THE EFFECTS OF SODIUM FLUORIDE AND CHLORHEXIDINE USE ON SALIVARY IL-6 AND MATRIX METALLOPROTEINASE LEVELS IN CHILDREN WITH ACTIVE CARIES (Dental caries)

Methods:The study will be conducted at Alanya Alaaddin Keykubat University, Faculty of Dentistry, Department of Pediatric Dentistry. The study will be initiated after obtaining ethics committee approval. The nature of the study will be explained to the children and their parents/guardians; informed consent will be obtained through patient information forms and informed consent forms. Investigator calibration will be conducted to minimize measurement error and eliminate inter-observer variability. In this context, the investigator will perform active caries assessment on a group of children excluded from the study at two different time points, and the consistency between the two measurements will be evaluated using Cohen's kappa method. If the agreement coefficient reaches a sufficient level, the investigator will be permitted to begin clinical data collection. Similarly, trial samplings will be conducted for the saliva collection protocol, and study procedures will commence once consistency in sample collection time, volume, and processing steps is ensured. Sample size calculations were performed assuming 80% power (1-β = 0.80), a significance level of α = 0.05, and Cohen's d ≈ 0.8 effect size for a two-independent-group comparison. Analyses were conducted using G*Power 3.1 software (Heinrich-Heine-Universität Düsseldorf, Germany), and the sample size was determined as 20 participants per group:Group A: 5% Sodium Fluoride (NaF) varnish - topical applicationGroup B: 2% Chlorhexidine (CHX) +5% NaF varnish - topical application. Group C: Standard care / oral hygiene education (negative control). Group D: Caries-free children (biological control; matched by age and sex; no intervention, baseline saliva sampling only). Measurement Time Points: T0 (Baseline): Pre-intervention (preferably between 09:00-11:00 AM; at least 1 hour after eating/tooth brushing/sugar consumption), dmft/ICDAS assessment and saliva collection. T1: Post-intervention , T2: 1 month (30 days). Saliva Collection, Processing, and Storage:Participants and their parents will be asked not to eat or drink for at least 1 hour prior to saliva collection. Morning hours will be preferred for sample collection, and the time of application will be recorded. First, the patient will be asked to accumulate unstimulated saliva, which will then be aspirated from the floor of the mouth using disposable syringes to prevent potential contamination. The sample will then be transferred to an Eppendorf tube and labeled. All procedures will be performed under isolation using sterile dental instruments, cotton rolls, and aspirators. Samples will be identified by code numbers assigned during collection and processing, and the same codes will be used for subsequent sample collections.Saliva samples will be re-collected after preventive applications. Patients will be recalled at 1 month for saliva sampling. Each sample will be transported immediately after collection, then centrifuged at 5,000 g for 10 minutes at 4 °C to remove insoluble particles. Eppendorf tubes will be stored at -80 °C in a freezer.Subsequently, IL-6, MMP-8, MMP-9, and TIMP-1 levels in saliva samples will be evaluated using a human saliva enzyme-linked immunosorbent assay (ELISA) kit. ELISA (Enzyme-Linked Immunosorbent Assay) will be used for the quantitative measurement of target biomarkers. All analyses will be conducted in accordance with the manufacturer's protocol, and the kits are CE-marked and/or FDA-approved.Aim: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.Inclusion Criteria:Age: 6-8 years, (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.(Group D):All surfaces caries-free and non-high-risk individuals (ICDAS II = 0). (Only baseline (T0) saliva sampling will be performed.)Exclusion Criteria:Professional topical fluoride application or continuous CHX use within the past 3 months., Systemic chronic disease, immunodeficiency, or severe neuromotor disorder., Presence of gingival redness, swelling, or bleeding., All first permanent molars not yet erupted., Known allergy to fluoride compounds., Behavioral problems that would prevent safe cooperation during application., Regular antibiotic use within the past 1 month., According to Ethics Committee regulations: individuals with infectious diseases, those at high risk of endocarditis, those with allergy to varnish components, individuals with a history of substance dependence, those with epilepsy, and those with renal failure or immunosuppression.

Study Overview

• Status: Recruiting
• Conditions: Dental Caries in Children; Dental Caries
• Intervention / Treatment: Other: 5% Sodium Fluoride Varnish; Other: 2% Chlorhexidine Pre-Treatment + 5% NaF Varnish; Behavioral: Standard Oral Hygiene Education

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: Interventional
• Enrollment (Estimated): 80
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: GÜL KESKİN, DDS, PhD; Phone Number: 905444468857; Email: gul.keskin@alanya.edu.tr
• Study Contact Backup: Name: BİNNUR B ÖZDEMİR, DDS; Phone Number: 905069033834; Email: busra.ozdemir@alanya.edu.tr

Study Locations

• Turkey (Türkiye)
  • Antalya
    • Alanya, Antalya, Turkey (Türkiye), 07450
      • Recruiting
      • Alanya Alaaddin Keykubat University, Faculty of Dentistry
      • Contact: GÜL KESKİN, DDS, PhD; Phone Number: 905444468857; Email: gul.keskin@alanya.edu.tr
      • Contact: BİNNUR B ÖZDEMİR, DDS; Phone Number: 905069033834; Email: busra.ozdemir@alanya.edu.tr
      • Principal Investigator: GÜL KESKİN, DDS, PhD
      • Sub-Investigator: BİNNUR B ÖZDEMİR, DDS
      • Sub-Investigator: ERKAN MAYTALMAN, DDS, PhD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• 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.

Exclusion Criteria:

• Professional topical fluoride application or continuous CHX use within the past 3 months Systemic chronic disease, immunodeficiency, or severe neuromotor disorder Presence of gingival redness, swelling, or bleeding All first permanent molars not yet erupted Known allergy to fluoride compounds Behavioral problems that would prevent safe cooperation during application Regular antibiotic use within the past 1 month According to Ethics Committee regulations: individuals with infectious diseases, those at high risk of endocarditis, those with allergy to varnish components, individuals with a history of substance dependence, those with epilepsy, and those with renal failure or immunosuppression

Study Plan

How is the study designed?

Design Details

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

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
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

Outcome Measure	Measure Description	Time Frame
Change in Salivary MMP-8 Levels	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), post-application (T1) , 1 month post-intervention (T2)
Change in Salivary IL-6 Levels	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), post-application (T1), 1 month post-intervention (T2)
Change in Salivary MMP-9 Levels	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), post-application (T1), 1 month post-intervention (T2)
Change in Salivary TIMP-1 Levels	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), post-application (T1), 1 month post-intervention (T2)

Collaborators and Investigators

• Sponsor: Gül Keskin
• Collaborators: Alanya Alaaddin Keykubat University
• Investigators: Study Director: GÜL KESKİN, DDS, PhD, Alanya Alaaddin Keykubat University

Study record dates

Study Major Dates

• Study Start (Actual): May 15, 2026
• Primary Completion (Estimated): January 1, 2027
• Study Completion (Estimated): June 30, 2027

Study Registration Dates

• First Submitted: May 20, 2026
• First Submitted That Met QC Criteria: May 20, 2026
• First Posted (Actual): May 27, 2026

Study Record Updates

• Last Update Posted (Actual): May 27, 2026
• Last Update Submitted That Met QC Criteria: May 20, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: Matrix metalloproteinase; Dental caries; Chlorhexidine; Interleukin-6; Sodium fluoride
• Additional Relevant MeSH Terms: Stomatognathic Diseases; Tooth Diseases; Tooth Demineralization; Dental Caries
• Other Study ID 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