This clinical trial respected the principles of the Declaration of Helsinki on human experimentation and was carried out in accordance with Good Clinical Practice. The Ethical Committee of the University Medical Center Freiburg verified this trial with a positive vote (EK No. 317/14). All enrolled participants signed an informed consent form as well as a data privacy statement. This report follows the criteria of the CONSORT statement .
This trial was part of a larger study which focused on clinical parameters (PD, CAL and BOP) comparing two treatment methods, air-polishing using a new trehalose powder and sonic scaling as control. Furthermore, participants rated both treatments in terms of comfort on a visual analogue scale. It was conducted as a single-blinded randomized clinical trial over six months at the Center for Dental Medicine, Department of Operative Dentistry and Periodontology, University Medical Center Freiburg, Germany . Using a split-mouth design, one tooth was treated subgingivally using an air-polishing device with trehalose powder (test group) while another tooth in the opposite quadrant was treated with a sonic scaler (control group). This microbiological part was added as a pilot study, so a subgroup was formed within the clinical study. Microbiological samples from ten patients were analyzed. The samples were taken from the periodontal site with the deepest pocket depth (PD). Samples were taken before and immediately after treatment, and after three and six months. The total number of bacteria and the change in the bacterial composition were analyzed. All patients received professional supragingival tooth cleaning after three months. Additionally, a follow-up treatment of persisting pockets with PD > 4 mm or 4 mm and bleeding on probing took place after three months. Treatment was performed by the same method used at baseline.
Fifty-two individuals were recruited during periodontal maintenance therapy from the patient population at the Department of Dental Medicine, University Medical Center Freiburg (see Fig. S1 CONSORT Flow Diagram). All participants needed to meet the following inclusion criteria:
chronic periodontitis patients undergoing maintenance therapy .
completed cause-related phase of periodontal therapy within the last 2 years.
2 single-rooted, non-adjacent teeth from different quadrants with PD = 5 mm and positive bleeding on probing or > 5 mm with or without positive bleeding on probing [22,23,24].
no systemic antibiotic treatment during the last 12 weeks.
good general health (no major disease, e.g., untreated heart disease, poorly controlled diabetes, HIV, cancer, hemorrhagic conditions).
no pregnant or breastfeeding women.
smokers were included to a maximum of 30%.
Out of all included participants, ten were randomly chosen for microbiological examination including a maximum of 20% smokers.
In the test group a new trehalose powder (Lunos® Prophylaxis Powder Perio Combi, Dürr Dental SE, Bietigheim-Bissingen, Germany) was applied using an air-polishing device.
(Perio-Flow® handpiece with Perio-Flow® Nozzle EMS, Nyon, Switzerland). For the control group a sonic device was used for subgingival instrumentation (Sonic Flex, KaVo, Biberach/Riß, Germany).
The procedure assigned by randomization was carried out on the respective teeth for 20 s each, irrespective of air-polishing or sonic instrumentation. 3 months after intervention all participants received oral hygiene instructions by the same dentist and received professional supragingival cleaning. If there was a need for follow-up treatment (PD = 4 mm and positive BOP or PD > 4 mm), the assigned intervention was repeated in terms of a state of the art supportive periodontal treatment [1, 24].
Collection and storage of samples
Following supragingival professional tooth cleaning and recording of clinical parameters, the microbiological samples were taken from the reference periodontal pocket using 40/04 sterile paper tips. The withdrawals were performed at baseline (before and immediately after treatment), at three, and six months. The samples were preserved in 0.75 mL reduced transport fluid and stored in a freezer at − 80 °C .
Processing of the samples
In a first step, the frozen samples were thawed in a water bath at 36 °C and then homogenized for 45 s by vortex. Subsequently, a dilution series was carried out and the samples were applied on three different nutrient media in order to culture the individual colonies of the different bacterial species. The following agar plates and liquid media were used:
Columbia Blood Agar (CoBl): for the cultivation of aerobic and facultative anaerobic bacteria
Yeast cysteine blood agar (HCB): for culturing anaerobic bacteria
Trypticase Soy Bacitracin Vancomycin Selective Agar (TSBV): for the cultivation of
Dilution medium (PY): this medium was used to prepare the dilution series
Isolation and identification of bacterial species
After an incubation period of 4–5 days for CoBl plates and TSBV Selective agar plates at 36 °C in a CO2 cabinet or an incubation period of 10–12 days for HCB plates at 36 °C under anaerobic conditions, the colony forming units (CFU) on agar plates were counted. Single colonies were assessed for shape and color. This served the temporary classification of the bacterial species. The number of bacteria was determined as follows:
Bacteria number / mL = colony number x dilution grade
For the final identification of the bacteria, the individual colonies were subcultured to obtain pure cultures of the germs.
