Diederik Hentenaar

34 Chapter 2 of the University Medical Center Groningen, with only healthy implants and free from periodontal inflammation (probing pocket depth (PPD) ≤ 3mm, no bleeding on probing (BoP) and no anatomical loss of periodontal structures), were consecutively asked to participate as control subjects. Healthy implants (HI) were defined as: PPD <5mm, no bleeding/suppuration on probing (BoP/SoP), and no marginal bone loss (MBL). Control group patients did not underwent a therapeutic intervention. The Medical Ethics Review Board of the University Medical Center Groningen (METc UMCG) has discussed and considered whether or not the sampling the study protocol falls within the scope of the Medical Research Involving Human Subjects Act (WMO) and decided that no ethics committee approval was needed for assessment of these patients (METc 2018.537). In both groups, all eligible implants present were included until 20 implants per group (according to our sample size calculation) were sampled. Peri-implant crevicular fluid (PICF) Biomarker sampling and volume quantification A 30-second sampling protocol following Wassall & Preshaw (2016) was applied. In brief, before sampling the sample site was isolated with cotton rolls and dried with a gentle stream of air. Two PICF samples were collected from the same implant pocket per included implant in the healthy and diseased group using Periopaper® strips (Oraflow Inc. Smithtown, NY, USA) (Stewart et al. 1993). These presterilized filter paper strips were placed 1-2mm into the sulcus/pocket and absorbed fluids up to 1.2μl. To minimize evaporation, volume quantification was performed immediately after sampling, using a Periotron 8000 device (Oraflow Inc. Smithtown, NY, USA). Volumes were used to calculate modified concentration levels. The Periotron 8000 was calibrated before commencing the study and recalibrated periodically, following the manufacturer’s recommendations. A calibration curve was generated accordingly. Directly after quantification, the two Periopaper® strips per implant were pooled in a dry Eppendorf tube ® (Eppendorf AG, Hamburg, Germany). The tubes were placed on ice for transport to the laboratory and stored at -80°C until antibody array quantification took place. Implants with peri- implantitis (PI) were sampled at baseline (T0) and additionally at 3 months after therapy (T3). Health implants were only sampled at baseline. Biomarkers of interest, determination and analysis (using Luminex™ xMAP multi-analyte profiling technology) Periopaper® strips were thawed at the day of analysis after being stored dry at -80°C. To extract PICF from the strips, Luminex ™ assay buffer (23µl) was added to each vial after which all the samples were vortexed for 30 minutes. Before centrifugation, the Periopaper® strips were fixed in the tube’s cap. The samples were then centrifuged for 60 min at 300 rpm (8.7 g ) at 4°C, followed by another 2 minutes of centrifugation