The Effects of Sodium Fluoride and Chlorhexidine Use on Salivary IL-6 and Matrix Metalloproteinase Levels in Children With Active Caries (DentalCaries)

June 1, 2026 updated by: Gül Keskin

This study aims to compare salivary IL-6, MMP-8, MMP-9, and TIMP-1 levels between healthy children and caries-active children, and to evaluate the effects of 5% sodium fluoride (NaF) varnish and 2% chlorhexidine (CHX) used in caries prevention on these biomarkers.

The study will be conducted at Alanya Alaaddin Keykubat University, Faculty of Dentistry, Department of Pediatric Dentistry. Informed consent will be obtained from children and their parents/guardians. Investigator calibration will be performed using Cohen's kappa method prior to data collection. Sample size was calculated assuming 80% power (1-β = 0.80), α = 0.05, and Cohen's d ≈ 0.8, resulting in 20 participants per group using G*Power 3.1 software.

Study groups: Group A: 5% NaF varnish. Group B: 2% CHX + 5% NaF varnish. Group C: Standard oral hygiene education (negative control). Group D: Caries-free children (biological reference; baseline saliva sampling only).

Saliva samples will be collected at T0 (baseline), T1 (30 minutes post-application), and T2 (1 month). Unstimulated whole saliva will be collected, centrifuged at 5,000 g for 10 minutes at 4°C, and stored at -80°C. IL-6, MMP-8, MMP-9, and TIMP-1 levels will be measured using CE-marked/FDA-approved human saliva ELISA kits.

Study Overview

Status

Status

Indicates the current recruitment status or the expanded access status.
Recruiting

Conditions

Conditions

The disease, disorder, syndrome, illness, or injury that is being studied. On ClinicalTrials.gov, conditions may also include other health-related issues, such as lifespan, quality of life, and health risks.

Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.

Detailed Description

Biomarkers to Be Measured in the Study and Expected Benefits:Biomarkers to Be Measured in the Study and Expected Benefits:

Dental caries is a multifactorial infectious disease with high prevalence that affects a large proportion of the world's population. The fundamental pathogenesis of the disease is characterized by tissue destruction caused by complex and synergistic biological processes that arise from the interaction of acids-produced through the fermentation of dietary carbohydrates by bacteria-with susceptible host factors such as dental hard tissues and saliva. The body develops an inflammatory response against dental caries, which is characterized by infection and tissue destruction; the purpose of this response is to eliminate the agent that initiated the inflammation and to restore tissue homeostasis.

As a result of bacterial stimulation and recognition, odontoblasts, pulp tissue fibroblasts, and immune cells such as dendritic cells, macrophages, and neutrophils collectively produce a large number of molecules; these include cytokines and chemokines such as interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and prostaglandins, which prolong the inflammatory state and thereby support the activation of innate and adaptive immune responses.

IL-6, produced by odontoblasts and immune cells, increases markedly in the inflamed pulp. IL-6 neutralizes bacterial cell wall components by enhancing the secretion of LBP (lipopolysaccharide-binding protein) and regulates the immune response by reducing pro-inflammatory cytokine production. It also contributes to edema formation by increasing vascular permeability. It has been reported that salivary IL-6 may serve as a potential biomarker for assessing caries severity.

As caries lesions progress toward the pulp, demineralization and collagen degradation in the dentin matrix accelerate. The decrease in pH exposes collagen fibers to proteolytic enzymes, facilitating the demineralization of the dentin matrix. This acidic environment also accelerates caries progression by increasing the activation of MMPs (zinc- and calcium-dependent endopeptidases). MMPs are classified into five subgroups based on their substrate specificity and structural similarities: collagenases, stromelysins, gelatinases, matrilysins, and membrane-type MMPs. These enzymes can be activated in an acidic environment or by lactate released by cariogenic bacteria. MMP-8 (neutrophil collagenase) can cleave triple-helical fibrillar collagens into characteristic 3/4 and 1/4 fragments. MMP-9 is a gelatinase capable of degrading type IV collagen. The activation of MMP-8 and MMP-9 plays a critical role in collagen degradation in dentin caries lesions.

The activity of MMPs, which play a central role in dentin collagen degradation, also stands out as an important target for maintaining tissue balance. The control of these enzymes is primarily achieved through tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 and TIMP-2 are the primary regulators of MMP activity and effectively prevent excessive extracellular matrix (ECM) degradation . TIMP-2 exhibits strong inhibition of polymorphonuclear cell (PMN)-derived MMPs, while TIMP-1 demonstrates more pronounced inhibition of fibroblast-derived MMPs.

