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.
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
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
Parallel
5% Sodium Fluoride Varnish
Other interventionTeeth 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.
2% Chlorhexidine Pre-Treatment + 5% NaF Varnish
Other interventionFollowing 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.
Standard Oral Hygiene Education
BehavioralChildren 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).
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.
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.
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.
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.
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Gül Keskin
Lead sponsor
Alanya Alaaddin Keykubat University
Sponsor institution
Alanya Alaaddin Keykubat University
Collaborator
