by many factors, among them cigarette smoking is an important known risk factor.[5] Current smokers have 3.1 times more chances of being infected with Aa and Pg and also it is more difficult to eliminate these bacteria in smokers.[6] Thus, the present study was carried out to evaluate clinically and microbiologically, the effects of local application of hyaluronic acid (HA) in the management of chronic periodontitis in smokers and nonsmokers.

MATERIALS AND METHODS

This was a randomized controlled trial in which 24 patients who have given consent and fulfilling inclusion criteria were selected by purposive sampling, from the outpatients presenting to the Department of Periodontology. It included patients of both genders in the age group of 35–65 years of which 12 were smokers and 12 were nonsmokers (Based on Centers for Disease Control classification for smokers). The study protocol was accepted and cleared by the Institutional Ethical Committee and was conducted in accordance with the Helsinki Declaration of 1975. A written informed consent was obtained from all the patients before the study.

Chronic periodontitis patients with ≥20 remaining teeth and contralateral pockets measuring ≥5 mm with evident CAL and bone loss shown in radiograph were selected. A person who smoked on an average of 15 cigarettes/day for a minimum of past 2 years was considered as a smoker and a person who has never smoked a cigarette in his life was considered nonsmoker.

Patients who had undergone any periodontal therapy or taken any antibiotics in the past 6 months, pregnant/lactating women, and patients having systemic diseases were excluded.

Sample size

Based on previous study results,(3) using power of 90% with a mean difference of 3 and standard deviation 1.4, calculated sample size was found to be 24 with 48 sites (including attrition rate of 20%). Sample size was calculated using the formula for “Comparison of Means,” i.e.,

Materials: Gengigel (Ricerfarma), which contains high-molecular-weight of HA, present as sodium hyaluronan (molecular weight: 1.03 ± 0.01 g/cm³ at 20°C) was the test gel.

The placebo gel was prepared in Pharmacy College of our institution using Hydroxypropyl Methylcellulose E-15 (5%) powder, cellulose gum, and deionized water and sterilized by autoclave at 121°C and ultraviolet radiation.

Both the test and placebo gels were packed in similar syringes with their respective labels for individual use by a third person.

Intra-examiner calibration

A single examiner recorded all the parameters. Before the study, 20 sites were examined twice, 24 h apart to achieve intra-examiner. A difference within 1 mm at the 95% level, at baseline, and 24 h was accepted.

Clinical procedure

All the subjects were categorized as smokers and nonsmokers. In each patient, the treatment sites were randomly divided into the test and control sites by computer generated random table [Figure 1].

Figure 1.

Flowchart showing selection of study population. n – number of patients; SRP – Scaling and Root Planing; HA – Hyaluronic acid

1. Site A (Test site): SRP followed by local application of HA gel.

2. Site B (Control site): SRP followed by application of placebo gel.

A double-blind, split-mouth design was considered for the study. Each of the sites was assessed for the following clinical parameters at baseline, 1 and 3 months: plaque index,[7] gingival index, bleeding on probing, PPD using UNC-15 probe, and Clinical attachment level (CAL) by Ramjford method.[8]

Acrylic stents were used to guide the probe penetration vertically in the same plane every time it was inserted for recording the measurements. The lower margin of the stent was used as fixed reference point (FRP) and clinical measurements were recorded at proximal line angle of the tooth [Figure 2]. The following measurements were recorded at baseline and all recall intervals:

Figure 2.

Measurement of pocket probing depth and clinical attachment level using acrylic stent

1. Stent (FRP) to Cemento-enamel Junction (CEJ)

2. Stent (FRP) to Gingival Margin (GM)

3. Stent (FRP) to base of the pocket (BP).

Based on the above measurements PPD and CAL were calculated as follows:

1. PPD = FRP to BP– FRP to GM

2. CAL = FRP to BP– FRP to CEJ

Two weeks after supragingival scaling, the patient was recalled after 2 weeks. Clinical parameters were recorded and subgingival plaque was collected. Full mouth root planing was done using area-specific gracey curettes under local anesthesia to prevent cross infection. “Gengigel” was placed in the test site and placebo gel was placed in the control site. Both the gels, “Gengigel” in the test site and placebo in the control site were inserted deep into the periodontal pocket using a 24 gauge needle [Figure 3] and periodontal dressing was given. Oral hygiene instructions included only normal brushing and rinsing. At 1-week interval, both the gels were re-inserted into the pocket after recording indices. Clinical parameters were recorded at 1 and 3 months; microbiological parameters were recorded at only 1 month.

Figure 3.