Inhibition of MMP activity is considered a potential strategy for slowing caries progression and preventing dentin erosion. In this context, sodium fluoride (NaF) and chlorhexidine (CHX) are cited in the literature as the principal agents capable of reducing MMP activity. NaF has been shown to inhibit the catalytic activity of recombinant MMP-8 and MMP-9. Similarly, CHX can largely prevent collagen degradation in demineralized dentin and can demonstrate long-lasting anti-MMP effects by binding electrostatically to the dentin matrix.

All of these findings demonstrate that inflammation can be objectively measured through both local and systemic biomarkers. The dynamic interaction between pro-inflammatory signals and matrix degradation, along with the endogenous inhibitors involved in this process, is of critical importance for the preservation of dentin tissue and the control of caries progression. However, studies that comprehensively reveal the molecular-level interactions of this axis, particularly in caries-active children, are limited in the literature. In this context, the aim of our study is to compare the biomarkers IL-6, MMP-8, MMP-9, and TIMP-1-representing the key components of the inflammation-matrix degradation axis-between healthy children and caries-active children, and to evaluate the effects of materials such as NaF and CHX used in caries prevention on these parameters.

Biomarker Analysis The levels of IL-6, MMP-8, MMP-9, and TIMP-1 in saliva samples will be evaluated using human saliva enzyme-linked immunosorbent assay (ELISA) kits. Biomarker analyses (ELISA) of the samples will be performed at the ALKU Faculty of Medicine Pharmacology Laboratory.

The obtained data will be statistically analyzed. Descriptive statistics: Quantitative variables will be expressed as mean ± standard deviation or median and interquartile range (IQR), and qualitative variables will be expressed as frequencies and percentages.

Between-group comparisons and within-group changes over time will be evaluated using appropriate parametric or non-parametric tests.

Results will be interpreted by correlating changes in salivary biomarker levels with clinical findings.

Study Type

Study Type

Describes the nature of a clinical study. Study types include interventional studies (also called clinical trials), observational studies (including patient registries), and expanded access.
Interventional

Enrollment (Estimated)

Enrollment

The number of participants in a clinical study. The "anticipated" enrollment is the target number of participants that the researchers need for the study.
80

Phase

Phase

The stage of a clinical trial studying a drug or biological product, based on definitions developed by the U.S. Food and Drug Administration (FDA). The phase is based on the study's objective, the number of participants, and other characteristics. There are five phases: Early Phase 1 (formerly listed as Phase 0), Phase 1, Phase 2, Phase 3, and Phase 4. Not Applicable is used to describe trials without FDA-defined phases, including trials of devices or behavioral interventions.
  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Contact

The name and contact information for the person who can answer enrollment questions for a clinical study. Each location where the study is being conducted may also have a specific contact, who may be better able to answer those questions.

Study Contact Backup

Study Locations

  • Turkey (Türkiye)
    • Antalya
      • Alanya, Antalya, Turkey (Türkiye), 07450
        • Recruiting
        • Alanya Alaaddin Keykubat University, Faculty of Dentistry
        • Contact:
        • Contact:
        • Principal Investigator:
          • GÜL KESKİN, DDS, PhD
        • Sub-Investigator:
          • BİNNUR B ÖZDEMİR, DDS
        • Sub-Investigator:
          • ERKAN MAYTALMAN, DDS, PhD

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Eligibility Criteria

The key requirements that people who want to participate in a clinical study must meet or the characteristics they must have. Eligibility criteria consist of both inclusion criteria (which are required for a person to participate in the study) and exclusion criteria (which prevent a person from participating). Types of eligibility criteria include whether a study accepts healthy volunteers, has age or age group requirements, or is limited by sex.

Ages Eligible for Study

  • Child

Accepts Healthy Volunteers

Yes

Description

Inclusion Criteria:

  • Children aged 6-8 years with all first permanent molars erupted
  • For active caries groups (Groups A, B, C): participants must meet at least one of the following criteria: dmft ≥ 6, or presence of at least one active caries lesion with an ICDAS code ≥ 3, or clinical evidence of active caries on ≥ 10 surfaces
  • For the dmft = 0 reference group (Group D): all surfaces caries-free and non-high-risk individuals (ICDAS II = 0); only baseline (T0) saliva sampling will be performed
  • No professional topical fluoride application or continuous CHX use within the past 3 months
  • No systemic chronic disease, immunodeficiency, or severe neuromotor disorder
  • No gingival redness, swelling, or bleeding
  • Written parental informed consent obtained

Exclusion Criteria:

  • Known allergy to fluoride compounds or varnish components
  • Behavioral problems that would prevent safe cooperation during application
  • Regular antibiotic use within the past 1 month
  • Individuals with infectious diseases
  • Those at high risk of endocarditis
  • History of substance dependence
  • Epilepsy
  • Renal failure or immunosuppression

