Effect of Non-surgical Periodontal Therapy on Gingival Crevicular Fluid Interleukin-21 Levels
Condition: Chronic Peridontitis Patients; Chronic Gingivitis; Healthy
MeSH terms: Gingivitis
Study type: Interventional

Summary: 
      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
    

Detailed description: 
      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.
    

Intervention name(s): non surgical periodontal therapy
Intervention type(s): Procedure
Intervention description(s): and root planing (SRP) was performed in two to four appointments lasting approximately 60 minutes each under local anaesthesia (2% lignocaine hydrochloride with 1:2,00,000 adrenaline) using area specific Gracey periodontal curettes and an ultrasonic device. The treatment was concluded in 3 weeks. Within the duration of the study, all subjects received supportive therapy, which included professional plaque control and reinstruction of oral hygiene.
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: A total of 34 patients with 19 males and 15 females in the age group of 20-60 years were included. Patients were divided into 3 groups to collect Gingival Crevicular Fluid (GCF) samples for the study.
Masking: Double (Investigator, Outcomes Assessor)
Primary purpose: Treatment