Evaluation of Adiponectin and Resistin Levels in Gingival Crevicular Fluid in Patients with Chronic Periodontitis Before and After Treatment: A Clinico-Biochemical Study.

AIM: The aim of this study was to find out the association of adiponectin and resistin levels in gingival crevicular fluid (GCF) with the clinical parameters before and after scaling and root planing in periodontitis patients.
METHODS: A total of 50 patients were recruited from the Outpatient Department of Periodontics, JKK Nattraja Dental College and Hospital, Komarapalayam, Tamil Nadu, India. The protocol was reviewed and approved by the institutional ethical board. Informed consent was obtained from the patients and the study-related procedures were explained. Patients were divided into two groups. Group I (25 patients) consisted of healthy patients. Group II (25 patients) consisted of periodontitis patients. The periodontitis patients after treatment were considered as Group III. GCF samples were collected before treatment and 21 days after treatment. The levels of adiponectin and resistin were evaluated using Immunotag™ Human RETN enzyme-linked immunosorbent assay (ELISA) kit (ITEH0269) and Immunotag™ Human ADP (adiponectin) ELISA kit (ITEH2593). The levels were correlated with the clinical parameters.
RESULTS: There were an increase in the mean adiponectin level and a decrease in the mean resistin level at the end of periodontal therapy.
CONCLUSIONS: Adiponectin which is an anti-inflammatory component was found to increase after periodontal therapy, and resistin which is an inflammatory component has been decreased after periodontal therapy. Copyright:

INTRODUCTION

Chronic periodontitis is an infectious disease affecting the supporting tissues of the teeth that may result in tooth loss if untreated.[1] Periodontitis is usually caused by interactions between many factors such as bacteria and their byproducts, immune cells, and inflammatory mediators. The activation of host immune inflammatory process by the bacterial products initiates the release of inflammatory mediators such as cytokines, chemokines, adipokines, and arachidonic acid metabolites which play an important role in disease progression and tissue destruction.[2]

Adipokines are a group of biologically active molecules produced by the adipose tissue. Adipokines such as adiponectin and resistin play an important role in periodontal inflammation. These adipokines and cytokines have been shown to play a vital role in the host response to infection and periodontal inflammation.[3] Adiponectin exhibits its anti-inflammatory property by stimulating interleukin-10 (IL-10) and heme oxygenase-1 which are known to be anti-inflammatory and can inhibit the lipopolysaccharide (LPS)-stimulated nuclear factor-kappa B (NF-κB) nuclear translocation. It also inhibits the production of pro-inflammatory and proteolytic molecules in periodontal cells.[4] Resistin increases the level of pro-inflammatory cytokines (IL-1, IL-6, tumor necrosis factor-alpha [TNF-α], IL-12, and monocyte chemoattractant protein-1) in human peripheral blood mononuclear cells, macrophages, and hepatic stellate cells via the NF-κB pathway. This shows that resistin may have a major role in inflammation.[5]

The primary objective of nonsurgical periodontal therapy is to achieve gingival health by completely removing elements that are responsible for the gingival inflammation (i.e. plaque, calculus, and endotoxins) in the oral environment. Both hand instruments and ultrasonic instruments are capable of dramatically reducing the numbers of subgingival microorganisms. Despite various treatment options, nonsurgical periodontal therapy still remains the gold standard of the periodontal treatment regimens.[6]

The literature is with limited information with respect to the association of clinical parameters with adiponectin and resistin levels in gingival crevicular fluid (GCF). The evaluation of adiponectin and resistin in GCF of chronic periodontitis patients could be used as a biomarker for prediction of active stage of the disease and periodontal destruction. However, the association between adiponectin and resistin levels in GCF and clinical parameters has not been studied in detailed yet.

Hence, this clinico-biochemical study was designed to estimate the levels of adiponectin and resistin in GCF of patients with clinically healthy periodontium and chronic periodontitis patients before and after scaling and root planing (SRP).

MATERIALS AND METHODS

The research protocols and guidelines were reviewed and approved by the Ethical Committee of JKK Nattraja Dental College and Hospital, Komarapalayam, Tamil Nadu, India. Informed consent was obtained from the patients, and the study-related procedures were explained. The study participants were recruited from the patients who came to the department of periodontics.

