Effect of Nonsurgical Periodontal Therapy Verses Oral Hygiene Instructions on Patients With Chronic Periodontitis
Condition: Chronic Periodontitis
MeSH terms: Periodontitis; Chronic Periodontitis
Study type: Interventional

Summary: 
      Periodontitis, a bacterial dental biofilm based infectious disease, is a chronic inflammatory
      disease of the periodontium which results in irreversible destruction of supporting
      structures of the teeth such as periodontal ligaments, connective tissues, cementum and
      alveolar bone. More than 700 species of bacteria are estimated to be found in the
      sub-gingival dental biofilm in which periodontopathogens are found to be responsible in
      initiating periodontal disease. Chronic periodontitis, although termed as 'chronic', starts
      as an acute inflammation. Low levels of bacteremia and endotoxins provides a stimulus for the
      systemic inflammatory response. In periodontitis, the interaction of lipopolysaccharide (LPS)
      from gram-negative bacteria with host cells initiates the secretion of cytokines and the
      expression of cell adhesion molecules in gingival tissue which leads to loss of alveolar bone
      and connective tissues supporting the teeth.

      In the management of chronic periodontitis, non surgical periodontal therapy (NSPT) which
      includes oral hygiene instruction (OHI), scaling and root planing, is administered to improve
      clinical parameters as well as controlling the bacterial count responsible in initiating the
      disease and the resultant inflammatory response. Success of the treatment can be analyzed
      when the clinical parameters, counts of the microbes is reduced and inflammatory response is
      controlled. Studies which have evaluated the microbiological and immunological response
      following NSPT clearly demonstrates an improvement in clinical parameters, reduction in
      inflammatory mediators present as well as a decrease in microbial count.

      OHI which includes tooth brushing and inter-dental cleaning is found beneficial in removing
      microbial plaque, thus, preventing periodontal diseases such as gingivitis and periodontitis.
      Studies have evaluated the role of OHI on clinical and immunological parameters and
      microbiological profiles of periodontal maintenance subjects, while none of them evaluated
      its role alone in the management of chronic periodontitis subjects. Therefore, the purpose of
      the following investigation was to compare the changes in the clinical parameters,
      inflammatory mediators as well as level and frequency of detection of periodontopathogens
      that take place after 3-months of NSPT versus OHI and to evaluate if clinical parameters have
      an effect on these inflammatory mediators and microbiological changes.
    

Detailed description: 
      Subjects with moderate to advanced chronic periodontitis were selected for this longitudinal
      randomized clinical trial. Ethical clearance for the study was granted by the Medical Ethics
      Committee, Faculty of Dentistry, University of Malaya [Medical Ethics Committee Number: DF
      PE1002/0045(P)]. The study was conducted in accordance with the Helsinki Declaration of 1975,
      as revised in 2000. The CONSORT guidelines for clinical trials were followed.

      Out of 112 screened patients 56 patients fulfilled the inclusion and exclusion criteria.
      Inclusion criteria were minimum of 12 teeth present with pocket of 5mm or more and probing
      attachment loss of 4mm or more in at least 2 different quadrants which bled on probing.
      Exclusion criteria included history of cardiovascular problems, pregnancy, smokers, and
      subjects receiving antibiotics in past 4 months or NSPT in past 6 months. Recruited patients
      were treated at the Periodontology clinic, Faculty of Dentistry, University of Malaya. Using
      Cohen's d formula it was calculated that at least 26 patients would be needed per treatment
      group to detect this difference with 80% power.

      Prior to baseline examination, patients were assigned in the NSPT and OHI group using block
      randomization. Patients in NSPT group received OHI, scaling and root planing followed by
      0.12% Chlorhexidine mouth rinses while patients in OHI group received OHI only. At each month
      recall visit, participants in both groups were reviewed and motivated. Professional
      prophylaxis was only received by NSPT group. Two calibrated examiners (RPCR and WNAWA)
      performed all treatment on the patients. Probing pocket depth ( PPD), probing attachment loss
      (PAL), gingival bleeding index (BI) and visible plaque index (PI) were determined on all
      present teeth except third molars at baseline and 3 months after therapy. For PI and BI, 4
      sites were measured (mesial, distal, buccal, lingual) and for PPD and PAL 6 sites were
      recorded (disto-buccal, mid-buccal, mesio-buccal, mesio-lingual, mid-lingual and
      disto-lingual). Utilizing Kappa statistics, good agreements (>0.8) were obtained for intra
      and inter operator reproducibility of all recorded clinical parameters.

      Identification of subgingival microbiota using quantitative polymerase chain reaction
      technique.

      Sub-gingival plaque scraping samples were obtained at baseline and 3-months post therapy with
      sterile curettes from the deepest sites at each quadrant and were pooled together for each
      subject. Prior to sampling, isolation was maintained with cotton rolls and supra-gingival
      plaque was removed using cotton pellets. Plaque scrapings were re-suspended in 1.5 ml of
      Phosphate Buffered Saline and stored at -80⁰C freezer prior to DNA extraction. 100µl of
      plaque sample was used for automated DNA extraction in the lab using the Qiacube machine
      (Qiagen®, Biotechnology, Netherlands). The tubes containing the plaque samples were
      centrifuged at a speed of 5,000 × g for 10 minutes on a tabletop centrifuge machine to obtain
      the pellet. The pellet was then used for automated DNA extraction with the Qiacube machine.
      100µl of eluted DNA was stored in -20⁰C for bacterial detection using quantitative polymerase
      chain reaction (Applied Biosystem, USA).

