Study design
The study was approved by the Ethics Commission (#AZ436/16-ek) of the Medical Faculty of the University of Leipzig. It is registered in the German register of clinical trials (DRKS00015239), and the full study protocol is deposited there. The clinical study was conducted as a randomized controlled trial with parallel design in a private dental practice. The examiner as well as the laboratory personnel were blinded. Seventy-two patients were randomly selected, screened and asked to participate in the study. Fourty-nine patients (30 male and 19 female) were willing and gave their written informed consent. The clinical trial was conducted in a private dental practice (Berlin, Germany). The principles outlined in the Declaration of Helsinki, as revised in 2008, were followed to obtain the informed consents and to conduct the clinical study. Patients with moderate to severe chronic periodontitis were included in the study (Armitage 1999) corresponding to stage II - III, grade B of the new classification scheme for periodontal and peri-implant diseases and conditions (Tonetti et al. 2018).
The same experienced dentist (C. F.) performed all SIs in both study groups. All assessment of the clinical data as well as the collection of biofilm and gingival crevicular fluid (GCF) was performed by a second experienced dentist (B. K.) blinded to the study groups. The intra-examiner calibration for reliability testing resulted in κ = 0.91 for repeated measurements of PD and AL in two quadrants of eight patients. A computer-generated randomization table was used for the recruitment and blinded the randomization of 49 participants either to the test (n = 24) or control groups (n = 25) with a 1:1 allocation ratio. An assistant of the dental practice performed the assignment to interventions and the documentation. To obtain the allocation concealment a sealed opaque envelope was used. The envelope contained the treatment number of the allocation table to the specific subject. The examiner did not perform the treatment procedures and was unaware of the treatment assignment.
Participants of the study
The inclusion criteria were age between 40 and 65 years, at least 16 natural teeth in function, periodontitis with probing depth ≤ 6 mm, at least 16 teeth with need for SI, interproximal plaque index (API, Lange et al. 1977) ≤ 35% at the baseline appointment after two appointments of professional prophylaxis with motivation and instruction, no diseases with influence on the periodontal disease, no diabetes mellitus, arthritis or allergies on used substances or products. Patients were excluded if they were pregnant or in the period of lactation, if they were smokers who smoked more than seven cigarettes per day or if they had a treatment with antibiotics within the six months prior to the study. Other exclusion criteria were: need for periodontal surgery or adjunctive antibiotic treatment to the SI and any periodontal treatment during the last year before the study.
Clinical procedures and sampling methods
In the test and control groups the clinical variables were recorded at three appointments: at baseline before SI (t1) as well as three (t2) and six months (t3) after SI. The GCF samples and the samples of subgingival biofilm were collected at the same time. The participants received full-mouth SI at sites with PD ≥ 4 mm in two sessions carried out within 24 hours using hand and sonic instruments (Hu-Friedy Manufacturing Co., Chicago, IL, USA and Dentsply Sirona, Bensheim, Germany) under local anaesthesia with articaine hydrochloride/epinephrine hydrochloride (Ultracain D-S, Sanofi-Aventis, Frankfurt/Main, Germany). The criterium was a bioacceptable root surface without clinically detectable nonmineralized and mineralized material on the root surface after SI. In addition in the test group, erythritol powder (Air-Flow® Plus Powder, EMS Nyon, Switzerland) was applied for five seconds per site using the Perioflow®handpiece with the Perioflow® nozzle with the Air-Flow® Master apparatus (all EMS Nyon, Switzerland) respecting the recommendations of the manufacturer. The nozzle was introduced into the periodontal pocket as described by Hägi et al. (2015). The powder was directed perpendicular to the root surface. Via inclination of the handpiece as well as via the flexibility of the plastic nozzle device the access of interproximal buccal and oral sites was possible and the bottom of the pocket (inclusion criteria ≤ 6 mm) could be reached.
During the first seven days after SI all patients used a chlorhexidine digluconate mouthrinse (Chlorhexamed forte 0.2%, GlaxoSmithKline Healthcare, Bühl, Germany) for one minute twice daily. After SI careful normal oral hygiene using toothbrush and interdental brushes was performed. An appointment of comprehensive supportive periodontal therapy with removal of biofilm and calculus, applying the erythritol powder with the perioflow handpiece as well as re-motivation and re-instruction took place three months after SI. In Fig. 1 the timeline of the study is presented.
In a six-point measurement per tooth (mesiobuccal, buccal, distobuccal, mesiooral, oral and distooral) the clinical variables PD, CAL and BOP of all teeth were recorded. For the assessment a manual periodontal probe (PCP-UNC 15, Hu-Friedy Manufacturing Co., Chicago, IL, U.S.A.) using a pressure of 0.25 N was used. The oral hygiene was recorded by using the API (Lange et al., 1977).
At each appointment, samples of the GCF and the subgingival biofilm were taken from the deepest site per quadrant at baseline. To sample GCF for the analysis of the biomarkers, sterile paper strips (Periopaper; Oraflow Inc., Smithtown, New York, U.S.A.) were placed at the entrance of the periodontal pocket for 30 s. As described by Griffiths (2003) this intracrevicular superficial method avoids the destruction of the subgingival biofilm in the periodontal pocket. After pooling the paper strips were placed into a tube with 100 µl protease inhibitor solution (Sigma Aldrich Chemie GmbH, München, Germany). To collect the subgingival biofilm at the same sites as for GCF sampling endodontic paper points (ISO 60, Roeko GmbH, Langenau, Germany) were inserted into the pocket until resistance was felt. The paper points were left in place for 30 s. The GCF samples were stored at -80 °C and the biofilm samples at -20 °C until analysis.
Laboratory analysis
Before analysis, the GCF samples were eluted overnight into 650 µl phosphate-buffered saline (+ proteinase inhibitors solution) at 4 °C. From the eluates, the levels of interleukin (IL)-1β and matrix-metalloproteinase (MMP)-8 were determined by using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems Europe Ltd., Abingdon, UK) according to the manufacturer’s instructions. The detection levels were 2 pg/site for IL-1β and 100 pg/site for MMP-8.
For microbiological analysis, DNA was extracted and a multiplex-realtime qPCR for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola was performed as described recently (Jentsch et al. 2020). The results are given as bacterial counts log10.
Statistical analysis
Based on the results of Chondros et al. (2009) for BOP with a difference of 11% at baseline a minimum of 20 volunteers per group would be necessary to detect a significant difference (p ≤ 0.05) with a test power of 80%. The primary outcome variable of the study is BOP six months after SI. Secondary outcome variables were changes of BOP after three months as well as PD, AL, of the number of sites with PD ≥ 5 mm, counts of the four selected periodontopathogenic bacteria, the levels of IL-1β and MMP-8 three and 6 months after the SI.
The statistical analysis of all clinical and laboratory data was performed with the help of the software SPSS ® Statistics 24.0 (IBM Corporation, New York, NY, U.S.A.). Non-parametric tests (Friedman-test for comparing several time-points within a group, Wilcoxon test for paired samples and Mann Whitney U–test for inter-group comparisons, respectively) were used for intra- and inter-group comparisons. The unit of analysis in all statistical tests was the individual participant. The level of significance was α ≤ 0.05.