<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT02220751</url>
  </required_header>
  <id_info>
    <org_study_id>2008/11033-9</org_study_id>
    <secondary_id>2008/11033-9</secondary_id>
    <nct_id>NCT02220751</nct_id>
  </id_info>
  <brief_title>Diagnostic Biomarkers Related to Periodontal Disease Activity in Diabetic</brief_title>
  <official_title>Diagnostic Biomarkers Related to Periodontal Disease Activity in Diabetic</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>University of Sao Paulo</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
    <collaborator>
      <agency>Fundação de Amparo à Pesquisa do Estado de São Paulo</agency>
      <agency_class>Other</agency_class>
    </collaborator>
  </sponsors>
  <source>University of Sao Paulo</source>
  <oversight_info>
    <has_dmc>Yes</has_dmc>
  </oversight_info>
  <brief_summary>
    <textblock>
      The purpose of the study was to monitor the activity of periodontal disease and suggest&#xD;
      potential biomarkers related to active periodontal disease in patients with chronic&#xD;
      periodontitis (PD) associated or not with type 2 diabetes mellitus (DM), based on the&#xD;
      evaluation of the profile of gene expression of periodontal sites and the evaluation of&#xD;
      inflammatory salivary proteins. Two hundred and five periodontal patients were enrolled, but&#xD;
      only 41 exhibited ≥ 1 mm attachment loss in at least three periodontal site (active sites) 2&#xD;
      months after non-surgical periodontal therapy. The final sample was: 21 patients with chronic&#xD;
      periodontitis (PD group) and 20 with chronic periodontitis and diabetes (PD+DM group).&#xD;
      Fifteen periodontal- and systemically healthy patients were included as control group. Saliva&#xD;
      collection, glycated hemoglobin measurement, periodontal examination and radiographs were&#xD;
      conducted before and 2 months after non-surgical periodontal therapy. Radiographic&#xD;
      subtraction was performed from pairs of the radiographs. Measurements of the areas with&#xD;
      density loss were recorded. Gingival biopsies of active and non-active sites with similar&#xD;
      clinical parameters were harvested for Real Time Polymerase Chain Reaction Array gene&#xD;
      expression analysis. Saliva samples were analyzed by Multiplex Cytokine Profiling Immunoassay&#xD;
      for analysis of protein expression. The clinical attachment loss mean was higher in the PD+DM&#xD;
      group (p&lt;0.05). There was a high correlation between clinical attachment loss and darkened&#xD;
      radiographic areas in active sites of the PD group and PD+DM group. When compared PD group to&#xD;
      PD+DM, patients with diabetes had an up-regulated profile. Active sites of the PD group&#xD;
      showed nine genes (specific chemokines, interleukins and receptors) differentially expressed&#xD;
      with an up-regulated profile. Active sites of the PD+DM group showed six genes (specific&#xD;
      chemokines, interleukins and receptors) differentially expressed with an up-regulated&#xD;
      profile. After periodontal therapy, there was a reduction of some salivary proteins in both&#xD;
      periodontal groups, but not significant. In conclusion, it was possible to identify genes&#xD;
      differentially expressed in active sites from both groups, which may be considered useful in&#xD;
      indicating potential biomarkers for the diagnosis of periodontitis; salivary proteins show a&#xD;
      trend in distinguishing the standard of health and disease and may be used in the future as&#xD;
      potential biomarkers of periodontitis with or without diabetes.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      This case-control longitudinal study was carried out at University of São Paulo between March&#xD;
      2009 and June 2012 as a joint collaboration of the Department of Oral Surgery and&#xD;
      Periodontology and the Department of Internal Medicine, Division of Endocrinology and&#xD;
      Metabolism. It was reviewed and approved by the Institutional Human Ethics Research Committee&#xD;
      of the Ribeirão School of Dentistry - University of Sao Paulo (process n. 2009.1.88.58.7).&#xD;
&#xD;
      The clinical attachment loss above 1 mm was determined according to the tolerance method&#xD;
      adapted to the computerized periodontal probe, considering the standard deviation of 0.3 mm&#xD;
      for the electronic probe multiplied by 3. Teeth with prosthesis or furcation lesions were not&#xD;
