Effect of Non-surgical Periodontal Therapy on Gingival Crevicular Fluid Interleukin-21 Levels
Effect of Non-surgical Periodontal Therapy on Gingival Crevicular Fluid Interleukin-21 Levels
Human IL-21 is present in gingival crevicular fluid in periodontal health, gingivitis and
 chronic periodontitis. A significant increase in the concentration of IL-21 in gingival
 crevicular fluid is observed with an increase in the amount of periodontal destruction. Non-
 surgical periodontal therapy aided in decrease of GCF IL-21 levels in clinical gingivitis and
 chronic periodontitis
Introduction

 Periodontal disease is a poly-microbial disease that affects tooth-supporting tissues.
 Integral part of periodontal disease involves destruction of periodontal tissues that results
 from stimulation of the bacterial challenge to host immune-inflammatory response. Chronic
 periodontitis is the most common form of periodontal disease in adult population. The
 bacterial plaque biofilm is the primitive factor for periodontitis, but the majority of
 destruction of the periodontal tissues would eventually conclude from a sequence of
 immune-inflammatory reactions. As a result of cellular activation, inflammatory mediators
 include cytokines, chemokines etc. collectively contribute to tissue destruction and bone
 resorption. Cytokines can be grouped as Th1, Th2, Th17 and T regulatory (Treg) based on their
 expression pattern and effects on target cells or tissues.5 Classically, periodontal disease
 has been explained by Th1/Th2 pathway. Th1 cells are more frequently detected in the early
 stage of periodontal lesions suggesting that Th1 cells are associated with a protective
 response against bacterial infection whereas Th2 cells predominate in the later and advanced
 period of the disease, having a role in the destruction and progression of periodontal
 lesions. Alternative type of host response to pathogenic bacteria has been described by the
 IL-23/IL-17 pathway. This alternative pathway occurs when bacteria induce synthesis of
 IL-23/17 rather than IL-12, which plays a pivotal role in initiation and maintenance of
 inflammatory response. IL-23/17 pathway as compared to classical Th1/Th2 pathway is a much
 fine tuned cellular immune response that has a multitude of functions that bridges the innate
 and adaptive arm of the immune response.9 Th17 cytokines (IL-17, IL-21, IL-22, IL-6, Tumor
 necrosis factor {TNF}-a, etc.)8 participate in periodontal disease, but whether their
 dominant role is host-protective or destructive is questionable. These cytokines have not
 been elucidated at different inflammatory status in periodontitis.

 IL-21 is a type-I cytokine, structurally it appears similar to IL-2, IL-4, and IL-15
 proteins. IL-21 is principally composed by activated T cells, but it directs a vast spectrum
 of myeloid and lymphoid cells of the immune system, which facilitate IL-21 to modulate the
 acquired and innate immunity.10 IL-21 compete in the immunity against tumor cells11 and
 chronic viral infections,12 however, enormous accomplishment of IL-21 has been correlated
 with the advancement of immune inflammatory diseases in various organs.13 IL-21 is heighten
 in dermatological conditions like skin biopsies14 of patients with systemic lupus
 erythematosus, psoriasis, and atopic dermatitis. In addition, IL-21 interpretation correspond
 with the presence of Th17 cells in synovial fluid and peripheral blood of rheumatoid
 arthritis patients.

 Role of IL-21 in inflammation has been extensively studied; the treatment aspect on its
 levels in periodontal diseases needs to be further explored. Therefore, in the light of the
 above findings, the investigators determine the levels of interleukin-21 (IL-21) in GCF from
 chronic gingivitis, chronic periodontitis and control patients before and after non-surgical
 periodontal therapy along with the co-relation of clinical parameters of periodontal tissues
 destruction.

 MATERIAL AND METHODS:

 PATIENTS:

 Thirty four patients (19 males and 15 females, aged 20-60 years) were consecutively enrolled
 over a six month period (April 2014 to September 2014) from the outpatient department of
 periodontology, Krishnadevaraya College of Dental Sciences, Bangalore, and Karnataka. Ethical
 clearance for the study was obtained from the institutional ethical committee
 (02-D012-36773). The study was conducted in accordance with the ethical principles described
 in the Declaration of Helsinki 2008. Procedure of the study was explained and informed
 written consent was obtained from all the participants before their inclusion in the study.
 Among them, 24 patients having diseased periodontium were grouped into 12 chronic gingivitis
 and 12 chronic periodontitis patients whereas 10 healthy individuals were included as
 control. Inclusion criteria were: patients having more than or equal to 14 functional teeth,
 systemically healthy patients who had not received any form of surgical and non surgical
 periodontal therapy or received antibiotics or non-steroidal anti-inflammatory therapy within
 the past 6 months of the study. Chronic gingivitis was defined as having probing depth (PD)
 less than or equal to 4mm and more than to 25% sites with gingival bleeding present (BOP).15
 Chronic periodontitis was defined as having probing depth more than or equal to 5mm, RAL more
 than or equal to 8mm, with more than or equal to 10% sites with BOP positive and evidence of
 bone loss determined radiographically.16 Patients who volunteered with no evidence of
 periodontal disease determined by the absence of increased PD or AL were considered as
 healthy control group.