      Reference bacterial strains used in this study were P.gingivalis (W83),
      A.actinomycetemcomitans (NCTC9710), P.intermedia (ATCC25611) and T.forsythia (CCUG 21028AT).
      All the bacterial strains were grown as recommended by American Type Culture Collection
      (ATCC). After the growth of bacteria till late exponential phase they were washed with
      sterile distilled water and DNA extraction was carried out using the same protocol as for
      plaque samples.

      The concentration and purity of extracted DNA was determined using spectrophotometer machine
      (Nanodrop 2000, Thermo scientific, USA). DNA concentration from both plaque samples and
      reference strain were calculated and recorded accordingly. The extracted DNAs were stored in
      -20⁰C until the initiation of quantitative polymerase chain reaction procedure.

      The protocol given by Boutaga et al., (2005) was followed for quantitative polymerase chain
      reaction . Primers and probes selected for this study are shown in Table 1. A total reaction
      mixture of 20ul was used for amplification, which contains 2ul of template DNA from plaque
      sample/reference strain,10 ul of 2 x TaqMan fast advanced master mix (Applied biosystems,
      USA), 1ul of 20x gene expression assay and 7ul nuclease free water. All the components of
      reaction mixture except for DNA sample were added in 1.5ml micro centrifuge tube. The tube
      was vortexed and than centrifuged to eliminate air bubbles. Mixture was transferred to
      96-well plate with addition of 2ul DNA sample. The Plate was then covered with optical
      adhesive and centrifuged again to make sure air bubbles are eliminated. The cycling
      conditions used were as follows: 50oc for 2min and 95oc for 10min followed by 45 cycles at
      95oc for 15s and 60oc for 1min.

      Dilutions of known amounts of reference strains of DNA were used to generate standard curves
      with correlation coefficient (R2: 0.99). For quantification/copy number determination, the
      results from unknown samples were projected on the standard curves obtained from the pure
      cultures using the formula: Quantity DNA=10(ct-b)/m.

      Determination of Interleukin-1, Interleukin-6, Adiponectin and Tumour Necrosis Factor-alpha
      level in Serum.

      All reagents were allowed to reach room temperature before use and were gently mixed prior to
      the experiments. Number of 8-well strips needed for the assay was determined. 100 ul of the
      Standard Diluent Buffer were added to zero wells and 1 well was reserved for chromogen blank.
      100 ul of standards, samples and controls were added to the appropriate microtiter wells. 50
      ul of biotinylated anti-Interleukin-6 (Biotin Conjugate) solution were pipetted into each
      well except the chromagen blank and then gently tapped on the side of the plate to mix. Next,
      plates were covered with plate covers and incubated for 2 hours at room temperature.
      Solutions were thoroughly aspirated from wells and the liquid discarded. The wells were
      washed 4 times. 100 ul streptavidin-histidine-rich protein working solution were added to
      each well except the chromogen blank. Again, the plates were covered with the plate covers
      and incubated for 30 minutes at room temperature. Solution from wells was thoroughly
      aspirated and the liquid discarded. The wells were washed 4 times. 100ul of Stabilized
      Chromogen was added to each well. The plates were incubated for 30 minutes at room
      temperature in the dark. 100 ul of stop solution was added to each well and then gently
      tapped on the side of the plate to mix. The absorbance of each well was read at 450 nm having
      blanked the plate reader against a chromogen blank composed of 100 ul each of Stabilized
      Chromogen and Stop Solution. The absorbance of the standards against the standard
      concentration was then plotted on graph paper.
    

Intervention name(s): Nonsurgical periodontal therapy; Oral hygiene instructions
Intervention type(s): Procedure; Procedure
Intervention description(s): All patients were instructed in oral hygiene methods using a soft bristled toothbrush, a compact-tuft toothbrush, interdental brushes and dental floss utilizing the modified Bass technique. Full mouth debridement, which consisted of scaling and root planing, was done in a single visit for all subjects using an ultrasonic scaler and gracey curettes. Additionally, all patients were given a 0.12% Chlorhexidine mouthrinse (Hexipro®). They were instructed to rinse three times a day using 15ml each time for a period of 14 days commencing immediately after completion of full mouth debridement. Thereafter at each recall visit, all participants were re-motivated and professional prophylaxis was performed.; All patients were instructed in oral hygiene methods using a soft bristled toothbrush, a compact-tuft toothbrush, interdental brushes and dental floss utilizing the modified Bass technique.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: Chronic periodontitis; periodontal pathogens; inflammatory mediators; Non surgical periodontal therapy; Oral hygiene instructions; Polymerase chain reaction; Elisa