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Basic Science
  • Allocation: Randomized
  • Interventional Model: Parallel Assignment
  • Masking: Single

Arms and Interventions

Participant Group / Arm

Participant Group / Arm

A group or subgroup of participants in a clinical trial that receives a specific intervention/treatment, or no intervention, according to the trial's protocol.
Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.
Experimental: 5% Sodium Fluoride (NaF) Varnish
Caries-active children aged 6-8 years. Teeth are gently air-dried for 30 seconds under isolation with cotton rolls and aspirator. Approximately 0.5 mL of 5% NaF varnish is applied to all teeth sequentially by quadrant starting from the upper jaw using an applicator tip and left in place for 1 minute. Parents are instructed to restrict water and hard food intake for 1 hour post-application. Saliva samples collected at T0, T1 and T2. n=20.
Other: 5% Sodium Fluoride Varnish
Teeth are gently air-dried for 30 seconds under isolation with cotton rolls and aspirator. Approximately 0.5 mL of 5% NaF varnish is applied to all tooth surfaces sequentially by quadrant starting from the upper jaw using an applicator tip and left in place for 1 minute. Parents are instructed to restrict water and hard food intake for 1 hour post-application. This is a routine preventive application; no off-label use is involved.
Experimental: 2% Chlorhexidine Pre-Treatment + 5% NaF Varnish
Caries-active children aged 6-8 years. Teeth are gently air-dried for 30 seconds under isolation. 2% CHX is applied to the lesion/tooth surfaces using an applicator tip and left for 30 seconds. Excess CHX is absorbed, an additional 30 seconds was allowed to pass.NaF varnish was then applied and left for 1 minute. Precautions are taken to prevent CHX ingestion. Parents are instructed to restrict water and hard food intake for 1 hour post-application. Saliva samples collected at T0, T1 and T2. n=20.
Other: 2% Chlorhexidine Pre-Treatment + 5% NaF Varnish
Following isolation and air-drying, 2% chlorhexidine solution is applied to the lesion/tooth surfaces using an applicator tip and left in place for 30 seconds. Excess CHX is then absorbed. Precautions are taken to prevent ingestion. This pre-treatment is performed immediately prior to 5% NaF varnish application. This is a routine preventive application; no off-label use is involved.
Active Comparator: Standard Care / Oral Hygiene Education
Caries-active children aged 6-8 years. No professional topical agent is applied. Children and parents receive standardized oral hygiene education including twice-daily brushing with fluoride toothpaste, reduction of sugar frequency, and a brief parent information brochure. Necessary treatment will be offered at the end of the study upon request (wait-list design). Saliva samples collected at T0 and T2. n=20.
Behavioral: Standard Oral Hygiene Education
Children and parents receive standardized oral hygiene education including instruction on twice-daily brushing with fluoride toothpaste, reduction of sugar intake frequency, and a brief parent information brochure. No professional topical agent is applied. Necessary treatment will be offered at the end of the study upon request (wait-list design).
No Intervention: Caries-Free Healthy Control
Age- and sex-matched caries-free children (ICDAS II = 0, dmft = 0) aged 6-8 years. No intervention is applied. Clinical assessment (dmft/ICDAS) is performed and recorded. Saliva sampling is performed at T0 only. This group serves as a non-randomized biological reference group. n=20.

What is the study measuring?

Primary Outcome Measures

Primary Outcome Measures

In a clinical study's protocol, the planned outcome measure that is the most important for evaluating the effect of an intervention/treatment. Most clinical studies have one primary outcome measure, but some have more than one.
Outcome Measure
Measure Description
Time Frame
Change in Salivary MMP-8 Levels
Time Frame: Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)
Change in salivary matrix metalloproteinase-8 (MMP-8) concentration measured by ELISA from baseline (T0) to post-application (T1) and 1 month post-intervention (T2) , and comparison between caries-active and caries-free groups at baseline. Unstimulated whole saliva samples will be analyzed using CE-marked/FDA-approved human saliva ELISA kits. Results expressed as ng/mL.
Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)
Change in Salivary IL-6 Levels
Time Frame: Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)
Change in salivary interleukin-6 (IL-6) concentration measured by ELISA from baseline (T0) to post-application (T1) and 1 month post-intervention (T2) , and comparison between caries-active and caries-free groups at baseline. Unstimulated whole saliva samples will be collected, centrifuged at 5,000 g for 10 minutes at 4°C, stored at -80°C, and analyzed using CE-marked/FDA-approved human saliva ELISA kits. Results expressed as pg/mL.
Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)
Change in Salivary MMP-9 Levels
Time Frame: Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)
Change in salivary matrix metalloproteinase-9 (MMP-9) concentration measured by ELISA from baseline (T0) to post-application (T1) and 1 month post-intervention (T2), and comparison between caries-active and caries-free groups at baseline. Unstimulated whole saliva samples will be analyzed using CE-marked/FDA-approved human saliva ELISA kits. Results expressed as ng/mL.
Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)
Change in Salivary TIMP-1 Levels
Time Frame: Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)
Change in salivary tissue inhibitor of metalloproteinase-1 (TIMP-1) concentration measured by ELISA from baseline (T0) to post-application (T1) and 1 month post-intervention (T2), and comparison between caries-active and caries-free groups at baseline. Unstimulated whole saliva samples will be analyzed using CE-marked/FDA-approved human saliva ELISA kits. Results expressed as ng/mL.
Baseline (T0), 30 minutes post-application (T1), 1 month post-intervention (T2)

