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Trial Review
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Trial registered on ANZCTR
Registration number
ACTRN12625000202460
Ethics application status
Approved
Date submitted
24/01/2025
Date registered
20/02/2025
Date last updated
20/02/2025
Date data sharing statement initially provided
20/02/2025
Type of registration
Prospectively registered
Titles & IDs
Public title
Host and microbial derived salivary extracellular vesicles in periodontitis
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Scientific title
Host and microbial derived salivary extracellular vesicles in periodontitis before and after periodontal therapy
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Secondary ID [1]
313798
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None
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Universal Trial Number (UTN)
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Trial acronym
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Linked study record
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Health condition
Health condition(s) or problem(s) studied:
Periodontitis
336433
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Condition category
Condition code
Oral and Gastrointestinal
332955
332955
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0
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Other diseases of the mouth, teeth, oesophagus, digestive system including liver and colon
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Inflammatory and Immune System
332954
332954
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0
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Other inflammatory or immune system disorders
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Intervention/exposure
Study type
Observational
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Patient registry
False
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Target follow-up duration
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Target follow-up type
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Description of intervention(s) / exposure
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)
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Intervention code [1]
330387
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Early Detection / Screening
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Intervention code [2]
330386
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Diagnosis / Prognosis
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Comparator / control treatment
Individuals in the periodontal health and gingivitis groups will serve as controls, as they are do not require additional treatment beyond oral hygiene instruction and professional plaque removal. Individuals in this group are patients who are assessed as not having any active periodontal disease but were treated at the same clinic for other dental conditions.
Unstimulated saliva, GCF and plaque samples (from upper right first molar, lower right lower incisor) will be collected from these healthy controls and pooled
The duration of the control group treatment is 1 session up to 1 hour
The control participants will only have samples collected at a single time point (baseline)
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Control group
Active
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Outcomes
Primary outcome [1]
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Cytokine and EV associated protein expression in GCF
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Assessment method [1]
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Cytokine and EV associated protein detection by ELISA flow cytometry. The above methods will apply to all separate samples i.e. applied to samples from plaque, GCF and saliva . This assessment method will also apply to host and non host EVs
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Timepoint [1]
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Baseline, 3 , 6, 12 , 18 and 24 month post routine periodontal treatment
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Primary outcome [2]
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Levels of EVs particle numbers and associated proteins e.g. cytokines, chemokines in unstimulated saliva
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Assessment method [2]
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Nanopartical tracking analysis (NTA) , Fourier transform infrared spectroscopy (FTIR )to detect EV's biochemical components, EV surface marker characterisation using commercial MACSPLex Exosome Kit (Miltenyi Biotec) will be assessed together as a composite primary outcome , The above methods will apply to all separate samples i.e. applied to samples from plaque, GCF and saliva and for EVs from host and non host sources
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Timepoint [2]
340492
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Baseline, 3 , 6, 12 , 18 and 24 month post routine periodontal treatment
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Primary outcome [3]
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Cytokine and EV associated protein expression in plaque
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Assessment method [3]
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Cytokine and EV associated protein detection by ELISA flow cytometry. The above methods will apply to all separate samples i.e. applied to samples from plaque, GCF and saliva . This assessment method will also apply to host and non host EVs
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Timepoint [3]
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Baseline, 3 , 6, 12 , 18 and 24 month post routine periodontal treatment
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Secondary outcome [1]
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Number of deep pockets (>5mm)
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Assessment method [1]
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Clinical examination including periodontal probing by calibrated examiners
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Timepoint [1]
444483
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Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
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Secondary outcome [2]
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Plaque index
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Assessment method [2]
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Clinical examination including periodontal probing by calibrated examiners
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Timepoint [2]
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Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
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Secondary outcome [3]
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Number of moderate (4-5mm) pockets
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Assessment method [3]
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Clinical examination including periodontal probing by calibrated examiners
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Timepoint [3]
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Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
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Secondary outcome [4]
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periodontal probing depths
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Assessment method [4]
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Clinical examination including periodontal probing by calibrated examiners
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Timepoint [4]
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Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
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Secondary outcome [5]
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percentage of sites with bleeding on probing (BOP),
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Assessment method [5]
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Clinical examination including periodontal probing by calibrated examiners
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Timepoint [5]
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Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
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Eligibility
Key inclusion criteria
Patients 18 years of age or older.
For the periodontal health group (n=30):
BOP<=10% and PPD<4mm
For the gingivitis group* (n=20):
>10% BOP and PPD<4mm
For the periodontitis group* (n=40):
Stage III-IV periodontitis patients will have interdental CAL=5mm at 2 or more non adjacent teeth, PPD=6mm, and significant radiographic bone loss ie to middle or apical third of the root **
*Based on Chapple et al 2018
**based on Papapanou et al 2018, Tonetti et al 2018
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Minimum age
18
Years
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Maximum age
No limit
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Sex
Both males and females
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Can healthy volunteers participate?
