Non-surgical Periodontal Treatment and Periostin Levels
Condition: Periodontal Treatment, Cytokines
Study type: Interventional

Summary: 
      Two different studies were planned from the recruited samples. The aim of these studies was
      to investigate the effect of non-surgical periodontal treatment on the inflammatory markers
      levels in serum and gingival crevicular fluid (GCF) in patients with periodontitis.

      There were two groups consisting of individuals who had periodontitis and those who were
      healthy, with 24 individuals in each group.

      Periostin levels were assessed at baseline, at the 24th h, 48th h, 14th day 1st month, 3rd
      month after non-surgical periodontal treatment in serum and GCF samples by the Elisa method
      in the first study. In parallel, interleukin 1 (IL)-beta and fibroblast growth factor levels
      were measured.

      Progranulin levels were assessed at baseline, at the 24th h, 48th h, 14th day 1st month, 3rd
      month after non-surgical periodontal treatment in serum and GCF samples by the Luminex method
      in the second study. In parallel, vascular endothelial growth factor, IL-1 beta, tumor
      necrosis factor-alpha, and IL-10 levels were measured.
    

Detailed description: 
      The First Study

      The observational prospective study was designed to characterize periostin changes over time
      after non-surgical periodontal treatment in serum and GCF in the Department of Periodontology
      in the Faculty of Dentistry at Hatay Mustafa Kemal University, Hatay, Turkey. The study
      protocol was approved by the Ethics Committee for the Use of Human Subjects in Research of
      Hatay Mustafa Kemal University (Protocol No: 2019/118). The study was carried out in
      accordance with the tenets of the Declaration of Helsinki and conducted in CONSORT guidelines
      (Schulz, Altman, & Moher, 2010). This study project was financially supported by Hatay
      Mustafa Kemal University Scientific Research Projects Coordinator (19M051).

      Individuals were included in the study from December 2019 through August 2021. Written
      informed consent was obtained from each participant before the clinical periodontal
      examination.

      Study Population

      Forty-eight adult individuals were enrolled in the study. Participants were recruited into 2
      sex matched groups with 24 participants in each: periodontally healthy (PH) and periodontitis
      (P) groups. In accordance with the demographic variables of the patients included in the P
      group, the patients in the PH groups were included.

      Periodontal Parameters

      Periodontal clinical parameters were recorded by a single calibrated examiner (k =0.95)
      (author AS). Intra-examiner agreement was determined for CAL. The intra-examiner
      reproducibility was determined through repeated examinations of 10 subjects with a one-hour
      interval. Clinical periodontal measurements were assessed using the following periodontal
      measurements for periodontal diagnosis in both groups and also at the 1st month, 3rd month
      after non-surgical periodontal treatment in the periodontitis group.

      The measurements were performed using a Williams periodontal probe (Hu-Friedy, Chicago, IL,
      USA) and included probing pocket depth (PPD), clinical attachment level (CAL), plaque index
      (PI) (Silness & Loe, 1964), gingival index (GI) (Loe & Silness, 1963), and percentage
      bleeding on probing (BOP) (Ainamo & Bay, 1975) at six sites per tooth (mesio-buccal, buccal,
      disto-buccal, mesio-lingual, lingual and disto-lingual) on each tooth.

      Diagnosis of periodontal diseases and conditions was made according to the radiographic and
      clinical diagnostic criteria proposed by the 2017 World Workshop on Classification of
      Periodontal and Peri-implant Diseases and Conditions (Caton et al., 2018). Individuals with
      BOP < 10% without attachment loss and radiographic bone loss were considered to have
      periodontal health (Tonetti & Sanz, 2019). The criteria for periodontitis included patients
      with CAL ≥ 5 mm in two or more interproximal sites and PPD ≥ 6 mm in one or more
      interproximal sites. Only stage III-IV (severe) periodontitis was included in the present
      study (Tonetti, Greenwell, & Kornman, 2018).

      Periodontal Intervention

      Periodontitis patients were treated by one-stage full-mouth disinfection. Full-mouth
      disinfection included full-mouth scaling and root planning procedures within 24 h in two
      sessions. Each session was practiced for 60 min on two consecutive days. Subgingival
      irrigation and tongue brushing were made with 1% CHX gel for 1 min. Mouthwashes with 0.12%
      CHX were performed for 30 s at the beginning and at the end of each session and twice a day
      for 2 weeks (Quirynen et al., 1995).

      Collection of GCF Samples

      GCF samples were collected following 8 hours of night fasting and one week after clinical
      periodontal measurements at baseline in two groups and then at the the 24th h, 48th h, 14th
      day 1st month, 3rd month after non-surgical periodontal treatment (Akalin, Baltacioglu,
      Alver, & Karabulut, 2007). The samples were collected from the teeth side with PPDs ≥6 mm,
      and BOP positive in patients with periodontitis. Six GCF samples were taken per participant.
      Samples were collected from a mesial and a distal site on each of three teeth (incisors,
      premolars, and molars). Saliva contamination was prevented by isolation with cotton rolls and
      gently air-drying the sampling area. Samples contaminated with saliva or blood were not
      included. Plaque was gently removed from the sampling area by the periodontal curette. The
      samples were collected within 30 seconds with standardized paper strips (Periopaper; Oraflow
      Inc., Plainview, NY) by the orifice method.(Rudin, Overdiek, & Rateitschak, 1970) The volumes
      were measured on a pre-calibrated electronic gingival fluid measuring device (Oraflow Inc.,
      Plainview, NY)|| (Chapple, Landini, Griffiths, Patel, & Ward, 1999). The values of the
      electronic device were referenced to a standard curve and converted to an actual volume (µl).
      All of the Periopaper strips were pooled in plastic Eppendorf microcentrifuge tubes. They
      were stored at -80 °C until the biochemical analysis.

