Comparative Evaluation of Salivary Visfatin Levels in Healthy and Periodontally Diseased Patients before and after Scaling and Root Planing.

AIM: The aim of this study was to compare the levels of salivary visfatin in healthy controls, patients with gingivitis and patients with chronic periodontitis and also to assess the outcome of scaling and root planing (SRP) on the levels of salivary visfatin in patients with gingivitis and chronic periodontitis.
MATERIALS AND METHODS: A total of 48 patients, both males and females, in the age group of 25-50 years were enrolled into three groups, based on their clinical parameters: Group I (Healthy controls - 16), Group II (Patients with gingivitis - 16), and Group III (Patients with chronic periodontitis - 16). Groups II and III were examined 2 months after SRP. The clinical parameters that were recorded include plaque index, modified gingival index, gingival bleeding index, pocket depth, and clinical attachment level. The samples of saliva were collected from each patient and the levels of visfatin were evaluated using enzyme-linked immunosorbent assay kit.
RESULTS: The mean concentration of salivary visfatin at baseline was found to be highest in Group III and lowest in Group I. A statistically significant reduction (P < 0.01) in the mean concentration of salivary visfatin and the clinical parameters were observed after 2 months of SRP.
CONCLUSION: The levels of salivary visfatin can thus be considered as an inflammatory biomarker for periodontal diseases. However, future longitudinal prospective studies are needed to support these findings. Copyright:

INTRODUCTION

Periodontitis is defined as “an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms or groups of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with increased probing depth formation, recession, or both.”[1] In response to these infection causing microorganisms, the host releases several pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, and prostaglandin E2 (PGE2) which in turn promotes the action of matrix metalloproteinase (MMPs) in the periodontium, resulting in the degradation of connective tissue matrix and stimulation of alveolar bone resorption. These cytokines circulate to gingival crevicular fluid (GCF) and ultimately reach and accumulate in saliva,[2] and serve as possible salivary biomarkers of periodontal disease.[3]

Salivary biomarkers are now used for early detection, risk assessment, and monitoring of treatment response in periodontitis.[45] Clinically, saliva is a very useful biological liquid that contains various biomarkers such as IL-1b, IL-6, PGE2, and MMP-8 which provides added information than the classic clinical and radiographic results.[6] Unlike GCF, saliva can be collected easily by a noninvasive method. Thus, saliva can be used as a diagnostic tool for the assessment of periodontal disease.

Adipose tissue is a very active and complex metabolic endocrine organ[7] which expresses several adipokines and adipocytokines. Visfatin (pre-B-cell colony-enhancing factor) is a newly identified adipokine that is secreted by the visceral adipocytes. It was introduced by Fukuhara et al.[8] in 2005. It is a pro-inflammatory adipocytokine that is also produced by neutrophils following inflammatory stimuli.[9] It upregulates the release of several cytokines by monocytes. The multiple inflammatory actions of visfatin make this cytokine a suitable biomarker and as a diagnostic tool in periodontitis.

The aim of the present study was to compare the levels of salivary visfatin in healthy individuals and participants with gingivitis and chronic periodontitis and also to assess the effects of scaling and root planing (SRP) on the levels of salivary visfatin in participants with gingivitis and chronic periodontitis.

MATERIALS AND METHODS

From February 2015 to July 2016, 48 patients aged 25–50 years were selected from the outpatient section of the Department of Periodontics, PMS College of Dental Science and Research, Trivandrum, Kerala, India. All the patients provided their consent to the protocol. The Institutional Ethical Committee of PMS College of Dental Science and Research (IEC No. PMS/IEC/2014/04(A)) provided the ethical clearance.

The included patients had ≤20 natural teeth and no other diseases. The patients with the following conditions were excluded from the study: (1) received periodontal treatment in the past 6 months, (2) smoking and alcoholism, (3) chronic inflammatory diseases such as rheumatoid arthritis and bowel disease, (4) systemic diseases such as hypertension, diabetes mellitus (5) aggressive periodontitis, (6) consumption of antibiotics and anti-inflammatory drugs since 6 months, and (7) pregnancy and lactation.

