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
Scientific title
Host and microbial derived salivary extracellular vesicles in periodontitis before and after periodontal therapy
Secondary ID [1] 313798 0
None
Universal Trial Number (UTN)
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Periodontitis 336433 0
Condition category
Condition code
Oral and Gastrointestinal 332955 332955 0 0
Other diseases of the mouth, teeth, oesophagus, digestive system including liver and colon
Inflammatory and Immune System 332954 332954 0 0
Other inflammatory or immune system disorders

Intervention/exposure
Study type
Observational
Patient registry
False
Target follow-up duration
Target follow-up type
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)
Intervention code [1] 330387 0
Early Detection / Screening
Intervention code [2] 330386 0
Diagnosis / Prognosis
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)
Control group
Active

Outcomes
Primary outcome [1] 340660 0
Cytokine and EV associated protein expression in GCF
Timepoint [1] 340660 0
Baseline, 3 , 6, 12 , 18 and 24 month post routine periodontal treatment
Primary outcome [2] 340492 0
Levels of EVs particle numbers and associated proteins e.g. cytokines, chemokines in unstimulated saliva
Timepoint [2] 340492 0
Baseline, 3 , 6, 12 , 18 and 24 month post routine periodontal treatment
Primary outcome [3] 340569 0
Cytokine and EV associated protein expression in plaque
Timepoint [3] 340569 0
Baseline, 3 , 6, 12 , 18 and 24 month post routine periodontal treatment
Secondary outcome [1] 444483 0
Number of deep pockets (>5mm)
Timepoint [1] 444483 0
Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
Secondary outcome [2] 444481 0
Plaque index
Timepoint [2] 444481 0
Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
Secondary outcome [3] 444482 0
Number of moderate (4-5mm) pockets
Timepoint [3] 444482 0
Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
Secondary outcome [4] 444251 0
periodontal probing depths
Timepoint [4] 444251 0
Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment
Secondary outcome [5] 444480 0
percentage of sites with bleeding on probing (BOP),
Timepoint [5] 444480 0
Baseline, 3 , 6, 12, 18 and 24 month post routine periodontal treatment

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
Minimum age
18 Years
Maximum age
No limit
Sex
Both males and females
Can healthy volunteers participate?
No
Key exclusion criteria

periodontal treatment or antibiotics therapy six months prior to investigation
pregnancy, cardiovascular disease, active infectious disease

Study design
Purpose
Screening
Duration
Longitudinal
Selection
Convenience sample
Timing
Both
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.

Recruitment
Recruitment status
Not yet recruiting
Date of first participant enrolment
Anticipated
3/03/2025
Actual
Date of last participant enrolment
Anticipated
29/12/2028
Actual
Date of last data collection
Anticipated
31/01/2031
Actual
Sample size
Target
70
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
QLD

Funding & Sponsors
Funding source category [1] 318265 0
University
Name [1] 318265 0
School of Dentistry-University of Queensland
Country [1] 318265 0
Australia
Primary sponsor type
University
Name
School of Dentistry -University of Queensland
Address
Country
Australia
Secondary sponsor category [1] 320651 0
None
Name [1] 320651 0
Address [1] 320651 0
Country [1] 320651 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 316905 0
Metro North Health Human Research Ethics Committee B
Ethics committee address [1] 316905 0
Ethics committee country [1] 316905 0
Australia
Date submitted for ethics approval [1] 316905 0
15/07/2020
Approval date [1] 316905 0
17/03/2021
Ethics approval number [1] 316905 0
54584

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 139386 0
Dr Pingping Han
Address 139386 0
The University of Queensland, School of Dentistry, 288 Herston Rd, Herston 4006 QLD
Country 139386 0
Australia
Phone 139386 0
+61 733658172
Fax 139386 0
Email 139386 0
[email protected]
Contact person for public queries
Name 139387 0
Dr Pingping Han
Address 139387 0
The University of Queensland, School of Dentistry, 288 Herston Rd, Herston, 4006 QLD
Country 139387 0
Australia
Phone 139387 0
+61 733658172
Fax 139387 0
Email 139387 0
[email protected]
Contact person for scientific queries
Name 139388 0
Prof Saso Ivanovski
Address 139388 0
The University of Queensland, School of Dentistry, 288 Herston Rd, Herston, 4006 QLD
Country 139388 0
Australia
Phone 139388 0
+61 733658064
Fax 139388 0
Email 139388 0
[email protected]

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.