 Clinical Measurements:

 Clinical parameters were measured and evaluated for all the teeth excluding 3rd molars, at
 all the six sites for each tooth (mesio-buccal, disto-buccal, disto-lingual, mid-lingual and
 mesiolingual). These parameters consisted of pocket depth (PD), assessment of gingival
 bleeding i.e gingival index (GI), dichotomous measurement of supragingival plaque
 accumulation i.e plaque index (PI), and bleeding on probing (BOP) to the base of the crevice.
 One calibrated examiner (MN) performed all the patient evaluations and measurements.

 GCF samples collection and analysis:

 In all three groups, GCF samples were collected at baseline and 6 weeks post treatment. Site
 selection was based on the highest score recorded for single site in the oral cavity. A
 single site in each subject that showed worst inflammatory manifestations (chronic
 gingivitis) and the highest RAL level (chronic periodontitis) was selected for gingival
 crevicular fluid collection. In our study, we selected a single site in each subject to avoid
 pooling of samples from multiple sites, as periodontitis is a site specific disease. Prior to
 the collection of GCF samples supragingival plaque was removed with cotton pellets avoiding
 contact with marginal gingiva. Standard paperstrips were carefully inserted to a depth of
 approximately 2mm, into the sulcus/pocket for 30 seconds for collection of GCF. Blood
 contaminated strips were discarded and alternative site was used to obtain replacement
 samples. In chronic gingivitis patients, alternative sample wre collected with the next
 highest GI scores and in chronic periodontitis patients the next deepest Probing pocket depth
 {PPD} was selected for the alternative sample site. A calibrated appliance was used to
 quantify the GCF sample volume and reading were converted to actual volume (μL) by reference
 to a standard curve, prepared using the periotron reading of volume of fluid (μL) distilled
 water in perio-col strips. A blank gingival fluid collection (perio-col) strip was placed
 between the periotron fluid meta sensors and the instrument was adjusted to display a reading
 of zero. A microlitre syringe was used to accurately deliver 0.25-1.25 μL fluid (distilled
 water) to perio-col strip. The strips were immediately placed between the periotron sensors.
 The periotron score volume displayed the known volume of fluid recorded. This step was
 repeated three more time with 0.25 μL of test fluid and the average score recorded. The above
 step was repeated using volume of 0.5, 0.75, 1.0, 1.25 μL, and in every instance the mean
 periotron value calculated and recorded. Once all the score were obtained, a standard curve
 was computed with known fluid volume (X-axis) and periotron score (Y-axis). In a similar way
 GCF volumes (from health, chronic gingivitis, chronic periodontitis) from study patients were
 obtained automatically with periotron score. The interpolation from standard calibration
 graph gave volume of fluid. After measurement of volume, the strips from the selected sites
 were placed immediately into individual microcentrifuge tubes containing 200 μL of phosphate
 buffer solution. The samples were stored at -80 °C until further analysis.

 ELISA

 The levels of IL-21 in the GCF were determined using ELISA according to the manufactures
 instruction. To elute the proteins, the tube containing the periopaper strips were vortexed
 and homogenized for 30 seconds and then centrifuged at 12,500 rpm at 4°C for 5 minutes. The
 kit used monoclonal antibody MT 21.3 biotin and human recombinant IL-21 standard (captured
 antibody). Samples were run in triplicate to authenticate the sensitivity of ELISA and all
 the samples were found to be within the detection limit of ELISA. An ELISA reader (spectramax
 190 Ab sorbance microplate reader; molecular devices; Sunnyvale, CA, USA) with 450 nm as
 elementary wavelength was used to measure the absorbance of the substrate. Conversion of the
 absorbance readings obtained, into definite volume (pg/mL) were performed using standard
 reference curve. The protein concentration at each site (pg/mL) were determined by dividing
 the total amount of IL-21 (pg) by gingival crevicular fluid volume (μL) and subsequently the
 (pg/μL) values were converted into (pg/mL).

 Periodontal treatment protocols:

 At baseline, clinical parameters and collection of GCF samples for all patients was done. All
 patients received a thorough oral hygiene instructions, a full mouth supragingival
 subgingival scaling along with root planing. Non-surgical periodontal therapy for group III
 was performed in 2-3 appointments. A single calibrated examiner (MN) provided treatment to
 all study patients.

 STATISTICAL ANALYSIS:

 The power of study and sample size calculation was determined on the basis of change in GCF
 IL-21 level. Current estimates as a pilot study which included 12 patients in each test group
 and 10 in control group with total sample size of 34 patients. Type II error level of β =
 0.20 (80% power) and type I error level of α=0.05 (5% probability) was calculated. The
 distribution of the samples with respective values of PI, GI, BOP, PD, RAL and IL-21 levels
 of pre-operatively and post-operatively was analyzed statistically. Data was entered in
 Microsoft excel and analysed using SPSS {statistical package for social science}, ver.10.05)
 package. Proportions were compared using Chi-square test (χ2) test of significance. Normality
 of data was tested using Shapiro-Wilk test. A student t-test was performed to determine pre
 and post treatment difference values. One way analysis of variance (ANOVA) was used to test
 the difference between the groups. Comparison of the biochemical and clinical parameters were
 performed using Kruskal-Wallis non-parametric test. P<0.05 was considered statistically
 significant.
Inclusion Criteria:

 -  patients having more than or equal to 14 functional teeth, systemically healthy
 patients who had not received any form of surgical and non surgical periodontal
 therapy or received antibiotics or non-steroidal anti-inflammatory

 Exclusion Criteria:

 -  smokers pregnant females