Based on Plaque Index (PI), Modified Gingival Index (MGI), Gingival Bleeding Index (GBI), Probing Pocket Depth (PD), and Clinical Attachment Level (CAL), the patients were categorized into the following three groups: Group I: 16 (clinically healthy subjects who had no bleeding on probing(BOP), absence of gingival inflammation (GI=0), PD ≤ 3mm, and absence of clinical attachment loss (CAL=0));Group II: 16 (patients with gingivitis - BOP ≥10%, GI > 1, CAL =0 and PD ≤ 3 mm); Group III: 16 (patients with chronic periodontitis - GI > 1, PD ≥ 5mm in 30 % of sites, CAL > 3mm (AAP Classification) and radiographic evidence of alveolar bone loss). Group II and III patients received SRP and were examined after 2 months.

Periodontal examination

The periodontal status of all the patients was evaluated before performing SRP and again after 2 months, which included PI, MGI, GBI, PD, CAL, and gingival recession. A standard periodontal probe (PCP-UNC 15, Hu-Friedy, Chicago, IL, USA) was used to measure all clinical parameters.

Collection of saliva

Saliva samples were collected according to the circadian rhythm. Participants were refrained from eating or rinsing 60 min before the collection of saliva sample. The whole saliva was collected into a sterilized vial by letting the saliva accumulate in the mouth and then spitting out into an Eppendorf tube. About 2 ml of unstimulated saliva was collected from each participant and samples were immediately placed on cryobox maintained at −20°C and taken to the Biogenix Research Centre, Trivandrum, Kerala, and stored at −80°C until further determinations were done. Samples were thawed and analyzed within 6 months of collection.

Visfatin analysis

Each sample of saliva was pipetted into a fresh microcap tube and subjected to centrifugation for 10 min at 4000 revolutions per minute (rpm) at 4°C. The supernatant was transferred to clean microcap tubes using sterilized micropipette [Figure 1] and an enzyme-linked immunosorbent assay (ELISA) was performed immediately. The concentrations of visfatin were determined using an ELISA kit (Human Visfatin ELISA Kit, Bioassay Technology Laboratory). In the test well, 40 μl saliva samples, 10 μl visfatin antibodies, and 50 μl Streptavidin – HRP were added, covered with membrane, and incubated at 37°C for 60 min. About 30 ml of washing solution was prepared and the wells were washed five times with it. Human Visfatin Chromogen A (50 μl) and Chromogen B (50 μl) were added and shook gently, incubated for 10 min at 37°C. The color changed to blue. Stop solution (50 μl) was added to each well and blue color changed to yellow [Figure 2]. The values of the optical density were measured within 10 min.

Figure 1

Micropipette tips and micropipette

Figure 2

Addition of stop solution changes blue color to yellow

The absorbance of the substrate color reaction was read on ELISA reader 3 using 450 nm as the primary wavelength. The total visfatin was expressed in nanograms (ng), and its concentration in each sample was calculated by dividing the amount of visfatin by the volume of sample (ng/ml).

Statistical analysis

The intragroup and intergroup comparison of mean salivary visfatin levels and mean of outcome parameters were performed using paired and unpaired t-tests, respectively. For post hoc multiple comparisons between groups, Scheffe multiple comparisons along with ANOVA were performed. For all statistical evaluations, P < 0.01 was considered statistically significant.

RESULTS

The mean salivary visfatin level at baseline was found to be highest in Group III and lowest in Group I [Table 1]. The mean salivary visfatin level at baseline among three groups was 19.2, 30.7, and 56.8 pg/mL, respectively (P < 0.01). Comparison of salivary visfatin levels before and after SRP in Groups II and III, and intergroup comparisons after SRP are shown in Tables 2 and 3. After 2 months of performing SRP in Groups II and III, the mean visfatin concentrations reduced to 20.9 pg/mL in Group II and 24.8 pg/mL in Group III. The intergroup comparisons between Groups I and III, and Groups II and III after 2 months of SRP were statistically significant. There was no significant difference between Groups I and II.

Table 1

Comparison of salivary visfatin levels at baseline based on group

Group	n	Mean±SD	F	P	Scheffe multiple comparisons
					Pair	F	P
I	16	19.2±0.9	177.38**	0.000	I and II	15.6**	0.000
II	16	30.7±5.7			I and III	168.7**	0.000
III	16	56.8±8.0			II and III	81.8**	0.000

** Significant at 0.01 level. F: ANOVA, F: Pairwise comparison. SD: Standard deviation

Table 2

Comparison of salivary visfatin levels before and after scaling and root planing in gingivitis and chronic periodontitis groups

Group	Salivary visfatin level	Mean±SD	n	Mean difference	Paired t	P
II	Baseline	30.7±5.7	16	9.8	7.35**	0.000
	After 2 months	20.9±2.9	16
III	Baseline	56.8±8.0	16	32.0	14.18**	0.000
	After 2 months	24.8±2.1	16