      considered. The periodontal sites that had this clinical attachment were called active sites.&#xD;
&#xD;
      Before beginning the initial clinical examination, a supragingival scaling with ultrasonic&#xD;
      device was performed to facilitate the examination. The clinical parameters evaluated were:&#xD;
      probing pocket depth, relative clinical attachment level and bleeding on probing were&#xD;
      recorded at six sites per tooth with the aid of a computerized periodontal probe. To reduce&#xD;
      the variations between baseline and 12-month evaluations, an acrylic stent was used to&#xD;
      standardize the position of the computerized periodontal probe. Bleeding on probing was&#xD;
      assessed according to presence or absence of bleeding up to 20 seconds after probing. The&#xD;
      plaque index, presence or absence of biofilm, was recorded at four sites per tooth. It was&#xD;
      also verified the furcation involvement with the aid of a manual periodontal probe.&#xD;
&#xD;
      All clinical parameters were recorded two weeks after supragingival scaling (baseline) and&#xD;
      two months after non-surgical periodontal therapy by one-blinded calibrated examiner. A&#xD;
      calibration exercise was performed to achieve acceptable intraexaminer reproducibility.&#xD;
&#xD;
      For the metabolic control assessment, the HbA1c levels were analyzed in patients from both&#xD;
      groups at baseline and two months after non-surgical periodontal therapy, at the&#xD;
      Endocrinology Clinic of the Ribeirão Preto School of Medicine, University of São Paulo. All&#xD;
      diabetics were under the supervision of an endocrinologist advised to communicate any change&#xD;
      in medicine intake or diet.&#xD;
&#xD;
      A program of plaque control with dental prophylaxis and oral hygiene instruction, and the&#xD;
      scaling and root planning sessions were performed by the same operator using curettes and an&#xD;
      ultrasonic device in PD and PD+DM groups. All scaling and root planning procedures were&#xD;
      inspected for a second operator. Oral hygiene was reviewed after a week and after a month of&#xD;
      periodontal disinfection, followed by dental prophylaxis.&#xD;
&#xD;
      In PD+DM group, the non-surgical periodontal therapy was associated with systemic doxycycline&#xD;
      100 mg/day, for two weeks after an initial dose of 200 mg, started on the day before&#xD;
      periodontal therapy. Patients of the PD group had no access to information about antibiotics&#xD;
      administration to patients of the PD+DM group.&#xD;
&#xD;
      Non-stimulated whole expectorated saliva was collected (~ 3 ml) from each subject into&#xD;
      sterile tubes. Subjects were refrained from eating, drinking, and oral hygiene for 2 hours&#xD;
      prior to saliva collection. Saliva samples were placed on ice immediately and aliquoted prior&#xD;
      to freezing at -80º Celsius. Samples were thawed and analyzed within 6 months of collection.&#xD;
&#xD;
      A complete series of radiographs was taken in each patient at baseline, using the paralleling&#xD;
      technique. Two months after periodontal therapy, radiographs were taken with the same&#xD;
      technique in teeth with active periodontal sites. Thereafter, the radiographs were digitized&#xD;
      in tagged image file format (TIFF) on a scanner. Digital subtraction radiographs were&#xD;
      performed with the baseline and 2-month radiographs using specific software. Only teeth with&#xD;
      interproximal sites with periodontal disease activity were included in this analysis. Changes&#xD;
      between radiographs were depicted as a darkened area for loss of alveolar bone mass. These&#xD;
      areas were measured (mm2) using specific measurements software. As in the clinical&#xD;
      examination, it was performed intra-examiner calibration for the measurements of areas of&#xD;
      radiographic density loss.&#xD;
&#xD;
      Gingival tissue samples were obtained from active sites of the each patient in both groups&#xD;
      during regular periodontal surgery. The excised gingival collar was then carefully removed&#xD;
      from the roots and the alveolar process. Gingival biopsy comprised epithelial and connective&#xD;
      tissues. The samples are immediately submerged into liquid nitrogen to be then stored at -80º&#xD;
      Celsius for RNA extraction and gene expression analysis.&#xD;
&#xD;
