Evaluation of the Effects of Immune Cells on Periodontal Healing

Study Overview
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Brief Summary
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Periodontitis is an inflammatory disease with an infectious character, where, as the result of the host response to a dysbiotic microflora, attachment and bone loss occur. The host response and the healing period following the treatment differs among individuals, but the reason behind is not fully understood. The macrophages and T cells play an important role in the immune response and in the pathogenesis of periodontal diseases, but their role in the healing following periodontal therapy is not known. In this study, we aim to reveal the effects of initial macrophage and T cell activities in the gingival tissue on the differences of the response to phase I periodontal treatment. 42 individuals will be included in the study. Granulation tissue samples will be collected from two separate deep pockets of each individual, initially. At the same session, full-mouth scaling and root debridement will be conducted. Saliva, subgingival biofilm and gingival crevicular fluid (GCF) samples will also be collected, initially, and at the 2nd, 6th, 12th and 24th weeks. At the same appointments, periodontal parameters will be recorded. When the clinical procedures are concluded, the samples will be sent to Turku University with dry ice. Tissue and GCF concentrations of related cytokines will be analyzed with Luminex. The density of macrophage types will be defined by immunoblot analysis of related markers. Macrophage subpopulations in tissues will be specified by proteomics. Likewise, quantities of periodontal pathogens will be evaluated with DNA isolation and next generation sequencing.

Official Title
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Evaluation of the Effects of Initial Macrophage and Th17 Cell Activities on the Healing Following Periodontal Phase I Therapy

Conditions
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Periodontitis

Intervention / Treatment
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* Procedure: Initial periodontal treatment


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: moderate to severe periodontitis having at least two pockets ≥ 6 mm systemic healthy Exclusion Criteria: received periodontal treatment prior to study received antibiotic or antiinflammatory drugs in the last 6 months pregnant or in lactation

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Screening
Intervention Model: Single Group Assignment
Interventional Model Description: Healing will be monitored of periodontitis patients who receive conventional initial treatment. The patients will be grouped according to their healing capacity at the end of the study.
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Other: Periodontitis patients<br>Single-group receiving periodontal treatment. The data will be evaluated according to the healing potential of individuals in the group and also site-specifically. | Procedure: Initial periodontal treatment<br>* Conventional periodontal treatment (scaling and root debridement) will be conducted<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in Probable pocket depth (PPD) | distance between the gingival margin and pocket base (mm); full-mouth scores, 6 sites per tooth | baseline, 2nd, 6th, 12th and 24th weeks following therapy | 
| Change in Bleeding on probing (BoP) | visual inspection, dichotomous; full-mouth, 6 sites per tooth; change in positive sites % | baseline, 2nd, 6th, 12th and 24th weeks following therapy | 
| Baseline macrophage related cytokine concentrations in granulation tissue samples | two pockets (≥6mm; BoP+) of each individual; Luminex: monocyte chemoattractant protein (MCP)-1, -2, -3, -4; macrophage derived chemokine (MDC); macrophage inhibitory factor (MIF), monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP)-1α, interferon γ-induced protein (IP)-10, CD163, TWEAK, BAFF concentrations (pg/mL); each will be correlated with the change in probable pocket depth and bleeding on probing scores | Samples will be obtained during the intervention and will be kept in -80oC until immunohistochemical analysis which will be carried out through study completion, 6-9 months | 
| Baseline Th17-pathway related cytokine concentrations in granulation tissue samples | two pockets (≥6mm; BoP+) of each individual; Luminex: Interleukin (IL)-1β, -4, -6, -10, -17A, -17F, -21, -22, -23, -25, -31, -33, interferon (IFN)-γ, soluble cluster of differentiation 40 ligand (sCD-40L), tumor necrosis factor (TNF)-ɑ concentrations (pg/mL); each will be correlated with the change in probable pocket depth and bleeding on probing scores | Samples will be obtained during the intervention and will be kept in -80oC until immunohistochemical analysis which will be carried out through study completion, 6-9 months | 
| Baseline density of macrophage types in granulation tissue samples | two pockets (≥6mm; BoP+) from each individual; Immunoblot analysis: cluster of differentiation(CD)68 and CD163; cell number / optic field (cells/mm2); each will be correlated with the change in probable pocket depth and bleeding on probing scores | Samples will be obtained during the intervention and will be kept in -80oC until immunohistochemical analysis which will be carried out through study completion, 6-9 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in macrophage and Th17-pathway related cytokine concentrations in saliva | MCP-1, -2, -3, -4, MDC, MIF, MIG, MIP-1α, IP-10, IL1β, -4, -6, -10, -17A, -17F, -21, -22, -23, -25, -31, -33, IFN-γ, sCD-40L, TNF-ɑ, sCD163, TWEAK, BAFF, sST2 concentrations (pg/mL); will be correlated with the change in probable pocket depth and bleeding on probing scores | baseline, 2nd, 6th, 12th and 24th weeks following therapy; immunohistochemical analysis will be carried out through study completion, 6-9 months | 
| Change in macrophage and Th17-pathway related cytokine concentrations in gingival crevicular fluid (GCF) | MCP-1, -2, -3, -4, MDC, MIF, MIG, MIP-1α, IP-10, IL1β, -4, -6, -10, -17A, -17F, -21, -22, -23, -25, -31, -33, IFN-γ, sCD-40L, TNF-ɑ, sCD163, TWEAK, BAFF, sST2 concentrations (pg/mL); will be correlated with the change in probable pocket depth and bleeding on probing scores | baseline, 2nd, 6th, 12th and 24th weeks following therapy; immunohistochemical analysis will be carried out when through study completion, 6-9 months | 
| Change in plaque index | Silness-Löe plaque index (scored as 0-3); full-mouth, 6 sites per tooth; will be monitored for oral health assessment during the healing period | baseline, 2nd, 6th, 12th and 24th weeks following therapy | 
| Change in clinical attachment level (CAL) | distance between the cement-enamel junction and pocket base (mm); full-mouth, 6 sites per tooth; will be correlated with pocket depth reduction | baseline, 2nd, 6th, 12th and 24th weeks following therapy | 
| Change in biofilm microbiota | DNA isolation and next generation sequencing: P. gingivalis, T. denticola, T. forsythia, P. intermedia, F. nucleatum and A. actinomycetemcomitans; bacterial count (log10 scale) | baseline, 2nd, 6th, 12th and 24th weeks following therapy; microbiological analysis will be carried out through clinical phase completion, 6-9 months | 
| Change in neutrophil-associated cytokines in saliva | MMP-2, MMP-7, MMP-8, active MMP-8, MMP-9, MMP-13, TIMP, myeloperoxidase, PMN elastase (IFMA and ELISA methods) concentrations (pg/mL) in saliva | baseline, 6th, 12th and 24th weeks following therapy | 
| Change in neutrophil-associated cytokines in and oral rinse | MMP-2, MMP-7, MMP-8, active MMP-8, MMP-9, MMP-13, TIMP, myeloperoxidase, PMN elastase (IFMA and ELISA methods) concentrations (pg/mL) in oral rinse samples | baseline, 6th, 12th and 24th weeks following therapy | 
| active MMP-8 point-of-care test results in oral rinse | Dichotomous aMMP-8 test results in oral rinse samples (according to the manufacturer, a concentration below 20 ng/mL gives a negative test result, otherwise positive - Periosafe (R) Dentognostics GmHb, Jena, Germany) | baseline, 6th, 12th and 24th weeks following therapy | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Periodontitis, Macrophages, T-Lymphocytes, Treatment Outcome