No
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Key exclusion criteria
periodontal treatment or antibiotics therapy six months prior to investigation
pregnancy, cardiovascular disease, active infectious disease
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Study design
Purpose
Screening
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Duration
Longitudinal
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Selection
Convenience sample
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Timing
Both
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Statistical methods / analysis
The primary outcome is the level of host EVs cytokine expression. Secondary outcomes include changes in clinical periodontal parameters: probing pocket depth (PPD), bleeding on probing (BOP), plaque index (PI), number of moderate and deep residual pockets at baseline, 3, 6, 9, 12, `18,24 months post non surgical periodontal therapy (NSPT).
A non-responder is defined as a participant who did not reach the clinical endpoint of treatment of less or equal to 4 sites with PPD equal to 5mm remaining (Feres et al 2020 PMID PMID: 32224549) Data will be analysed and presented as scatter plots displaying mean ±standard deviation (SD) using GraphPad Prism 10.0. Data normality was determined by the Shapiro Wilk test with a cut off at 0.05. If the data has an abnormal distribution, Friedman tests with Dunn’s multiple comparison test will be used to compare clinical parameters between each time point. If the distribution is normal, a one way ANOVA will be used .
Data on host EV surface marker expression, host EV protein and host-EV cytokine levels will be analysed for normality using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA). As the data had an abnormal distribution, the Friedman test with Dunn’s multiple comparison was also used to compare surface marker expression and cytokine expression of EXO-NET EVs at different time points. Statistical significance was defined as a two tailed F value <0.05.
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Recruitment
Recruitment status
Not yet recruiting
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Date of first participant enrolment
Anticipated
3/03/2025
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Actual
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Date of last participant enrolment
Anticipated
29/12/2028
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Actual
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Date of last data collection
Anticipated
31/01/2031
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Actual
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Sample size
Target
70
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Accrual to date
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Final
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Recruitment in Australia
Recruitment state(s)
QLD
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Funding & Sponsors
Funding source category [1]
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University
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Name [1]
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School of Dentistry-University of Queensland
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Address [1]
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Country [1]
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Australia
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Primary sponsor type
University
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Name
School of Dentistry -University of Queensland
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Address
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Country
Australia
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Secondary sponsor category [1]
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None
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Name [1]
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Address [1]
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Country [1]
320651
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Ethics approval
Ethics application status
Approved
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Ethics committee name [1]
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Metro North Health Human Research Ethics Committee B
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Ethics committee address [1]
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https://metronorth.health.qld.gov.au/research/ethics-and-governance/human-research-ethics-committee
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Ethics committee country [1]
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Australia
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Date submitted for ethics approval [1]
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15/07/2020
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Approval date [1]
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17/03/2021
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Ethics approval number [1]
316905
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54584
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Summary
Brief summary
This pilot study aims to reveal the profiles of extracellular vesicles (EVs) and their associated proteins in periodontal diseases (refers to diseased groups) before and after treatment follow-up (3, 6 , 12, 18 and 24 months). Host and non-host EVs from periodontally healthy (without follow-ups as no need to follow up) will be used as controls. Whole oral samples (saliva, GCF and plaque) will be used as controls. There are two general aims for this project: Aim 1: Diagnosis and prognosis values of EVs and their associated proteins in periodontal disease groups Compare the differences in host and microbial derived EVs and their EV content expressions between periodontally healthy periodontitis and periodontitis undergoing treatment over a 1-year observation period Aim 2: EV profiles after in vitro biofilm culture Examine the microbial EV microbiome and proteome profiles in samples from both healthy and diseased groups following in vitro biofilm culturing It is hypothesised that host and microbial EVs and EV content are differentially expressed in diseased patients compared with periodontally healthy patients, and correlates with the severity of periodontitis. Furthermore, it is hypothesised that EVs will be positively correlated with improvements in clinical parameters after periodontal treatment.
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Trial website
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Trial related presentations / publications
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Public notes
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Contacts
Principal investigator
Name
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Dr Pingping Han
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Address
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The University of Queensland, School of Dentistry, 288 Herston Rd, Herston 4006 QLD
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Country
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Australia
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Phone
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+61 733658172
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Fax
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Email
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[email protected]
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Contact person for public queries
Name
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Dr Pingping Han
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Address
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The University of Queensland, School of Dentistry, 288 Herston Rd, Herston, 4006 QLD
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Country
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Australia
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Phone
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+61 733658172
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Fax
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Email
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[email protected]
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Contact person for scientific queries
Name
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Prof Saso Ivanovski
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Address
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The University of Queensland, School of Dentistry, 288 Herston Rd, Herston, 4006 QLD
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Country
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Australia
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Phone
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+61 733658064
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Fax
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Email
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[email protected]
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Data sharing statement
Will the study consider sharing individual participant data?
No
What supporting documents are/will be available?
No Supporting Document Provided
Results publications and other study-related documents
Documents added manually
No documents have been uploaded by study researchers.
Documents added automatically
No additional documents have been identified.
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