      Total RNA from biopsies was extracted using the Trizol reagent according to the directions&#xD;
      supplied by the manufacturer. From 1 µg of total RNA, a strand of complementary DNA (cDNA)&#xD;
      was synthesized through a reverse transcription reaction according to the directions supplied&#xD;
      by the manufacturer. At the end of this reaction, cDNA was stored at -20º Celsius for later&#xD;
      use. The Real Time Polymerase Chain Reaction (PCR) Array allowed simultaneous analysis of 84&#xD;
      genes involved in specific signaling pathways. The genes included in PCR array plates for&#xD;
      human inflammatory cytokines and receptors.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date>March 2009</start_date>
  <completion_date type="Actual">June 2012</completion_date>
  <primary_completion_date type="Actual">September 2011</primary_completion_date>
  <phase>Phase 3</phase>
  <study_type>Interventional</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <allocation>Non-Randomized</allocation>
    <intervention_model>Parallel Assignment</intervention_model>
    <primary_purpose>Diagnostic</primary_purpose>
    <masking>Single (Participant)</masking>
  </study_design_info>
  <primary_outcome>
    <measure>clinical attachment level</measure>
    <time_frame>baseline and two months</time_frame>
    <description>relative clinical attachment level (rCAL) was recorded at six sites per tooth with the aid of a computerized periodontal probe.</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Gene expression</measure>
    <time_frame>Two months</time_frame>
    <description>Gingival tissue samples were obtained from periodontal sites (active or non-active) of the each patient in both groups during regular periodontal surgery. The samples are immediately submerged into liquid nitrogen to be then stored at -80º Celsius for RNA extraction and cDNA synthesis. The Real Time-PCR Array allowed simultaneous analysis of 84 genes involved in specific signaling pathways, including specific human inflammatory cytokines and receptors.</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Salivary proteins levels</measure>
    <time_frame>baseline and two months</time_frame>
    <description>Non-stimulated whole expectorated saliva was collected (~ 3 ml) from each subject into sterile tubes and stored at -80º Celsius. The salivary inflammatory proteins levels were identified simultaneously using Multiplex Cytokine Profiling Assay, which allows the simultaneous detection of multiple analytes in saliva sample size.</description>
  </secondary_outcome>
  <other_outcome>
    <measure>Probing pocket depth</measure>
    <time_frame>baseline and two months</time_frame>
    <description>It was recorded at six sites per tooth with the aid of a computerized periodontal probe.</description>
  </other_outcome>
  <other_outcome>
    <measure>Bleeding on probing</measure>
    <time_frame>baseline and two months</time_frame>
    <description>It was recorded at six sites per tooth with the aid of a computerized periodontal probe, and it was assessed according to presence or absence of bleeding up to 20 seconds after probing.</description>
  </other_outcome>
  <other_outcome>
    <measure>Plaque index</measure>
    <time_frame>baseline and two months</time_frame>
    <description>It was recorded at four sites per tooth to assess presence or absence of dental biofilm.</description>
  </other_outcome>
  <other_outcome>
    <measure>Glycated hemoglobin level</measure>
    <time_frame>baseline and two months</time_frame>
    <description>Metabolic control assessment</description>
  </other_outcome>
  <other_outcome>
    <measure>Density loss</measure>
    <time_frame>baseline and two months</time_frame>
    <description>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. 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. Changes between radiographs were depicted as a darkened area for loss of alveolar bone mass. These areas were measured (mm2) using specific measurements software.</description>
  </other_outcome>
  <number_of_arms>3</number_of_arms>
  <enrollment type="Actual">56</enrollment>
  <condition>Periodontitis</condition>
  <condition>Type 2 Diabetes Mellitus</condition>
  <arm_group>
    <arm_group_label>chronic periodontitis</arm_group_label>
    <arm_group_type>Placebo Comparator</arm_group_type>
    <description>Non-surgical periodontal therapy.</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Chronic periodontitis + type 2 diabetes</arm_group_label>
