Diagnostic Biomarkers Related to Periodontal Disease Activity in Diabetic
Condition: Periodontitis; Type 2 Diabetes Mellitus
MeSH terms: Periodontitis; Periodontal Diseases; Diabetes Mellitus, Type 2
Study type: Interventional
Phase: Phase 3

Summary: 
      The purpose of the study was to monitor the activity of periodontal disease and suggest
      potential biomarkers related to active periodontal disease in patients with chronic
      periodontitis (PD) associated or not with type 2 diabetes mellitus (DM), based on the
      evaluation of the profile of gene expression of periodontal sites and the evaluation of
      inflammatory salivary proteins. Two hundred and five periodontal patients were enrolled, but
      only 41 exhibited ≥ 1 mm attachment loss in at least three periodontal site (active sites) 2
      months after non-surgical periodontal therapy. The final sample was: 21 patients with chronic
      periodontitis (PD group) and 20 with chronic periodontitis and diabetes (PD+DM group).
      Fifteen periodontal- and systemically healthy patients were included as control group. Saliva
      collection, glycated hemoglobin measurement, periodontal examination and radiographs were
      conducted before and 2 months after non-surgical periodontal therapy. Radiographic
      subtraction was performed from pairs of the radiographs. Measurements of the areas with
      density loss were recorded. Gingival biopsies of active and non-active sites with similar
      clinical parameters were harvested for Real Time Polymerase Chain Reaction Array gene
      expression analysis. Saliva samples were analyzed by Multiplex Cytokine Profiling Immunoassay
      for analysis of protein expression. The clinical attachment loss mean was higher in the PD+DM
      group (p<0.05). There was a high correlation between clinical attachment loss and darkened
      radiographic areas in active sites of the PD group and PD+DM group. When compared PD group to
      PD+DM, patients with diabetes had an up-regulated profile. Active sites of the PD group
      showed nine genes (specific chemokines, interleukins and receptors) differentially expressed
      with an up-regulated profile. Active sites of the PD+DM group showed six genes (specific
      chemokines, interleukins and receptors) differentially expressed with an up-regulated
      profile. After periodontal therapy, there was a reduction of some salivary proteins in both
      periodontal groups, but not significant. In conclusion, it was possible to identify genes
      differentially expressed in active sites from both groups, which may be considered useful in
      indicating potential biomarkers for the diagnosis of periodontitis; salivary proteins show a
      trend in distinguishing the standard of health and disease and may be used in the future as
      potential biomarkers of periodontitis with or without diabetes.
    

Detailed description: 
      This case-control longitudinal study was carried out at University of São Paulo between March
      2009 and June 2012 as a joint collaboration of the Department of Oral Surgery and
      Periodontology and the Department of Internal Medicine, Division of Endocrinology and
      Metabolism. It was reviewed and approved by the Institutional Human Ethics Research Committee
      of the Ribeirão School of Dentistry - University of Sao Paulo (process n. 2009.1.88.58.7).

      The clinical attachment loss above 1 mm was determined according to the tolerance method
      adapted to the computerized periodontal probe, considering the standard deviation of 0.3 mm
      for the electronic probe multiplied by 3. Teeth with prosthesis or furcation lesions were not
      considered. The periodontal sites that had this clinical attachment were called active sites.

      Before beginning the initial clinical examination, a supragingival scaling with ultrasonic
      device was performed to facilitate the examination. The clinical parameters evaluated were:
      probing pocket depth, relative clinical attachment level and bleeding on probing were
      recorded at six sites per tooth with the aid of a computerized periodontal probe. To reduce
      the variations between baseline and 12-month evaluations, an acrylic stent was used to
      standardize the position of the computerized periodontal probe. Bleeding on probing was
      assessed according to presence or absence of bleeding up to 20 seconds after probing. The
      plaque index, presence or absence of biofilm, was recorded at four sites per tooth. It was
      also verified the furcation involvement with the aid of a manual periodontal probe.

      All clinical parameters were recorded two weeks after supragingival scaling (baseline) and
      two months after non-surgical periodontal therapy by one-blinded calibrated examiner. A
      calibration exercise was performed to achieve acceptable intraexaminer reproducibility.

