This randomized controlled clinical pilot study was conducted at Dr. Peset University Hospital in Valencia (Spain). Study protocol was approved by Dr. Peset Hospital Clinic Research Committee, and participants signed an approved consent form to participate in the study. Fifty three subjects with type 1 diabetes and moderate to severe periodontitis (25 females and 28 males) ranging in age from 19 to 60 (mean 35 ± 9 years) were recruited from the endocrinology division for this single-blind study. Patients with type 1 diabetes were diagnosed according to the criteria published by the American Diabetes Association in 1997 (18
), and they were treated by insulin, diet, and physical exercise recommendations.
Participants had diabetes for more than 1 year, and none of them had other major illnesses or severe diabetic complications. Patients had not taken antibiotics for at least 3 months prior to baseline and did not have any active infection. A panoramic radiograph was taken to assure that neither extensive caries nor periapical lesions were present. Eligible subjects had 14 or more natural teeth, of which at least five had a site with probing pocket depth (PPD) ≥ 5 mm and clinical attachment level (CAL) ≥ 3 mm. From this point, subjects with moderate to severe periodontal disease were included. They had not had periodontal treatment or professional cleaning of the teeth for at least 1 year prior to the study. Pregnant and breast feeding women were excluded. Twenty patients (38%) had good diabetic control (HbA1c < 7%), 12 individuals (22%) had moderate control (HbA1c between 7% and 8%), and 40% of the sample (21 diabetic individuals) had poor metabolic control (HbA1c > 8%) according to the American Diabetes Association criteria (19
). Most of the patients selected were nonsmokers (32 patients), some smoked less than 15 cigarettes per day (11 patients), and the rest were heavy smokers consuming 15 or more cigarettes per day (10 patients) ().
Laboratory and periodontal examination
Fasting venous blood was collected in vacuum tubes early in the morning, and high-sensitivity C-reactive protein (hs-CRP) was measured. Plasma hs-CRP levels were assessed using a kit with specific high-sensitivity methodology in a spectrophotometer according to the manufacturer’s instructions. The test samples were treated with a specific antibody to human CRP in a suitable buffer. The turbidity induced by the formation of immune complexes was measured at 546 nm, and the values were automatically calculated from a known standard. The lower detection limit for the assay was 0.1 mg/l. A commercial control serum was used to verify the assay performance.
Patients also received an oral soft tissue examination including periodontal measurements of plaque index (PI), bleeding on probing (BOP), PPD, and CAL for all teeth present. O’Leary PI was measured in four areas per tooth (mesiobuccal, midbuccal, distobuccal, and midlingual) (20
), and the other periodontal parameters were registered on six sites by tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual).
All patients with type 1 diabetes from Dr. Peset Hospital were screened (One hundred thirty-six), and 72 were found to match the selection criteria for this pilot study. They were told not to change their diet, exercise, or insulin dose unless absolutely necessary and to inform investigators if any change occurred. Hs-CRP values at the screening time, 3 month previous to the beginning of the study, were obtained from the medical records of the patients to evaluate changes in this parameter before the initiation of the investigation. These hs-CRP values were obtained in the same center and were made by the same laboratory that was used for this clinical protocol. The sample was randomized, allowing the subjects to self-select a coded number contained in an envelope; this number identified the group to which the patient was assigned (group 1 or 2). Baseline examination was performed 3 months after screening and within the 30 days prior to the beginning of the periodontal treatment. All periodontal measurements were taken by only one trained periodontist. Intra-examiner reproducibility was calculated and showed that periodontal clinical attachment measurements were in agreement within 2 mm more than 90% of the time. This clinician was blinded to the treatment applied in each patient and care was taken that subjects did not disclose their group category (21
Group 1 had baseline Hs-CRP measured just before the beginning of the periodontal treatment. Subjects were instructed on the modified Bass brushing technique and inter proximal cleaning. After that, scaling and root planning (SRP) under local anesthesia was performed by two trained dental hygienists using ultrasonic devices and manual Gracey curets (Hu-Friedy®, Chicago USA). SRP was scheduled in one or two sessions 1 week apart according to the periodontal disease severity and the number of teeth present. No less than 30 min were assigned to each quadrant. Chlorhexidine rinses 0,2% were prescribed after SRP (20 ml during 30 s, twice daily) and maintained for 12 weeks to the end of the clinical protocol. No other rinses or toothpaste was used during the study. Individuals were placed on doxycycline 100 mg (b.i.d. for the first day and then one capsule per day thereafter) for 15 days.
Group 2 had the same treatment as group 1 with the exception of the doxycycline which was not used in this group.
Twelve weeks after treatment, blood samples were taken again, and hs-CRP was analyzed. At the same time, periodontal parameters were measured again. Periodontal surgical treatment was recommended to patients with probing pocket depths ≥6mm. Compliance with use of oral hygiene devices, chlorhexidine, and doxycycline was assessed with a personal oral interview with the participants. It was classified as good (instructions were followed), fair or poor (prescriptions were not followed).
At the end of the study, 19 subjects were dropped out. Nine patients did not follow postoperative anti-infectious treatment (clorhexidine rinses and doxycycline if prescribed), 3 subjects had active acute infections during post-treatment period and 7 patients had an inadequate baseline laboratory exam (high-sensitivity C-reactive protein test could not be performed). Finally, baseline hs-CRP could be obtained from 53 patients and post-treatment hs-CRP was measured in 47 individuals (24 patient from group 1 and 23 patients from group 2).
First, laboratory and periodontal data were analyzed for distribution. Kolmogorov-Smirnov test showed that hs-CRP values before and after periodontal treatment (primary variables) did not follow a normal distribution. Consequently, hs-CRP data was expressed as median with minimum and maximum and non-parametric statistics were used to analyze the results. Periodontal parameters (secondary variables) followed a normal distribution and mean with standard deviation values were given.
Hs-CRP changes after non surgical periodontal treatment were studied by using the Wilcoxon test, and differences were considered significant when p<0.05. Power analysis was performed.
Baseline correlations between non-parametric hs-CRP values and periodontal measurements were analyzed with the Spearman test. Correlations were considered statistically significant when p<0.05. Correlation between hs-CRP and independent variables such as age, sex, diabetes duration, diabetes control, diabetes complications and smoking habits was performed with the same test.