Insertion of gengigel at the test site

Microbiologic analysis

The collection of sample was done in the Department of Microbiology. Gracey curette was used to obtain subgingival plaque sample. It was inserted sub-gingivally parallel to long axis of the tooth and scraped coronally. Two samples were collected from each patient from two sites. Each sample was separately mixed in the thioglycolate broth and divided into 2, for identification of Aa and Pg. Blood agar and Tryptone Soya Serum Bacitracin Vancomycin agar along with other biochemical tests were used for identification of P. g and A. a, respectively [Figures 4 and 5]. The Colony-forming unit per ml was counted and they were categorized as: 1+ =10,000–25,000 CFU/ml, 2+ = 25,000–50,000 CFU/ml, 3+ = 50,000–75,000 CFU/ml and 4+ ≥75,000 CFU/ml.

Figure 4.

Microbiological analysis of Porphyromonas gingivalis; (a and b); Statistically significant reduction in the growth of black colonies on blood agar indicating reduction of Porphyromonas gingivalis at 1 month; (c and d); Porphyromonas gingivalis in gram stain smear can be identified as a gram-negative, nonmotile, that usually exhibit coccal to short rod morphology

Figure 5.

Microbiological analysis of Aggregatibacter actinomycetemcomitans; (a and b); Statistically significant reduction in the growth of white colonies on Tryptic Soy-serum-Bacitracin-Vancomycin agar indicating reduction of Aggregatibacter actinomycetemcomitans at 1 month; (c and d); Aggregatibacter actinomycetemcomitans in gram stain smear can be identified as small, nonmotile, gram-negative and round ended rod

Statistical analysis

The Statistical Package for Social Science (IBM SPSS, version 18.5, USA) for Windows software was used. The normality of the data was assessed using Shapiro–Wilk test. The data for indices, PPD, and CAL were not normally distributed so comparison between the groups was carried out using nonparametric tests. Mann–Whitney test was applied to find the intergroup difference in indices, PPD and CAL. The Chi-square test was used to determine the intergroup differences in colony counts of Aa and Pg. P < 0.05 were considered significant.

RESULTS

A total of 48 sites in 24 patients (12 smokers and nonsmokers) were included in the study. Two patients (1 smoker and nonsmoker) were lost to follow-up; hence, 44 sites from 22 patients were analyzed. Postoperative healing was uneventful and no adverse complications were seen or reported during the procedure or at subsequent postoperative appointments. No significant difference was noted at baseline in any of the parameters assessed (P > 0.05).

In smoker and nonsmoker groups, a statistical increase in plaque, gingival, and balance of payments (BOP) scores was seen at 1 week, 1 and 3-month intervals in both the test and control sites (P < 0.05). However, there was no difference in the scores between the test and control sites at any time interval (P > 0.05). In both the test and control sites, no difference in the plaque, gingival, and BOP scores was seen between smokers and nonsmokers at any time interval (P > 0.05) [Table 1].

Table 1.

Comparison of indices at various intervals between test and control groups

	Baseline		1 week		1 month		3 months
	Control	Test	Control	Test	Control	Test	Control	Test
Smokers Group
Plaque index	0	0	0.068±0.162	0.027±0.090	0.455±0.688	0.455±0.445	0.727±0.720	0.545±0.568
P*	1.000		0.748		0.438		0.519
Gingival index	0	0	0.068±0.162	0.045±0.151	0.273±0.395	0.318±0.476	0.432±0.476	0.318±0.405
P*	1.000		0.748		0.898		0.562
Bleeding index	0	0	4.5±10.113	4.5±15.076	18.2±22.613	13.6±17.189	29.5±21.847	20.5±26.968
P*	1.000		0.797		0.748		0.300
Nonsmokers Group
Plaque index	0	0	0.068±0.117	0.068±0.162	0.455±0.590	0.432±0.593	0.545±0.522	0.545±0.522
P*	1.000		0.797		0.949		1.000
Gingival index	0	0	0.068±0.162	0.068±0.162	0.318±0.298	0.318±0.389	0.455±0.472	0.477±0.480
P*	1.000		1.000		0.797		0.949
Bleeding index	0	0	6.8±16.167	6.8±16.167	22.7±23.597	25.0±22.361	40.9±37.538	38.6±37.689
P*	1.000		1.000		0.847		0.898

Mann-Whitney test, *P<0.05 considered statistically significant. Highlighted values indicate statistically significant values

Within the smoker and nonsmoker groups, there was no statistical difference in the pocket probing depth (PPD) values between the test and control sites at any time interval (P > 0.05) [Table 2]. Within the test sites, there was no statistical difference in the PPD between smokers and nonsmokers at baseline and 3 months. At 1 month, there was significant alteration between smokers and nonsmokers, with smokers showing greater reduction (P < 0.05) [Table 3].