      All samples were thawed and 375 μl of phosphate buffered saline (pH:7.00, 10 mM Na2PO4, 137
      mM NaCl, and 2.7 mM KCl) was added into the eppendorf tubes containing the sample strips.
      Subsequently, samples were eluted for 30 minutes at room temperature and (Chapple et al.,
      1997) centrifuged at 11000×g for 15 min at 4 °C. The measurement of periostin, IL-1β, and FGF
      levels were assayed.

      Collection of Serum Samples

      Peripheral venous blood samples were taken from the patients. The serum was separated from
      the cells by centrifugation at 2000 rpm for 10 min, after which it was stored at -80°C, until
      the biochemical analysis was performed.

      Laboratory Analyses

      Periostin, IL-1β, and FGF levels were measured by the enzyme-linked immunosorbent assay
      (ELISA) using commercially available kits at 450 nm wavelength in ELISA Reader (Thermo Fisher
      Scientific Multiscan Go-Finland).

      Measurement of periostin levels in GCF samples

      Periostin levels were measured by ELABSCIENCE Human POSTN/OSF-2 (Kit Cat No: E-EL-H6113). The
      assay ranges for the periostin were 3.13-200 ng/ml; sensitivity, 1.88 ng/ml, intra and inter
      assay coefficients of variance (CV%) were < 10%.

      Measurement of IL-1β levels in GCF samples

      IL-1β levels were measured by ELABSCIENCE Human IL-1β (Kit Cat No: E-EL-H0149). The assay
      ranges for the IL-1β were 7.81-500 pg/ml; sensitivity, 4.69 pg/ml, intra and inter assay
      coefficients of variance (CV%) were < 10%.

      Measurement of FGF levels in GCF samples

      FGF levels were measured by ELABSCIENCE Human FGF21 (Kit Cat No: E-EL-H0074). The assay
      ranges for the FGF were 31.25-2000 pg/ml; sensitivity, 18.75 pg/ml, intra and inter assay
      coefficients of variance (CV%) were < 10%.

      The Second Study

      The study aimed to evaluate the effect of non-surgical periodontal treatment on serum and
      gingival crevicular fluid progranulin levels.

      3 paper strips and serum samples for each patient recruited from the same patients and same
      methods as back-up samples were transferred to King's College London Dental Institute,
      Periodontology Unit, Centre for Host-Microbiome Interactions, London, UK; after a material
      transfer agreement signed between Hatay Mustafa Kemal University, Hatay, Turkey and King's
      College London, London, UK in May 2022 for another study. T The study protocol was approved
      by the Ethics Committee for the Use of Human Subjects in Research of Hatay Mustafa Kemal
      University (Protocol No: 2022/90). The study was carried out in accordance with the tenets of
      the Declaration of Helsinki and conducted in the CONSORT guidelines (Schulz, Altman, & Moher,
      2010). This study project was financially supported by King's College London internal
      departmental funds.

      GCF Samples Elution

      50 μL of PBS/protease inhibitor cocktail (PBS with protease inhibitors 1X, Complete ULTRA
      tablets, Mini; EDTA-free) were added to a sterile microcentrifuge tube included 3 paper
      strips and centrifuged at 11000 rpm for 15 min at 4 C for GCF elutiation. This procedure was
      repeated two times and obtained a total volume of 100 μL. They were stored at -80 °C until
      the biochemical analysis.

      Serum and GCF Samples Analyses

      Luminex bead-based multiplex immunoassay (Luminex, R&D systems, Minneapolis, MN) was used to
      carry out the analysis (in duplicate) of each sample. A custom-made 5-plex kit included
      inflammatory and regenerative markers: Progranulin (PGRN), VEGF, interleukin (IL)-1 beta,
      tumor necrosis factor-alpha (TNF-alfa), and IL-10.

      Each 50-μL sample was resuspended in a dedicated well with a microparticle cocktail of
      antibodies covered with a foil plate sealer on magnetic beads and incubated for 2 h on a
      horizontal orbital microplate shaker. Following washes, the biotin antibody cocktail was
      added and incubated for 1 h as previously reported. After the washes,
      streptavidin-phycoerythrin (Streptavidin-PE) incubation was carried out for 30 min. Then, the
      samples were put into the Luminex MAGPIX analyzer (Luminex; R&D systems) to read. The marker
      values were obtained as pg/mL.

      Statistical Analysis

      The normality of the distribution of continuous variables was tested by the Shapiro-Wilk
      test. Descriptive statistics were presented as mean and standard deviation (mean±sd) for
      parametric distributed variables and median (interquartile range) for the non-parametric
      distributed variables.

      The chi-square test was used to analyze the associations between categorical variables. The
      Student t-test for normally distributed numerical variables and Mann Whitney u test for
      non-normally distributed numerical variables were performed to compare 2 independent groups
      for numerical data. Freidman test was performed for different time points with non-parametric
      variables. Dunn multiple comparison tests were performed for post hoc pairwise multiple
      comparison analyses.

      Statistical analysis was performed with SPSS for Windows version 24.0 and P value <0.05 was
      accepted as statistically significant.
    

Intervention name(s): Non-surgical periodontal treatment
Intervention type(s): Procedure
Intervention description(s): Full month non-surgical periodontal treatment procedure was applied to the periodontitis group.
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Masking: Single (Participant)
Primary purpose: Treatment
Keywords: Periodontitis; Periostin; Periodontal treatment; Gingival crevicular fluid