The final identification of the bacteria was mainly done using MALDI-TOF MS (Matrix Assisted Desorption Ionization Time of Flight Mass Spectrometry) analysis in a MALDI Biotyper Microflex LT as described in detail earlier . In brief, bacteria from single colonies were used for MALDI-TOF. The acquisition of the mass spectra was conducted according to the manufacturer’s recommendations. The obtained spectra were compared using the BioTyper 3.0 software with a reference database containing 3740 reference spectra (representing 319 genera and 1946 species). The resulting similarity value was expressed as a log score. Species level identification was indicated by a score of ≥2000, whereas a score of ≥1700 indicated identification on the genus level. No significant similarity of the obtained spectrum with any database entry was obtained by score value under 1700. The procedure was repeated, if the results were questionable.
If identification by MALDI-TOF-MS was unsuccessful, identification by polymerase chain reaction (PCR) and subsequencing of the 16S rDNA genes was used. The identification of bacterial species by 16S rDNA sequencing was conducted as described earlier in detail (Schirrmeister et al., 2009). In brief, DNA was extracted from pure bacterial isolates of Gram-negative bacteria by using lysis buffer (10 mmol/L Tris-HCl buffer, 1 mmol/L ethylenediaminetetraacetic acid, 1% Triton X-100, pH 8.0) and boiling for 10 min. After centrifugation at 12,000 g for 10 min, 1 μL of the supernatant was used for amplification of the 16S rRNA gene. DNA from Gram-positive bacterial isolates was extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 16S rRNA gene amplification was performed in a total volume of 50 μL, containing 5 μL 10X PCR-buffer (Qiagen), MgCl2 (2.5 mmol/L), 200 mol/L of each deoxyribonucleoside triphosphate (dNTP), 2 U Taq Polymerase (Qiagen), and 300 nmol/L of reverse- and forward-primer. For amplification of the 16S rRNA gene, a set of primers (forward-primer TP16U1: 5′-AGAGTTTGATC [C/A]TGGCTCAG-3′ and reverse-primer RT16U6: 5′-ATTGTAGCACGTGTGT [A/C]GCCC-3′) was used. The 1018 base pair-long PCR products were extracted and purified by using the GFX PCR DNA and gel band purification kit (Amersham Biosciences Europe GmbH, Freiburg, Germany). Purified PCR Products were sequenced by using the BigDye terminator kit v1.1 cycle sequencing kit (Applied Biosystem, Darmstadt, Germany) and the ABI 310 Genetic Analyzer (GMI, Inc., Ramsey, MN). TP16U1 was used as a sequencing primer. To identify then bacterial species sequences were analyzed by using the BLAST program from the NCBI (http://www.ncbi.nih.gov/BLAST). This was done in the present work for the identification of only 4 bacterial isolates, which were not identifiable in MALDI-TOF-MS. These bacterial species were Prevotella tannerae, Anaeroglobus geminatus, Actinomyces sp. oral taxon, Filifactor alocis.
Blinding and randomization
Participants were randomized to determine which tooth belonged to one of the two treatment arms. A randomization list was computer-generated by a statistician (KV) after participants were enrolled by two dentists (DA and RM). Concealed envelopes for each participant were prepared according to the randomization list. All baseline and follow-up examinations and microbiological sampling were done by the same dentist (DA). Interventions were exclusively undertaken by RM opening the envelope shortly before the treatment to know which method to use in each quadrant. By this procedure DA was blinded as an examiner.
For descriptive analysis, mean and standard deviation were calculated. Boxplots were used for the graphical presentation. Paired t-tests compared the temporal changes to baseline in log10 transformed bacterial concentration between the two methods and within the two groups. The calculations were done with STATA 15.