Patient selection

A total of 50 patients were selected in which 25 patients were periodontal healthy. Twenty-five periodontal patients were based on the following inclusion criteria:

Minimum of 20 teeth to be present in every patient

Minimum of 2 sites with >5 mm of pocket depth as assessed by Williams periodontal probe

Minimum of 6 sites which show bleeding on probing

Individuals who had not received any periodontal treatment in the last 6 months

Systemically and periodontally healthy individuals with probing depth (PD) <3 mm for the healthy group.

Study design

Group I (healthy) consists of 25 patients with clinically healthy periodontium. Group II (chronic periodontitis) consists of 25 patients with clinical signs of gingival inflammation and PD ≥5 mm. Group III (after treatment group) consists of 25 patients of Group II treated by SRP.

Clinical parameters

The following variables plaque index (PI) (Silness P and Loe H, 1964), gingival index (GI) (Loe H and Silness P, 1963), sulcus bleeding index (SBI) (Muhlemann HR and Son S, 1971), probing pocket depth, and clinical attachment level (CAL) were measured at baseline and at 21 days after SRP.

Procedure for gingival crevicular fluid collection

The test site selected for sampling was air-dried, isolated with cotton roll, and supragingival plaque was removed without touching marginal gingiva. Samples of GCF were obtained by placing 1–5 μL calibrated volumetric microcapillary pipettes which were obtained from Sigma-Aldrich Chemicals Company Limited, USA [Figure 1]. From each test site, a standardized volume of 2 μL was collected using the calibration on micropipette and placing the tip of the pipette intracrevicularly for 30 s. GCF contaminated with saliva and blood was discarded. Samples of GCF were collected at the initial visit in Group I and Group II patients, as shown in Figure 2. Samples collected after 21 days of treatment were considered for Group III patients. Each GCF sample was immediately placed into a sterile, labeled Eppendorf tube and placed on ice and then transported to the laboratory for processing.

Figure 1

Microcapillary pipettes and Eppendorf tubes used for gingival crevicular fluid collection

Figure 2

Gingival crevicular fluid sample collection

Nonsurgical periodontal therapy

Periodontal treatment (SRP) was performed for periodontitis patients at the same appointment after GCF collection. At baseline, full-mouth ultrasonic scaling was done. Under local anesthesia with 2% lignocaine solution (1:80,000), root planing was done using area-specific double-ended Gracey curettes (Hu-Friedy, USA), as shown in Figure 3. After 21 days, GCF was collected from the same site of the patients in Group III. Clinical parameters were repeated after 21 days.

Figure 3

Scaling and root planing

Biochemical evaluation of adiponectin and resistin using enzyme-linked immunosorbent assay

The samples were then assayed for adiponectin and resistin levels by using enzyme-linked immunosorbent assay kit for adiponectin (ITEH2593) and resistin (ITEH0269) from Immunotag™, as shown in Figures 4 and 5, respectively.

Figure 4

Adiponectin ELISA kit

Figure 5

Resistin ELISA kit

Statistical analysis

The results obtained were analyzed statistically, and comparisons were made between groups using paired samples t-test. “P value” between Group II and Group III postoperatively was evaluated. Further Pearson correlation analysis was done to identify the correlation coefficient between the levels of adiponectin, resistin, and clinical parameters. P < 0.001 denotes statistically significant and also denotes <1 in a thousand chance of being wrong. P < 0.05 denotes that there is only 5% chance that the null hypothesis is true. The statistical analysis was done using SPSS software version 19, IBM Company, Armonk, New York, USA.

RESULTS

The PI score in Group II was 2.86 ± 0.57 and reduced in Group III to 1.76 ± 0.24, as shown in Table 1. The comparison of PI scores between Group II and Group III shows a statistically significant difference with P < 0.001.