**Significant at 0.01 level. SD: Standard deviation

Table 3

Comparison of salivary visfatin levels in Group II and Group III 2 months after scaling and root planing to that of Group I

Group	Mean±SD	n	F	P	Scheffe multiple comparisons
					Pair	F	P
I	19.2±1.9	16	24**	0.000	I and II	1.9	0.158
II	20.9±2.9	16			I and III	22.7**	0.000
III	24.8±2.1	16			II and III	11.4**	0.000

**Significant at 0.01 level. F: ANOVA. SD: Standard deviation

All the clinical parameters (PI, MGI, GBI, PD, and CAL) demonstrated a statistically significant (P < 0.01) improvement in groups II and III by 2 months after SRP when compared to that at baseline [Table 4].

Table 4

Comparison of clinical parameters at baseline and 2 months after scaling and root planning

Clinical parameters	Mean±SD						P
	Gingivitis			Chronic periodontitis
	Baseline	2 months	Paired t	Baseline	2 months	Paired t
PI	2.0±0.6	1.1±0.3	6.64**	2.6±0.3	1.2±0.1	19.67**	0.00
MGI	1.9±0.6	1.0±0.2	6.49**	2.5±0.3	1.2±0.1	17.05**
GBI	58.3±5.6	6.8±1.0	37.74**	92.8±6.8	12.7±3.2	44.4**
PD	2.8±0.1	2.6±0.1	5.24**	7.1±0.7	5.9±0.5	7.2**
CALs		-		7.5±0.7	6.3±0.5	8.4**

**Significant at 0.01 level. SD: Standard deviation, CAL: Clinical attachment level, PD: Pocket depth, GBI: Gingival bleeding index, MGI: Modified gingival index, PI: Plaque index

DISCUSSION

Visfatin is a biological mediator that acts as a growth factor, cytokine, as well as an enzyme and has several pro-inflammatory functions. The existing evidence has demonstrated increased levels of visfatin and its mRNA expression in several inflammatory diseases. It can upregulate the expression of other key inflammatory cytokines such as IL-6, IL-1 β, and TNF-α in human monocytes.

The present study is the first of its kind that compared the levels of salivary visfatin in healthy patients and patients with gingivitis and chronic periodontitis, before and after the procedure of SRP. The levels of salivary visfatin concentration were highest in patients with Group III, followed by patients with Group II and Group I. The significant difference could be due to variations in the severity of periodontal disease, flow of GCF, differences in the number of immune cells migrating into the crevice,[10] as well as the composition of microorganisms. The cells of the periodontal ligament can also secrete visfatin locally and is stimulated by key periodontal pathogens such as Fusobacterium nucleatum and Porphyromonas gingivalis.[11]

On intergroup comparison, a statistically significant difference in the mean salivary visfatin concentrations was obtained. The results were in accordance with the study by Tabari et al.[12] Raghavendra et al.[13] and Pradeep et al.[14] demonstrated the highest concentrations of visfatin in both GCF and serum in patients with chronic periodontitis. The increased levels of salivary visfatin in patients with chronic periodontitis confirm the active secretion of visfatin by immune cells associated with periodontal disease activity.

The present study found a statistically significant reduction (P < 0.001) in the levels of salivary visfatin in patients with gingivitis and chronic periodontitis after 2 months of performing SRP when compared to that at baseline. Furthermore, statistically significant reductions were observed in the clinical parameters (MGI, GBI, and PD) in Groups II and III. This is same as the results obtained by Tabari et al.[10]

In the present study, the concentrations of salivary visfatin increased with the severity of the disease and decreased after SRP. An increase in the levels of pro-inflammatory cytokines such as IL-6, IL-Iβ, and TNF-α can significantly enhance the expression of visfatin. Thus, salivary visfatin can be considered as a potential biomarker of periodontal diseases that can be used to monitor the response to periodontal treatment. However, very few studies have evaluated the impact of periodontal treatment on the levels of visfatin. This study did not consider the body mass index of the enrolled patients.

CONCLUSION

Based on the results of the present study, enhanced visfatin levels may play a role in the pathogenesis of periodontal diseases. Thus, salivary visfatin can be considered as an inflammatory biomarker in periodontal diseases that can assess the response to nonsurgical periodontal treatment. However, the role of visfatin in periodontal diseases can further be ascertained by conducting longitudinal studies with larger sample sizes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.