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

Overview

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.

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.