Table 1

Comparison of clinical parameters between Group II and Group III

Indices	Group II	Group III	P
PI	2.86±0.57	1.76±0.24	<0.001*
GI	2.62±0.63	1.73±0.36	<0.001*
SBI	3.47±0.45	1.92±0.52	<0.001*
Probing depth (mm)	5.37±0.43	3.24±0.54	<0.001*
CAL (mm)	4.83±0.32	2.67±0.41	<0.001*

*Statistically significant at 1% level. PI: Plaque index, GI: Gingival index, SBI: Sulcus bleeding index, CAL: Clinical attachment level

The GI score in Group II was 2.62 ± 0.63 and reduced in Group III to 1.73 ± 0.36, as shown in Table 1. The comparison of GI scores between Group II and Group III shows a statistically significant difference with P < 0.001.

The SBI score in Group II was 3.47 ± 0.45 and reduced in Group III to 1.92 ± 0.52, as shown in Table 1. The comparison of SBI scores between Group II and Group III shows a statistically significant difference with P < 0.001.

The PD score in Group II was 5.37 ± 0.43 and reduced in Group III to 3.24 ± 0.54, as shown in Table 1. The comparison of PD scores between Group II and Group III shows a statistically significant difference with P < 0.001.

The CAL score in Group II was 4.83 ± 0.32 and reduced in Group III to 2.67 ± 0.41, as shown in Table 1. The comparison of CAL scores between Group II and Group III shows a statistically significant difference with P < 0.001.

The mean adiponectin level in Group I was 20.18 ± 0.26 ng/ml. The mean adiponectin level in Group III was 15.46 ± 0.32 ng/ml and Group II was 9.96 ± 0.27 ng/ml, as shown in Table 2. A statistically significant difference was observed between Group II and Group III with P < 0.05.

Table 2

Comparison of adiponectin levels between groups

Adiponectin level (ng/ml)	Mean±SD	Comparison	P
Group I	20.18±0.26	Group I versus Group II	<0.05*
Group II	9.96±0.27	Group II versus Group III	<0.05*
Group III	15.46±0.32	Group I versus Group III	>0.05

*Statistically significant at 5% level; P>0.05 denotes statistically insignificant at 5% level. SD: Standard deviation

Further analysis was carried out to find out which pairs differ significantly at 5% level of significance. A statistically significant difference was observed between Group II and Group III and between Group I and Group II with P < 0.05. A statistically insignificant difference was obtained when the mean levels of adiponectin were compared between Group I and Group III with P > 0.05. The results suggested that adiponectin levels decreased progressively in GCF from health to periodontitis and increased after periodontal therapy.

The mean resistin level in Group I was 7.19 ± 0.38 pg/ml. The mean level of Group II was 18.12 ± 0.43 pg/ml and Group III was 11.28 ± 0.39 pg/ml, as shown in Table 3. A statistically significant difference was observed between Group II and Group III with P < 0.05.

Table 3

Comparison of Resistin levels between groups

Resistin level (pg/ml)	Mean±SD	Comparison	P
Group I	7.19±0.38	Group I versus Group II	<0.05*
Group II	18.12±0.43	Group II versus Group III	<0.05*
Group III	11.28±0.39	Group I versus Group III	>0.05

*Statistically significant at 5% level; P>0.05 denotes statistically insignificant at 5% level. SD: Standard deviation

Further analysis was carried out to find out which pairs differ significantly at 5% level of significance. A statistically significant difference was observed between Group II and Group III and between Group I and Group II with P < 0.05. A statistically insignificant difference was obtained when the mean levels of resistin were compared between Group I and Group III with P > 0.05. The results suggested that resistin levels increased progressively in GCF from health to periodontitis and decreased after periodontal therapy.

DISCUSSION

Periodontitis is a chronic inflammatory state caused by the interactions between pathogenic bacteria and host defense system. Bacterial byproducts such as LPS, antigen, and virulence factors enter the gingival tissue to initiate the inflammatory reaction. As a result of this interaction, inflammatory mediators such as cytokines, chemokines, adipokines, and arachidonic acid metabolites are released, which play an important role in disease progression and tissue destruction.

Adipokines are a group of biologically active molecules produced by the adipose tissue. Adipokines such as adiponectin and resistin play an important role in periodontal inflammation.

In the present study, the mean levels of adiponectin in GCF were found to decrease progressively from health to periodontitis and later increased after treatment which is statistically significant. The results in our study were in accordance with Furugen et al.[7] who found that there were decreased levels of adiponectin in patients with periodontitis. Adiponectin levels were negatively associated with leukocyte counts which show that adiponectin was an anti-inflammatory mediator.