    <arm_group_type>Active Comparator</arm_group_type>
    <description>Non-surgical periodontal therapy + systemic doxycycline 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 first scaling and root planning session.</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Control</arm_group_label>
    <arm_group_type>No Intervention</arm_group_type>
    <description>Periodontal- and systemically healthy patients were included as control group.</description>
  </arm_group>
  <intervention>
    <intervention_type>Drug</intervention_type>
    <intervention_name>non-surgical periodontal therapy + systemic doxycycline</intervention_name>
    <description>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.&#xD;
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.</description>
    <arm_group_label>Chronic periodontitis + type 2 diabetes</arm_group_label>
    <other_name>Doxilegrand, Legrand, São Bernardo do Campo, SP, Brazil.</other_name>
  </intervention>
  <intervention>
    <intervention_type>Procedure</intervention_type>
    <intervention_name>Non-surgical periodontal therapy</intervention_name>
    <description>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.</description>
    <arm_group_label>chronic periodontitis</arm_group_label>
    <other_name>Scaling and root planning</other_name>
  </intervention>
  <eligibility>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  adults aged between 35 to 65 years old;&#xD;
&#xD;
          -  a minimum of 14 natural teeth, 10 of which should be posterior teeth;&#xD;
&#xD;
          -  periodontitis case definition was the presence of five teeth with a probing pocket&#xD;
             depth of ≥ 5 mm and clinical attachment loss of ≥ 3 mm;&#xD;
&#xD;
          -  type 2 diabetes for at least 5 years and with glycated hemoglobin level &gt; 7%.&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  smoking within the last 5 years;&#xD;
&#xD;
          -  pregnancy or lactating;&#xD;
&#xD;
          -  use of antibiotics or periodontal therapy in the previous six months;&#xD;
&#xD;
          -  concomitant medical therapy, except for diabetic condition;&#xD;
&#xD;
          -  other inflammatory conditions;&#xD;
&#xD;
          -  major diabetic complications such as retinopathy, nephropathy, neuropathy and&#xD;
             atherosclerosis.&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>35 Years</minimum_age>
    <maximum_age>65 Years</maximum_age>
    <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Priscila P Costa, PhD</last_name>
    <role>Principal Investigator</role>
    <affiliation>Department of Oral Surgery and Periodontology - Ribeirão Preto School of Dentistry, University of São Paulo</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>Mario Taba Jr</name>
      <address>
        <city>Ribeirao Preto</city>
        <state>Sao Paulo</state>
        <zip>14040-904</zip>
        <country>Brazil</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>Brazil</country>
  </location_countries>
  <verification_date>August 2014</verification_date>
  <study_first_submitted>August 18, 2014</study_first_submitted>
  <study_first_submitted_qc>August 18, 2014</study_first_submitted_qc>
  <study_first_posted type="Estimate">August 20, 2014</study_first_posted>
  <last_update_submitted>August 18, 2014</last_update_submitted>
  <last_update_submitted_qc>August 18, 2014</last_update_submitted_qc>
  <last_update_posted type="Estimate">August 20, 2014</last_update_posted>
  <responsible_party>
    <responsible_party_type>Principal Investigator</responsible_party_type>
    <investigator_affiliation>University of Sao Paulo</investigator_affiliation>
    <investigator_full_name>Priscila Paganini Costa</investigator_full_name>
    <investigator_title>PhD</investigator_title>
  </responsible_party>
  <keyword>Periodontal Attachment Loss</keyword>
  <keyword>Genes</keyword>
  <keyword>Saliva</keyword>
  <keyword>Diagnosis</keyword>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Periodontitis</mesh_term>
    <mesh_term>Periodontal Diseases</mesh_term>
    <mesh_term>Diabetes Mellitus, Type 2</mesh_term>
  </condition_browse>
  <intervention_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Doxycycline</mesh_term>
  </intervention_browse>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT02220751
org study id: 2008/11033-9
secondary id: 2008/11033-9
nct id: NCT02220751
lead sponsor: 
collaborator: 
has dmc: Yes
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.
