Changes of Oral Microbiome and Immunogenity of Saliva During Orthodontic Therapy With Aligners and Multibracket Therapy - a Pilot Study

Adult patients (18 years and older) from the Clinical Division of Orthodontics at the University Clinic of Dentistry Vienna will be invited to participate in this pilot microbiologic study according to defined inclusion/exclusion criteria after giving their consent. This study is a pilot project and therefore no sample size calculation will be made. Twenty patients will be treated with Invisalign® aligners and twenty patients will receive multibracket therapy. A previous microbiological study showed that lingual orthodontic therapy has a short-term effect on oral microflora. Another microbial study on colonization on dental implants also reported bacterial shift upon implant therapy.

All patients will get instructions in oral hygiene on the day when clinical records are gathered. On the day when the treatment plan will be presented to the patient, the Plaque Index will be evaluated by a trained orthodontist. For the start of the treatment a Plaque Index of < 20% is required. On the day before the orthodontic treatment is started, T0 sample collection will be conducted and periodontal indices will be assessed. A clinical examination including periodontal probing depth, bleeding on probing, and Plaque Index (PI) will be performed before (T0), after 3 months (T1) and after 6 months of the beginning of either aligner or bracket therapy. Microbiological sampling will be performed by stimulated saliva collection according to our protocol. Saliva samples will be divided in aliquots in stored in cryo-tubes at -80°C. Molecular detection of microorganisms by sequencing of 16S rDNA genes, sequencing analysis and taxonomic assignment will be performed at the Department of Laboratory Medicine at the Medical University of Vienna.

By sequencing of 16s rDNA genes with sequencing analysis and taxonomic assignment of bacteria in saliva we expect to reveal possible shifts in microbial phyla/genera between various time points. Metagenomic studies are commonly performed by analysing the prokaryotic 16S ribosomal RNA gene (16S rRNA), which is approximately 1,500 base pairs long and contains nine variable regions interspersed between conserved regions. Variable regions of 16S rRNA are frequently used in phylogenetic classifications such as genus or species in diverse microbial populations.

DNA Extraction:

Due to better quantitative and qualitative results in comparison to commercial DNA extraction kits, DNA will be isolated with a modified CTAB SDS chloroform-based method. Mechanical analysis will be carried out using 1mm silica spheres under addition of proteinase K (Qiagen, Venlo, Netherlands) as well as the detergents SDS (sodium dodecyl sulfate) and CTAB (cetyltrimethylammonium bromide). After extraction, the DNA quantity will be determined with Qubit® 2.0 via the dsDNA HS kit (Life Technologies, Carlsbad, California) and NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, Massachusetts). Additionally, the absorption ratios A260/280 and A260/230 will be used to estimate the purity of the DNA.