According to Tilg and Moschen,[8] expression of adiponectin was controlled by other pro-inflammatory mediators like IL-6 which inhibits adiponectin transcription and translation in adipocytes. Adiponectin induces the production of anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonist by human monocytes, macrophages, and dendritic cells and inhibits the release of interferon-γ by LPS-stimulated human macrophages. Through its receptor ADIPOR1, globular adiponectin inhibits TLR-induced NF-κB activation which shows that adiponectin negatively regulates macrophage responses to TLR ligands which is of importance in innate immune responses.

The findings of our study were in line with that of Ling et al.[9] who found that there was a significant decrease in serum adiponectin in chronic periodontitis compared to that of healthy controls. The increase in the levels of adiponectin after SRP could be due to the decrease of pro-inflammatory cytokines such as IL-6 and TNF-α and elevation of inducible factors such as IL-10 and IL-1RA which favor the anti-inflammatory role for adiponectin.

The correlation of adiponectin levels with the five clinical parameters of periodontal status was investigated. The adiponectin level was negatively correlated with PI, GI, and SBI scores in Group II indicating that the adiponectin levels are associated with the degree of periodontal tissue inflammation. This was in accordance with the study conducted by Ling et al.[9]

In the present study, the mean levels of resistin in GCF were found to increase progressively from health to periodontitis later decreased after treatment which is statistically significant.

The increase in resistin levels observed in our study was in accordance with Saito et al.[10] who found an increased level of serum resistin in association with periodontitis. It may be due to local involvement of monocytes and macrophages in periodontal inflammation. The increase in circulating resistin levels induced by periodontal disease might be derived from both periodontal and adipose tissues.

It was also in accordance with the study done by Karam and Al-Safi[11] who reported an increase in serum resistin and salivary resistin with conversion from periodontal health state to periodontal disease state. Periodontopathic bacteria produce LPS and/or leukotoxin which increases the circulating resistin concentration. Inflammatory cells such as monocytes and macrophages seen in the periodontal tissue appear to be the chief source of resistin. LPS produced by Porphyromonas gingivalis can induce resistin release from neutrophils. There was an association of periodontal conditions with serum resistin levels since its levels were dramatically increased by LPS stimulation in humans.

Our results with resistin were in accordance with Furugen et al.[7] who observed an increase in the serum resistin levels in patients with periodontitis when compared with controls. Resistin expression increases along with the maturation of monocytes into macrophages. LPS from periodontal pathogenic bacteria influences adipose tissues and macrophages through inflammatory cytokines which cause an increase in the resistin level.

The findings of our study were in line with that of Suresh et al.[12] who found that there was a significant decrease in GCF resistin levels in chronic periodontitis after treatment. The decrease in the levels of resistin after SRP could be due to the reduced expression of resistin by polymorphonuclear leukocytes and macrophages in inflammatory conditions. This reduces the periodontal inflammation and thereby reduces the pro-inflammatory cytokines such as IL-12 and TNF-α leading to decrease in GCF resistin levels.

The correlation of resistin levels with the five clinical parameters of periodontal status was investigated. The resistin level was positively correlated with PI, GI. and SBI scores in Group II indicating that resistin levels are associated with the degree of periodontal tissue inflammation. This was in accordance with the study conducted by Patel and Raju.[13] Moreover, the resistin levels were also positively associated with PD and CAL in Group III indicating that the resistin levels are positively associated with the degree of periodontal tissue destruction.

CONCLUSIONS

Based on the results obtained from our study, we can conclude that nonsurgical periodontal therapy (SRP) resulted in a statistically significant increase in adiponectin and a decrease in resistin in GCF proportionally. In addition, it was also found that all the clinical parameters of periodontal status also markedly improved, confirming its active role in periodontal attachment loss.

Since our data show that all the GCF samples tested positive to adiponectin and resistin, it can be considered as a biomarker of periodontal disease progression. However, controlled longitudinal, prospective studies involving larger population and more solid-phase assays are needed to verify this possibility.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.