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Sponsor

The organization or person who initiates the study and who has authority and control over the study.
Gül Keskin

Collaborators

Collaborators

An organization other than the sponsor that provides support for a clinical study. This support may include activities related to funding, design, implementation, data analysis, or reporting.
Alanya Alaaddin Keykubat University

Investigators

Investigators

A researcher involved in a clinical study. Related terms include site principal investigator, site sub-investigator, study chair, study director, and study principal investigator.
  • Study Director: GÜL KESKİN, DDS, PhD, Alanya Alaaddin Keykubat University

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

Study Start

The actual date on which the first participant was enrolled in a clinical study. The "anticipated" study start date is the date that the researchers think will be the study start date.
May 15, 2026

Primary Completion (Estimated)

Primary Completion

The date on which the last participant in a clinical study was examined or received an intervention to collect final data for the primary outcome measure. Whether the clinical study ended according to the protocol or was terminated does not affect this date. For clinical studies with more than one primary outcome measure with different completion dates, this term refers to the date on which data collection is completed for all the primary outcome measures. The "anticipated" primary completion date is the date that the researchers think will be the primary completion date for the study.
January 1, 2027

Study Completion (Estimated)

Study Completion

The date on which the last participant in a clinical study was examined or received an intervention/treatment to collect final data for the primary outcome measures, secondary outcome measures, and adverse events (that is, the last participant's last visit). The "anticipated" study completion date is the date that the researchers think will be the study completion date.
June 30, 2027

Study Registration Dates

First Submitted

First Submitted

The date on which the study sponsor or investigator first submitted a study record to ClinicalTrials.gov. There is typically a delay of a few days between the first submitted date and the record's availability on ClinicalTrials.gov (the first posted date).
May 20, 2026

First Submitted That Met QC Criteria

First Submitted That Met QC Criteria

The date on which the study sponsor or investigator first submits a study record that is consistent with National Library of Medicine (NLM) quality control (QC) review criteria. The sponsor or investigator may need to revise and submit a study record one or more times before NLM's QC review criteria are met. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
May 20, 2026

First Posted (Actual)

First Posted

The date on which the study record was first available on ClinicalTrials.gov after National Library of Medicine (NLM) quality control (QC) review has concluded. There is typically a delay of a few days between the date the study sponsor or investigator submitted the study record and the first posted date.
May 27, 2026

Study Record Updates

Last Update Posted (Actual)

Last Update Posted

The most recent date on which changes to a study record were made available on ClinicalTrials.gov. There may be a delay between when the changes were submitted to ClinicalTrials.gov by the study's sponsor or investigator (the last update submitted date) and the last update posted date.
June 2, 2026

Last Update Submitted That Met QC Criteria

Last Update Submitted That Met QC Criteria

The most recent date on which the study sponsor or investigator submitted changes to a study record that are consistent with National Library of Medicine (NLM) quality control (QC) review criteria. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
June 1, 2026

Last Verified

Last Verified

The most recent date on which the study sponsor or investigator confirmed the information about a clinical study on ClinicalTrials.gov as accurate and current. If a study with a recruitment status of recruiting; not yet recruiting; or active, not recruiting has not been confirmed within the past 2 years, the study's recruitment status is shown as unknown.
May 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

Other Study ID Numbers

Identifiers or ID numbers other than the NCT number that are assigned to a clinical study by the study's sponsor, funders, or others. These numbers may include unique identifiers from other trial registries and National Institutes of Health grant numbers.
  • 2025-KAEK-44

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

IPD Plan Description

Individual participant data will not be shared with external researchers. The informed consent form explicitly states that all personal data will be kept confidential, participant identities will be stored using coded numbers, and data will not be disclosed to third parties. As participants are children aged 6-8 years, additional data protection considerations apply.

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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