Can Saliva be the New Bio-fluid to Monitor Children's Oral Health?
Condition: Healthy
Study type: Observational

Summary: 
      Background: Salivary diagnosis is an emerging field; the potentially valuable biofluid
      contains many molecules of the systemic circulation. Still, its profile in health has been
      scarcely investigated.

      Aim: This study aimed to characterize the inflammatory profile of healthy children's saliva,
      and to associate the parameters with clinical diagnoses of caries and gingival inflammation.

      Study design: Unstimulated saliva was collected from 100 children who underwent clinical
      dental examinations. The saliva samples were analysed for total protein, specific cytokines
      (IL-10, IL-8, IL-6 and TNF- α) and bacteria using Bradford assay, ELISA and quantitative
      real-time polymerase chain reaction, respectively. Salivary values were analysed according to
      age, plaque index (PI) and gingival index (GI).
    

Detailed description: 
      Salivary diagnosis is an emerging field that has progressed through several important
      developments, including salivary proteome and the development of compact point-of-care
      devices for rapid analysis. Indeed, an increasing number of salivary markers have been
      identified; and some have been associated with cancer, cardiovascular diseases, autoimmune
      diseases, viral and bacterial diseases, and human immunodeficiency. Saliva is frequently
      studied due to the easy, non-invasive, and rapidly obtained collection, without the need for
      specialized equipment. Moreover, the prodigious fluid source of saliva provides many, if not
      most, of the molecules found in the systemic circulation, rendering it a potentially valuable
      bio-fluid for diagnoses.

      Several types of inflammatory biomarkers associated with oral diseases, as well as systemic
      diseases, have been detected in saliva. Cytokines are among the most investigated biomarkers
      in this context. Due to their involvement in inflammatory, infectious and immunological
      diseases, cytokines may serve as markers of local oral pathologies such as periodontal
      disease and carries. Indeed, in adults, evidence shows that such disease can be detected
      through saliva biomarkers such as interleukins (IL)-1b, -6, -8 and -10), tumor necrosis
      factor- α (TNF- α) and matrix metalloproteinases (MMP)-8 and -9 in adults. Additionally, as
      some etiologies of oral pathology as gingivitis include bacterial infection such as
      Streptococcus, Fusobacterium, Actinomyces, Veillonella, Treponema, Capnocytophaga and
      Eikenella, saliva may also be used to measure bacterial load.

      Dental caries is one of the most common childhood diseases worldwide, and is associated with
      polymicrobial colonization on tooth surfaces. Both acidogenic and aciduric bacteria, such as
      the Mutans group of Streptococci and Lactobacilli, are primary etiological agents of dental
      caries. Lactobacilli and S. Mutans are mostly found together in saliva . The diversity of
      microbes in saliva has been shown to increase in the caries active status. In children with
      cavitated carious lesions, altered concentrations of the cytokines IL-6 and IL-8 were
      reported.

      The objective of this study was to characterize the inflammatory and microbiological profile
      of the saliva of healthy children, and to associate those levels with clinical diagnosis of
      caries and gingival disease.

      Materials and methods Study population The study population comprised 100 participants, born
      during 2008-2016 (aged 4-12 years at the time of the study).

      Clinical oral examination

      Dental examinations were conducted by two dental students using a dental mirror, dental
      explorer and dental probe, and included the following parameters:

        -  Oral hygiene - measured by the plaque index (PI), as described previously,18 only on
           buccal surfaces.

        -  Periodontal status - measured by the gingival index (GI), as described previously

        -  Caries status measured by the DMFT/ dmft index (D=decay, M=missing, F=filling; T per
           tooth) in permanent/ primary dentition . All the values were collected and recorded in a
           data table.

      Saliva collection Saliva was collected in a quiet room between 0800 and 1230 hr. The children
      were asked to collect saliva in their mouths and to spit it into a wide test tube for 3 min.
      The saliva was immediately stored at 4°C and further kept at -80°C until analysis. Data were
      collected onto paper charts, which were transferred to a computer program.

      Bacteria testing Bacterial DNA was extracted from saliva using DNA extraction kit (Qiagen).
      The DNA was then tested using specific primers for total bacteria: Strep. Mutans,
      Lactobacillus and Fusobacterium nucleatum using quantitative real-time polymerase chain
      reaction.

      Salivary cytokine quantification Salivary levels of human TNF- α, IL-10, IL-8, and IL-6 were
      measured using ELISA kits according to manufacturer instructions (R&D systems, Minneapolis,
      MN, US). The saliva samples were transferred to the periodontal laboratory in Hadassah Ein
      Kerem Hospital for analysis. The ELISA method works by a cytokine carbonate-specific
      monoclonal antibody placed in a 96-well plate. After dilution of µL 83 saliva to 10 ml PBS of
      antibody capture, the active solution is placed with the antibody in the well, and covered
      overnight at room temperature. Subsequently, we wash with µL 400 (buffer wash) and repeat
      this step three times. We then block the wells by adding µL 300 diluent reagent for one hour
      at room temperature, and wash again as described above. We dilute at 100 µL, cover and
      incubate for 2 hours at room temp. We then rinse again, and add detection antibody at a
      concentration of 166 µL to 10 ml, cover and incubate for 2 hours. Next, we add biotin-related
      antibody, followed by HRP-streptavidin conjugate, and TMB as a substrate. The reaction is
      stopped by acid sulfuric and quantified using 650nm-OD490.

      Statistical analysis All the experiments were performed in triplicate. The data were
      stratified according to age, DMF, PI and GI. Three age groups were set according to dental
      age: primary dentition - ages 0-6 years; mixed dentition - ages 6-11 years; and permanent
      dentition - above age 11 years. DMFT was stratified by ≤2 and >2. The PI was stratified by ≤1
      and >1. GI was stratified by equal to zero versus above zero. The data were analysed using a
      statistical software package (SigmaStat, Jandel Scientific, San Rafael, CA, USA). One-way
      repeated measure analysis of variance (RM ANOVA) was applied to test the significance of the
      differences between the treated groups. When significant results were found, inter-group
      differences were tested for significance using the Student's t-test and the Bonferroni
      correction for multiple testing. Statistical significance was set at p<0.05.
    

Observational model: Case-Only
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        healthy children aged 4-12 visiting for routine dental treatment at hadassah medical center
      
Keywords: cytokines; saliva; gingivitis