      For the metabolic control assessment, the HbA1c levels were analyzed in patients from both
      groups at baseline and two months after non-surgical periodontal therapy, at the
      Endocrinology Clinic of the Ribeirão Preto School of Medicine, University of São Paulo. All
      diabetics were under the supervision of an endocrinologist advised to communicate any change
      in medicine intake or diet.

      A program of plaque control with dental prophylaxis and oral hygiene instruction, and the
      scaling and root planning sessions were performed by the same operator using curettes and an
      ultrasonic device in PD and PD+DM groups. All scaling and root planning procedures were
      inspected for a second operator. Oral hygiene was reviewed after a week and after a month of
      periodontal disinfection, followed by dental prophylaxis.

      In PD+DM group, the non-surgical periodontal therapy was associated with systemic doxycycline
      100 mg/day, for two weeks after an initial dose of 200 mg, started on the day before
      periodontal therapy. Patients of the PD group had no access to information about antibiotics
      administration to patients of the PD+DM group.

      Non-stimulated whole expectorated saliva was collected (~ 3 ml) from each subject into
      sterile tubes. Subjects were refrained from eating, drinking, and oral hygiene for 2 hours
      prior to saliva collection. Saliva samples were placed on ice immediately and aliquoted prior
      to freezing at -80º Celsius. Samples were thawed and analyzed within 6 months of collection.

      A complete series of radiographs was taken in each patient at baseline, using the paralleling
      technique. Two months after periodontal therapy, radiographs were taken with the same
      technique in teeth with active periodontal sites. Thereafter, the radiographs were digitized
      in tagged image file format (TIFF) on a scanner. Digital subtraction radiographs were
      performed with the baseline and 2-month radiographs using specific software. Only teeth with
      interproximal sites with periodontal disease activity were included in this analysis. Changes
      between radiographs were depicted as a darkened area for loss of alveolar bone mass. These
      areas were measured (mm2) using specific measurements software. As in the clinical
      examination, it was performed intra-examiner calibration for the measurements of areas of
      radiographic density loss.

      Gingival tissue samples were obtained from active sites of the each patient in both groups
      during regular periodontal surgery. The excised gingival collar was then carefully removed
      from the roots and the alveolar process. Gingival biopsy comprised epithelial and connective
      tissues. The samples are immediately submerged into liquid nitrogen to be then stored at -80º
      Celsius for RNA extraction and gene expression analysis.

      Total RNA from biopsies was extracted using the Trizol reagent according to the directions
      supplied by the manufacturer. From 1 µg of total RNA, a strand of complementary DNA (cDNA)
      was synthesized through a reverse transcription reaction according to the directions supplied
      by the manufacturer. At the end of this reaction, cDNA was stored at -20º Celsius for later
      use. The Real Time Polymerase Chain Reaction (PCR) Array allowed simultaneous analysis of 84
      genes involved in specific signaling pathways. The genes included in PCR array plates for
      human inflammatory cytokines and receptors.
    

Intervention name(s): non-surgical periodontal therapy + systemic doxycycline; Non-surgical periodontal therapy
Intervention type(s): Drug; Procedure
Intervention description(s): A program of plaque control with dental prophylaxis and oral hygiene instruction, and the scaling and root planning sessions were included in the non-surgical periodontal therapy. The scaling and root planning was performed by the same operator using curettes and an ultrasonic device, and it was inspected for a second operator. Oral hygiene was reviewed after a week and after a month of periodontal disinfection, followed by dental prophylaxis.
Non-surgical periodontal therapy was associated with systemic doxycycline 100 mg/day, for two weeks after an initial dose of 200 mg, started on the day before the first scaling and root planning session. Patients of the PD group had no access to information about antibiotics administration to patients of the PD+DM group.; A program of plaque control with dental prophylaxis and oral hygiene instruction, and the scaling and root planning sessions were included in the non-surgical periodontal therapy. The scaling and root planning was performed by the same operator using curettes and an ultrasonic device, and it was inspected for a second operator. Oral hygiene was reviewed after a week and after a month of periodontal disinfection, followed by dental prophylaxis.
Other intervention name(s): Doxilegrand, Legrand, São Bernardo do Campo, SP, Brazil.; Scaling and root planning
Intervention MeSH terms: Doxycycline
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Masking: Single (Participant)
Primary purpose: Diagnostic
Keywords: Periodontal Attachment Loss; Genes; Saliva; Diagnosis