Host and microbial derived salivary extracellular vesicles in periodontitis

o Baseline visit, treatment visits, and initial treatment visit , review visits at 3, 6 ,12, 18 and 24 months post completion of non surgical treatment o Pre-operative periodontal charting recorded by DClinDent postgraduate registrars, with a periodontal UNC probe. All examiners will be calibrated. o This will allocate participants into the group categories: Group #1: periodontal health (including stable periodontitis patients) and gingivitis; Group #2: periodontitis o Saliva, gingival crevicular fluid (GCF) and plaque samples will be collected from all participants at baseline. Saliva, plaque and GCF samples will be collected from Group 2 patients at 3, 6 , 12, 18 and 24 months following completion of periodontal treatment - For saliva collection: participants will be asked to refrain from eating and drinking for at least one hour prior to saliva sample collection. At the appointment, participants will rinse their mouths with ~10 mL of water to remove the food debris. Unstimulated whole saliva through spitting (5mL ideally; minimum 1-1.5ml) will be collected. - GCF will be collected prior to commencing treatment to avoid contamination via blood. This will be conducted using our established protocol (Han et al, 2023, PMID: 35771077) where paper strips (Oraflow) will be inserted into the deepest site of the maxillary and mandibular arch –excluding any tooth deemed likely to be lost within 12 months. Subgingival plaque will be collected from the same sites using hand instruments inserted into the pocket following removal of supragingival deposits. - For healthy and gingivitis patients: 2 healthy sites -the mesial buccal of 16 (right maxillary first molar), mesial buccal of 41 (right lower centre incisor) will be collected and pooled. If these teeth are absent, then either the contralateral tooth or nearest tooth will be used. Plaque, GCF and saliva samples will be placed in sterile glycerol (final concentrations: 15-30%), or stored immediately at -80 degrees at the Research Lab, Level 6, Oral Health Centre, until elution and extraction. Samples containing glycerol may be subjected to in vitro culturing for biofilm development prior to the extraction of bacterial EVs from the cultured media. EVs will be isolated, characterised and omics (proteome, methylome, microbiome, lipidome and metabolome or relevant omes), with saliva, GCF and plaque samples used as controls. Patients in the periodontitis group will be allocated the next available appointment for standard care, receiving oral hygiene instruction and supragingival and subgingival non-surgical debridement using hand scalers and ultrasonic instruments over 2 appointments (60-90 mins duration per appointment). Follow-up reviews (3-, 6- 12, 18 and 24-months post-periodontal treatment; 1-1.5 hours duration) o Periodontitis patients will be recalled 3 months following completion of debridement to re-assess periodontal parameters and recollect saliva and GCF (from the same sites as baseline appointment). Standard protocol of periodontal care based on the EFP S3 guidelines (Sanz et al 2020 PMID: 32383274 ) for treating periodontitis will be undertaken, including when surgical interventions for non-responsive sites if warranted. o Patients will then be recalled at the 6 , 12 , 18 and 24 months time points (since initial therapy). During observation periods, patients will be recalled for treatment and supportive care at an appropriately determined interval. o Patient compliance with treatment reviews will be recorded using a clinic attendance checklist. The profile and characteristics of extracellular vesicles (EVs) from both host and non-host sources in patients with periodontitis will be compared against those without disease to investigate the distinct mechanisms of disease pathogenesis related to EVs. Following our published protocols, host-derived EVs will be isolated using EXO-NET microbeads coated with host-specific antibodies (Liu et al 2023 PMID: 39697803 ). Non-host EVs will subsequently be isolated using in-house SEC columns, following our published protocols (Liu et al 2025 PMID: 39748613). Both host-derived and non-host-derived EVs will be characterized in accordance with ISEV guidelines before being subjected to further analysis. Host EVs will undergo cytokine profiling and subpopulation analysis using the MACS Exosome Multiplex Kit. For non-host-derived EVs, proteome, lipidome and metabolome by LC/MS, 16S sequencing and ITS sequencing will be performed to investigate the associated microbiome and fungal populations. Unstimulated saliva, GCF and plaque samples (from upper right first molar, lower right lower incisor) will be collected from these healthy controls and pooled. All analysed salivary EV profiles will be correlated with clinical parameters to identify potential associations. The participants will receive non surgical and/or surgical interventions for periodontitis described above regardless of their involvement of their involvement in the study. Non host EVs are EVs from bacteria or Outer Membrane Vesicles ( OMVs). These will be isolated from the same plaque, GCF and saliva samples collected in the methods above. They will be isolated in the laboratory using a separate EV extraction kit and method i.e. in house SEC columns following published protocols (Liu et al 2025 PMID: 39748613)