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.
allocation: Non-Randomized
intervention model: Parallel Assignment
primary purpose: Diagnostic
masking: Single (Participant)
measure: clinical attachment level
time frame: baseline and two months
description: relative clinical attachment level (rCAL) was recorded at six sites per tooth with the aid of a computerized periodontal probe.
measure: Gene expression
time frame: Two months
description: Gingival tissue samples were obtained from periodontal sites (active or non-active) of the each patient in both groups during regular periodontal surgery. The samples are immediately submerged into liquid nitrogen to be then stored at -80º Celsius for RNA extraction and cDNA synthesis. The Real Time-PCR Array allowed simultaneous analysis of 84 genes involved in specific signaling pathways, including specific human inflammatory cytokines and receptors.
measure: Salivary proteins levels
time frame: baseline and two months
description: Non-stimulated whole expectorated saliva was collected (~ 3 ml) from each subject into sterile tubes and stored at -80º Celsius. The salivary inflammatory proteins levels were identified simultaneously using Multiplex Cytokine Profiling Assay, which allows the simultaneous detection of multiple analytes in saliva sample size.
measure: Probing pocket depth
time frame: baseline and two months
description: It was recorded at six sites per tooth with the aid of a computerized periodontal probe.
measure: Bleeding on probing
time frame: baseline and two months
description: It was recorded at six sites per tooth with the aid of a computerized periodontal probe, and it was assessed according to presence or absence of bleeding up to 20 seconds after probing.
measure: Plaque index
time frame: baseline and two months
description: It was recorded at four sites per tooth to assess presence or absence of dental biofilm.
measure: Glycated hemoglobin level
time frame: baseline and two months
description: Metabolic control assessment
measure: Density loss
time frame: baseline and two months
description: 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. 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. Changes between radiographs were depicted as a darkened area for loss of alveolar bone mass. These areas were measured (mm2) using specific measurements software.
arm group label: chronic periodontitis
arm group type: Placebo Comparator
description: Non-surgical periodontal therapy.
arm group label: Chronic periodontitis + type 2 diabetes
arm group type: Active Comparator
description: Non-surgical periodontal therapy + systemic doxycycline 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 first scaling and root planning session.
arm group label: Control
arm group type: No Intervention
description: Periodontal- and systemically healthy patients were included as control group.
intervention type: Drug
intervention name: non-surgical periodontal therapy + systemic doxycycline
description: 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.
arm group label: Chronic periodontitis + type 2 diabetes
other name: Doxilegrand, Legrand, São Bernardo do Campo, SP, Brazil.
intervention type: Procedure
intervention name: Non-surgical periodontal therapy
description: 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.
arm group label: chronic periodontitis
other name: Scaling and root planning
criteria: 
gender: All
minimum age: 35 Years
maximum age: 65 Years
healthy volunteers: Accepts Healthy Volunteers
last name: Priscila P Costa, PhD
role: Principal Investigator
affiliation: Department of Oral Surgery and Periodontology - Ribeirão Preto School of Dentistry, University of São Paulo
facility: 
country: Brazil
responsible party type: Principal Investigator
investigator affiliation: University of Sao Paulo
investigator full name: Priscila Paganini Costa
investigator title: PhD
mesh term: Periodontitis
mesh term: Periodontal Diseases
mesh term: Diabetes Mellitus, Type 2
mesh term: Doxycycline