Table 2.

Comparison of clinical parameters at various intervals between control and test groups

	Baseline			1 month			3 months
	Control		Test	Control		Test	Control		Test
Smokers Group
PPD	7.00±1.673		6.55±1.036	5.27±1.737		3.91±1.221	5.36±1.567		4.36±1.362
P*		0.606			0.056			0.116
CAL	4.36±1.629		4.18±1.401	3.73±1.191		2.27±1.009	3.82±1.168		2.64±0.809
P		0.898			0.010*			0.019*
Nonsmokers Group
PPD	5.73±1.009		6.45±1.128	3.27±1.191		2.73±1.104	3.55±1.440		4.27±1.272
P*		0.116			0.300			0.193
CAL	2.45±1.036		3.27±1.168	2.00±0.775		1.64±1.027	2.27±0.905		1.91±0.831
P*		0.300			0.401			0.401

Mann-Whitney test, P<0.05 considered statistically significant. PPD - Pocket probing depth; CAL - Clinical attachment level

Table 3.

Comparison of clinical parameters at various intervals between smokers and nonsmokers

	Baseline			1 month			3 months
	Smokers		Nonsmokers	Smokers		Nonsmokers	Smokers		Nonsmokers
Control Group
PPD	7.00±1.673		5.73±1.009	5.27±1.737		3.27±1.91	5.36±1.567		3.55±1.440
P		0.076			0.007			0.013
CAL	4.36±1.629		2.45±1.036	3.73±1.191		2.00±0.775	3.82±1.168		2.27±0.905
P		0.003			0.001				0.004
Test Group
PPD	6.55±1.036		6.45±1.128	3.91±1.221		2.73±1.104	4.36±1.362		4.27±1.272
P		0.898			0.023			0.898
CAL	4.18±1.401		3.27±1.618	2.27±1.009		1.64±1.027	2.64±0.809		1.91±0831
P		0.193			0.193			0.088

Mann-Whitney test, P<0.05 considered statistically significant. PPD - Pocket probing depth; CAL - Clinical attachment level

Within the smoker group, there was no statistical difference in the CAL scores between test and control sites at baseline (P > 0.05). At 1 and 3 months, there was a significant alteration between test and control sites, with the test sites showing greater CAL gain (P < 0.05). Within the nonsmoker group, there was no statistical alteration in the CAL scores between test and control sites at any time interval (P > 0.05) [Table 2].

Within the control sites, there was statistical difference in the CAL scores between smokers and non-smokers at baseline, 1 and 3 months, with the smokers showing greater CAL gain (P < 0.05). Within the test sites, there was no statistical difference in the CAL scores between smokers and nonsmokers at any time interval (P > 0.05) [Table 3].

Within the smoker and nonsmoker groups, there was statistical difference in the colony counts of Aa and Pg between the test and control sites at 1 month, with more reduction seen in the test group (P < 0.05). In the intergroup comparison between smokers and nonsmokers, within the test and control sites, respectively, there was no statistical difference in the colony counts of Aa and Pg at 1 month (P > 0.05) [Table 4].

Table 4.

Comparison of colony counts of aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis arious intervals between control and test groups

	Baseline		1 month
	Control	Test	Control	Test
Smokers Group
Aa
0	1	1	2	1
1+	1	3	4	10
2+	7	6	5	0
3+	2	1	0	0
4+	0	0	0	0
P	0.703		0.019*
Pg
0	0	0	1	1
1+	0	0	1	8
2+	4	6	9	2
3+	4	5	0	0
4+	3	0	0	0
P	0.173		0.007*
Nonsmokers Group
Aa
0	0	0	1	2
1+	3	4	5	9
2+	7	6	5	0
3+	1	1	0	0
4+	0	0	0	0
P	0.896		0.039*
Pg
0	1	1	2	3
1+	2	3	4	8
2+	7	6	5	0
3+	1	1	0	0
4+	0	0	0	0
P	0.964		0.038*

Chi square test, *P<0.05 considered statistically significant. Highlighted values indicate statistically significant values. Aa - Aggregatibacter actinomycetemcomitans; Pg - Porphyromonas gingivalis

DISCUSSION

HA is a glycosaminoglycan made up of repeating disaccharide units of sugars N-acetyl-glucosamine and D-glucuronic acid, found in the connective tissue of vertebrates.[9] In our study, local application of HA gel was used an adjunct to SRP in the treatment of chronic periodontitis in smokers and nonsmokers.

