Using biological markers in saliva to diagnose and monitor treatment for gum disease around dental implants

Conditions observed – Participants will be allocated into three groups based on clinical diagnosis parameters, per the World Workshop Classification (Berglundh et al. 2018): • Group 1 (Peri-implant health) healthy controls: No signs of suppuration or bleeding on probing, absence of clinical signs of inflammation, absence of bone loss beyond crestal bone level changes resulting from initial bone remodelling • Group 2 (Peri-implant mucositis): Presence of bleeding and/or suppuration on probing, absence of bone loss beyond crestal bone level changes resulting from initial bone remodelling • Group 3 (Peri-implantitis): Presence of bleeding and/or suppuration in combination with PPD >=6 mm and a radiographic bone loss >=3 mm apical of the most coronal portion of the intraosseous part of the implant Frequency and duration of observation – • Group 1 (Peri-implant health): Baseline visit (T0) • Group 2 (Peri-implant mucositis): Baseline visit (T0), phone follow-up at 1 week • Group 3 (Peri-implantitis): Baseline visit (T0), phone follow-up at 1 week plus in-person follow-up visits at: 3 months post-treatment (T1), 6 months post-treatment (T2), 9 months (treatment only as part of standard of care), 12 months post-treatment (T3) Data collected – • Clinical examinations, sample collection, and treatments will be performed by registered periodontists or periodontal postgraduate students under supervision of a registered periodontist. All clinical providers will be calibrated. • Examination, sample collection and interventions will take place at Brisbane City Periodontics & Implants (BCPI) private practice clinics and Herston Oral Health Centre, The University of Queensland • All clinical measurements, biological samples will be collected at all timepoints (T0, T1, T2 and T3). PROMS will be collected at 1 week and 3 months (T1). Radiographic measurements will be made at T0, T2 and T3. Clinical measurements will include full mouth plaque score (FMPS), full mouth bleeding score (FMBS), probing pocket depth (PPD), clinical attachment level (CAL), recession, suppuration. Saliva collection: Participants are to refrain from eating/drinking for greater than or equal to 1 hour prior to collection and are to rinse with approximately 15mL water to remove food debris. Collection of unstimulated whole saliva by spitting and samples to be placed in 50ml falcon tube. Peri-implant crevicular fluid (PICF) collection: Sites dried and isolated to avoid saliva contamination. PerioPaper strips (Oraflow Inc) placed into the peri-implant sulcus/pockets for 30 seconds. Four sites per implant (Mesial, Distal, Buccal, Lingual/Palatal) collected. For peri-implant diseased patients: up to 6 paper strips per implant. Control GCF from a healthy tooth or PICF from a healthy implant (if applicable) will be collected. Samples pooled and eluted into 300µL of PBS. Plaque collection: Subgingival plaque collected using periodontal probe/scaler for 10-20 seconds per tooth surface and transferred to sterile microcentrifuge tube containing 100µL PBS. Treatment (standard routine treatment for all peri-implant patients) - Consistent with the standard of care, implant treatment is to be provided for all patients according to EFP S3 guidelines (Herrera et al. 2023). For group 3 (peri-implantitis/diseased group) non-surgical treatment will be provided at baseline and at each follow-up visit (T1, T2, 9 months, T3). If Peri-implant stability* is achieved at follow-up examinations oral hygiene instruction & PMPR will be provided instead of submarginal instrumentation. Surgical peri-implantitis therapy will be offered at T2 (6 months) if peri-implant stability is not achieved. Patients who undergo surgical treatment will continue to be followed at the scheduled timepoints (9 months and T3/12 months). *Peri-implant stability or patient responder is defined as residual probing depths <=5 mm with no BOP at more than one point and no suppuration (Herrera et al. 2023) Sample Storage - All samples (PICF, GCF, saliva and plaque) will be stored at -80 degrees C until the commencement of experiments at the Research Lab, Level 6, Oral Health Centre, University of Queensland. Sample processing, extraction and analysis will be conducted according to established protocols. Sample Analysis - Host-derived extracellular vesicles (EVs) will be isolated from saliva and PICF samples using immunoaffinity methods, while bacterial EVs (BEVs) will be isolated from the remaining supernatant using size-exclusion chromatography. Host EVs will undergo cytokine profiling to analyze inflammatory content, while BEVs will be subjected to microbiome profiling through 16S rRNA sequencing to identify associated bacterial populations. Titanium ions released from dental implants will be analyzed using inductive coupled plasma atomic emission spectrometry (ICP-AES). Inflammatory cytokines will be measured using a multiplex assay capable of simultaneously detecting multiple inflammation-related proteins to track changes following treatment.