This study was designed as double-blind, randomized, crossover study to evaluate the influence of a triclosan-containing toothpaste in the profile of osteo-immunoinflammatory mediators in the PICF of smoker individuals and in the clinical measurements during the progression of experimental peri-implant mucositis. This investigation was approved by the ethics committee of Paulista University . Patient recruitment started in July 2013 and was completed by the end of September 2014. Clinical procedures and evaluations were carried out between September 2013 and November 2014. Data entry and statistical analyses were performed in April 2015. All the patients in the study were recruited from the patients referred to Paulista University. Patients were thoroughly informed of the nature, potential risks and benefits of their participation in the study, and they each signed an informed consent document. During this 3-week period, participants were randomly assigned to two groups by a computer-generated list: Triclosan (n=13): triclosan/copolymer/fluoride toothpaste or Placebo (n=13): fluoride toothpaste, by filling the individual silicone stent with the respective toothpaste, according to the experimental group, and allowing it to come into contact with the implant area for 2 min, three times per day. Conventional tooth brushing was performed in the non-stent areas.

After 3 weeks, a professional prophylaxis was performed and a wash-out period of 30 days was established. All patients restarted their optimal mechanical plaque control practices to reach pre-experimental levels of oral cleanliness and gingival/mucosal health. Then, a second experimental 3-week period of undisturbed plaque accumulation around the implants was established and the experimental groups were exchanged. After that, a new professional prophylaxis was performed. Patients were blinded to the therapies.All evaluations (clinical and immunoenzimatic) were performed at baseline, 3, 7, 14 and 21 days of each period of experimental mucositis induction. Levels of osteo-immunoinflammatory mediators were considered primary outcome variable. The number of patients included was based on previous crossover investigations that found differences in the crevicular fluid levels of osteo-immunoinflammatory markers in different clinical status. The same examiner (SPP), who was blinded to the groups, performed all clinical measurements. To perform the intra-examiner calibration, 15 non-study individuals presenting dental implants were selected. The examiner measured the PD of all individuals twice within 24 hours. The intra-class correlation was calculated as 95% reproducibility.

Individual stents were prepared to standardize the location of periodontal probe in order to evaluate the following parameters at four sites of the experimental dental implants at baseline, 3, 7, 14, 21 days follow-ups: 1) plaque index (PI/%): dichotomous plaque index along the mucosal margin around implants, 2) Bleeding on probing (BOP,%):dichotomous index of bleeding during probing around implants, 3) Position of the peri-implant margin (PPM/mm): distance from the stent to the peri-implant margin; 4) Relative clinical attachment level (RCAL/mm): distance from the stent to the bottom of the peri-implant pocket; and 5) Peri-implant Probing depth (PD,mm): calculated by deducting PPM from RCAL.

Levels of interferon, interleukin (IL)-17, IL-1β, IL-10, IL-6, IL-8, tumor necrosis factor (TNF)-α , osteoprotegerin (OPG), osteocalcin (OC), osteopontin (OPN) , matrix metalloproteinase (MMP)-2, MMP-9 , transforming growth factor (TGF)-β , soluble receptor activator of nuclear factor ligand (RANKL) and crosslinked telopeptide of type I collagen (ICTP) in the PICF were determined using the MAGpix™ instrument and Xponent® software . The mean concentration of each mediator was calculated using the individual as a statistical unit and expressed as pg/ml.

All analyses were completed using SAS program release 9.1 . Data were examined for normality using the Kolmogorov-Smirnov test, and those that achieved normality were analysed using parametric methods. FMPS and FMBS measured before the beginning of each period of experimental mucositis in both groups were compared using the Wilcoxon test. For the other clinical parameters (PI, BoP, PPM, RCAL and PD), ANOVA two way/Tukey test was used to detect differences between groups and periods. Levels of osteo-immunoinflammatory markers between groups and among follow-ups were compared using the Wilcoxon and Friedman test, respectively. An experimental level of significance was determined at 5%.