Diagnostic Biomarkers Related to Periodontal Disease Activity in Diabetic

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.