Nitro-oxidative Stress in Periodontitis
Condition: Chronic Periodontitis; Aggressive Periodontitis
MeSH terms: Periodontitis; Periodontal Diseases; Chronic Periodontitis; Aggressive Periodontitis
Study type: Observational

Summary: 
      Periodontitis is a chronic inflammatory disease whose etio-pathogencity is not fully
      understood yet. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are
      involved in physiological and pathological processes. Nitro-oxidative stress has been
      implicated in Periodontitis.

      The aim of this study is to assess the levels of ROS and RNS in serum and gingival crevicular
      fluid (GCF) samples taken from periodontitis (chronic and aggressive) patients and healthy
      controls. Subsequently, correlating these levels with the severity of periodontal disease.

      Eighty subjects will be invited to participate in this study. Patients will be allocated into
      four groups (20 patients each). The biochemical parameters that will be investigated are
      Malondialdehyde (MDA) (using TBRSA assay) as a marker of oxidative stress and (NO- level
      using Griess reagent) as a marker of nitrosative stress.
    

Detailed description: 
      Introduction:

      Teeth and their supporting periodontal structures are of great importance in good oral
      health. Periodontitis is an inflammatory disease of the periodontium which affects the
      supporting tissues of the teeth. The disease is multifaceted and its etio-pathogenecity is
      still not fully understood and therefore the treatment of different types of periodontal
      disease can be very difficult. Numerous risk factors have been implicated in the disease
      process including risk determinants and risk indicators. Most recently, strong body of
      evidence has accumulated to support a role for reactive oxygen (ROS) and nitrogen species
      (RNS) either as trigger agents or more frequently, aggravators of the primary lesions in
      periodontitis.

      Oxygen and nitrogen reactive species are involved in a large number of physiological and
      pathological processes. ROS generated by monocytes and neutrophyles during inflammation are
      important aggression factors in the periodontal tissues. ROS play an important role in
      activation of osteoclasts and in bone resorption.

      Oxidative stress is a condition arising when there is a serious imbalance between the levels
      of free radicals in a cell and its antioxidant defenses in favor of the former. Thus, tissue
      damage can result when antioxidant systems are unable to counteract the free radicals actions
      efficiently. Inflammation when stimulated by bacterial pathogens, host cells release
      proinflammatory cytokines (IL-1α, IL-6, IL-8, and tumor necrosis factor-α) as part of immune
      response. These cytokines recruit polymorphonuclear cells (PMNs) that play a major role in
      the etiology of periodontal disease by producing proteolytic enzymes, such as elastase and O2
      (molecular oxygen) by the oxidative burst.

      In a recent study, Abou Sulaiman & Shehadeh found that serum total antioxidant capacity (TAS)
      was lower in non-smokers Syrian patients with chronic periodontitis compared to healthy
      controls. Also, they reported that periodontal treatment restored TAS levels to normal levels
      similar to healthy controls.

      The aim of this study is to assess the levels of ROS and RNS in serum and gingival crevicular
      fluid (GCF) samples taken from periodontitis patients (chronic and aggressive) and healthy
      controls. Subsequently, correlating these levels with severity of periodontal disease in
      Syrian patients.

      Materials and methods:

      A total of 80 subjects will be invited to participate in this study from the patients
      referred to the Department of Periodontology, Faculty of Dentistry, University of Damascus.
      The study has been approved by our local Review Board. Subjects will be recruited according
      to specific inclusion criteria after completion of medical and dental history questionnaires.
      Patients will sign a consent form after being advised about the nature of the study.

      The selection of patients will be made according of the criteria approved by the 1999
      international world workshop for a classification system of periodontal diseases and
      conditions using five clinical parameters and full mouth or panoramic radiographs for
      diagnosis.

      Subjects will be allocated into four groups:

        -  Chronic Periodontitis group (ChP): comprise 20 patients aged > 45 years and have
           presence of ≥2 non-adjacent sites per quadrant that were not first molars or incisors,
           with probing depth (PD) ≥5 mm, which bleed on gentle probing. The demonstrated
           radiographic bone loss ≥30% of the root length, patient with poor oral hygiene, the
           amount of accumulated plaque commensurate with the amount of clinical attachment level
           (CAL)

        -  Resistant Control group (R): comprise 20 age-sex matched patients who are > 45 years
           exhibit no signs of periodontal disease as determined by the absence of the evidence of
           interproximal (CAL ≤ 1mm), PD > 3 mm at any site, whole-mouth bleeding scores <10% and
           have no clinical signs of gingival inflammation .

        -  Aggressive Periodontitis group (AgP): comprise 20 patients who are aged < 35 years and
           diagnosed with rapid attachment loss with periodontal pocket depth (PD) > 4 mm around at
           least three teeth other than the first molars and incisors. Rapid bone destruction
           (>50%bone loss at diseased sites). Weak relationship between dental plaque and the
           severity of gingival inflammation.

        -  Young Control group (YC): comprise 20 age- and sex matched patients who are < 35 years
           and exhibit no signs of periodontal disease.

      Clinical measurements:

      A standard periodontal probe will be used for recording periodontal indices at six sites per
      tooth. The examined clinical parameters include bleeding on probing (BOP), plaque index (PI),
      clinical attachment loss (CAL) and periodontal pocket depth (PPD) and gingival index (GI).

      Collection of samples

      Subjects will be asked to refrain from brushing within 1 hour of sampling. (GCF) samples will
      be obtained using standard paper strips (Periocol strips, Oraflow, NY, USA). 6 samples from
      each individual (mesiofacial, distopalatal) sites from each examined tooth (incisor, premolar
      and molar) in the maxilla (Chapple et al. 2002). Strips will be put in PBS Buffer solution
      for 30 minutes then extracted and stored under liquid nitrogen.

      Venous blood samples will be collected from the antecubital fossa and stored in lithium
      heparin tubes and allowed to stand for 30 minutes before being centrifuged at 1000 ×g for 30
      minutes, samples will be aliquoted into cryogenic vials and will be stored under liquid
      nitrogen.

      Laboratory studies:

        -  TBARS assay (HPLC) will be used to assess the malondialdihyde levels as a marker of
           oxidative stress.

        -  Griess reagent to assess nitric oxide levels as a marker of nitrosative stress.
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        A total of 80 subjects will be invited to participate in this study from the patients
        referred to the Department of Periodontology, Faculty of Dentistry, University of Damascus.

        The study has been pproved by our local Review Board. Subjects will be recruited according
        to specific inclusion criteria after completion of medical and dental history
        questionnaires. Patients will sign a consent form after being advised about the nature of
        the study.

        The selection of patients will be made according of the criteria approved by the 1999
        international world workshop for a classification system of periodontal diseases and
        conditions (Armitage 1999) using five clinical parameters and full mouth or panoramic
        radiographs for diagnosis.
      
Keywords: oxidative stress; periodontal disease; nitric oxide