A statistically significant increase was seen in the plaque scores, gingival scores, and bleeding on probing indices with no inter and intragroup differences over a period of 3 months in all the four groups (P > 0.05). The increase in plaque, gingival, and BOP scores is observed over a period of time, as the baseline for the present study is considered after supragingival scaling, the score being Zero at baseline.

Hyaluronan has been proven to reduce inflammation in subjects with gingivitis. Jentsch et al. 2003[10] and Pistorius et al. 2005[11] found a significant decrease in the plaque, bleeding, and gingival index scores using hyaluronan gel along with conventional oral hygiene for 3 weeks.

When the periodontal status was assessed, a significant decrease in the PPD was seen in all the four subgroups at 3 months as compared to baseline. The changes seen were more significant in the test sites as compared to the control sites (P < 0.05). The reduction in PPD seen was as follows, smoker test group (mean reduction = 2.19 mm) > nonsmoker test group (2.17 mm) > nonsmoker control group (2.08 mm) > smoker control group (1.64 mm).

This significant decrease in PPD is in accordance with Johannsen et al.,[2] who also found statistically significant improvement in the PPD scores when test (SRP + HA gel) and control (SRP alone) sites were compared. However, it contradicts Xu et al.[12] and Gontiya and Galgali[13] who did not find any significant improvement in the PPD scores between test (SRP + HA gel) and control (SRP alone) sites.

A significant CAL gain was seen in all the four subgroups at 3 months as compared to baseline. The CAL gain was maximum in the smoker test (mean gain = 1.54 mm) > nonsmoker-test (1.36 mm) > smoker control (0.54 mm) > nonsmoker control (0.18 mm). This is in contradiction to previous studies reported in the literature, where a significant CAL gain was seen in the test sites when compared to the control sites.

Variations between these studies may be due to differences in hyaluronan formulations, treatment protocols, observation intervals, severity of disease and measurements. Xu et al.[12] and Gontiya and Galgali[13] have used only 0.2% hyaluronan in their study, whereas Johannsen et al.[2] and the present study have used 0.8% hyaluronan. This could possibly explain the differences in the results obtained.

Formation of new collagen fibers, resolution of inflammatory exudates, and decrease in gingival inflammation leads to the changes in PPD and CAL.[14] Hyaluronan promotes cell proliferation, granulation tissue organization and provides a scaffold through its interaction with the fibrin clot. It plays a role in phagocytosis and killing of invading microbes by influencing the migration and adherence of polymorphonuclear leukocytes (PMNs) and macrophages at the site of inflammation. Furthermore, it helps in production pro-inflammatory cytokines by osteoblasts, cementoblasts, keratinocytes, and fibroblasts.[15]

In the present study, the test sites of both groups show a significant PPD reduction and CAL gain at 3 months, with no significant difference on intergroup comparisons between the test sites. Various trials[16] have shown greater changes in PPD and CAL following NSPT in nonsmokers compared to smokers. However, in regard to PPD reduction and CAL gain, the present study found no difference between the smokers and nonsmokers receiving HA gel.

Smoking affects recruitment and adhesion of fibroblast to root surfaces. Nicotine also reduces type 1 collagen and fibronectin. Tobacco smoking has been known to affect the functions of neutrophils, shifting the activities into more destructive direction.[17] The results of the present study indicate that probably all these physiological and immunological variations caused by smoking were combated by the exogenous HA gel, through its various beneficial actions in the periodontal pocket.

Microbial analysis

Within the smoker and nonsmoker groups, there was a statistical difference in the colony counts of Aa and Pg between the test and control sites, with more reduction seen in the test group (P < 0.05). However, there was no statistical difference in the found between smokers and nonsmokers (P > 0.05).

Pirnazar et al.[18] has shown that HA has a bacteriostatic effect on various bacterial strains, particularly on Aa and Pg. Thus, HA membranes, gels, and sponges can be used during surgical therapy to reduce bacterial contamination. Furthermore, it plays a role in phagocytosis and killing of invading microbes by influencing the migration and adherence of PMNs and macrophages at the site of inflammation. Thus, explaining the low bacterial counts at the sites receiving HA gel.

The effects of mechanical therapy are more pronounced in nonsmokers than smokers; this can be due to higher levels of residual Aa and Pg in the pockets after therapy.[19,20] However, in the present study, the test sites of both smokers and nonsmokers have responded equally well. This can probably be attributed to the bacteriostatic effects of HA gel in the periodontal pockets.

CONCLUSION

Hyaluronan gel can be used as an adjunct to SRP in the treatment of chronic periodontitis in smokers and nonsmokers to enhance the short-term outcome of nonsurgical treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.