The goal of this observational cross-sectional study is to determine the salivary levels of E-cadherin, Calprotectin, and Matrix Metalloproteinase-9 (MMP-9) in patients with Recurrent Aphthous Stomatitis (RAS) and to compare them with those of healthy controls.

The main questions it aims to answer are:

Are the salivary levels of E-cadherin, Calprotectin, and MMP-9 significantly different in patients with minor RAS compared to healthy controls?

Can these salivary proteins serve as biomarkers involved in the pathogenesis of minor RAS?

Researchers will compare patients with minor RAS and healthy individuals to determine whether differences in salivary E-cadherin, Calprotectin, and MMP-9 levels are associated with the presence of disease.

Participants will:

Provide unstimulated saliva samples using a saliva swab

Undergo clinical evaluation for periodontal status (Community Periodontal Index - CPI) and caries experience (DMFT index)

Collected saliva samples will be stored at -80°C until analysis. The concentrations of the investigated proteins will be measured using ELISA kits, and total protein levels will be determined using a colorimetric protein assay.

The relationships among the salivary concentrations of the investigated proteins, as well as their associations with demographic variables such as age and sex, will be analyzed.

Hypotheses H₀ (Null Hypothesis): There is no statistically significant difference in salivary levels of E-cadherin, Calprotectin, and Matrix Metalloproteinase-9 (MMP-9) between patients with Recurrent Aphthous Stomatitis (RAS) and healthy controls.

H₁ (Alternative Hypothesis): There is a statistically significant difference in salivary levels of E-cadherin, Calprotectin, and MMP-9 between patients with RAS and healthy controls.

Sample Size Calculation Based on an effect size of 0.50, a power of 80%, and a type I error rate (α) of 5%, the required sample size for the study was calculated to be 102 participants using the "t tests - Means: Difference between two independent means (two groups)" module in G*Power (version 3.1).

Formation of Participant Groups Two study groups will be established: a patient group and a healthy control group. Patients clinically diagnosed with minor recurrent aphthous stomatitis (RAS) based on characteristic clinical appearance and medical history will be included in the patient group. For the diagnosis of minor RAS, a history of at least three episodes of RAS within the past year will be considered a required criterion. This lesion should be round or oval in shape, less than 5 mm in diameter, surrounded by an erythematous halo at the periphery, and covered by a grayish-white pseudomembrane. In order to obtain a salivary sample, at least one aphthous lesion must be present at the time of examination. Control group participants with a similar age and gender distribution and no lifetime history of RAS will be selected and included in the study.

In this study, the threshold value of DMFT < 5.0, defined by the World Health Organization (WHO) as a very low caries risk level in adults, will be used as an inclusion criterion for all participants.

DMFT Index:

D (Decayed): Number of decayed teeth M (Missing): Number of teeth lost due to caries F (Filled): Number of filled teeth due to past caries T (Teeth): Total number of teeth evaluated Participants with DMFT ≥ 5.0 will be excluded from the study. To assess periodontal status, the Community Periodontal Index (CPI) will be used. The CPI is a standardized system developed by the World Health Organization (WHO) to evaluate periodontal health. The index teeth (11, 16, 17, 26, 27, 31, 36, 37, 46, 47) will be examined to allow for rapid and standardized assessment of periodontal conditions. Periodontal measurements will be performed using the WHO Community Periodontal Index probe. Each index tooth will be assessed at four sites: buccal, lingual/palatal, mesial, and distal. The highest score recorded for each tooth or quadrant will be noted. The highest score in any quadrant will be used to represent the participant's overall CPI score.

0 - Healthy periodontal tissues

1. - Bleeding observed upon probing
2. - Calculus detected during probing
3. - Pocket depth of 4-5 mm
4. - Pocket depth of 6 mm or more If one of the two molars in the posterior quadrant is missing, it will not be replaced by another tooth. In cases where no suitable index teeth are available in a quadrant, all present teeth in that quadrant will be examined, and the highest score will be recorded as the quadrant's CPI score. The distal surfaces of third molars will not be included in the assessment. Only participants with a CPI score of 0-2 will be included in the study.

Collection and Analysis of Saliva Samples For saliva collection, the swab method, which is one of the unstimulated saliva collection techniques, will be used. Salivette® swab (Sarstedt, Germany) will be utilized to collect saliva samples from participants. In accordance with the manufacturer's instructions, samples will be taken at least 60 minutes after any food or drink intake (liquid or solid) or tooth brushing. Participants will be asked to rinse their mouths with water for 1-5 seconds approximately 10 minutes before sampling. They will then be instructed to hold the absorbent swab in their mouth without chewing for at least 2 minutes. After that, the swab will be removed and placed into the inner suspended tube of the Salivette container by the participant. Samples will be centrifuged within 4 hours using the A-PRF12 centrifuge (Germany) at 1000 × g for 2 minutes. During centrifugation, heavy particles (cells, debris, or other precipitable materials) will settle at the bottom of the tube, while the lighter, soluble components will remain in the upper phase (supernatant). Following centrifugation, the supernatant portion of the saliva will be carefully separated and transferred to sterile Eppendorf tubes. The samples will be coded and stored at -80°C until biochemical analysis. After all samples have been collected, they will be transferred to the Department of Biochemistry, Faculty of Medicine, Aydın Adnan Menderes University, and analyzed using the ELISA method on the same day. Human E-cadherin, Human MMP-9, and Human CALP (Calprotectin) ELISA kits, along with the BCA Protein Colorimetric Assay Kit, will be used according to the manufacturer's instructions. The concentrations of the investigated proteins as well as total protein levels will be measured in the saliva samples. The concentration of each investigated protein will be normalized by calculating its ratio to the total salivary protein concentration. This normalization will account for inter-sample variability due to differences in salivary flow rate, cellular content, or sample volume.

Inclusion Criteria:

• Participants aged 18 years or older
• Clinically diagnosed with minor recurrent aphthous stomatitis (RAS) (defined as having a history of at least three RAS episodes within the past year)
• Systemically healthy (i.e., without any diagnosed systemic disease)
• No history of tobacco use
• Presence of at least one active aphthous ulcer at the time of saliva sample collection
• No known mental or physical disabilities

Exclusion Criteria:

• Use of any pharmacological treatment within the past 3 months
• Use of antiseptic mouthwash within the past 3 months
• Pregnant or lactating individuals, or those currently using oral contraceptives
• History of systemic diseases associated with oral ulcerations such as Crohn's disease, ulcerative colitis, celiac disease, or Behçet's disease
• History of chemotherapy or radiotherapy
• Current or prior use of immunomodulatory drugs, cytotoxic agents, or systemic corticosteroids
• Undergoing orthodontic treatment (to avoid misdiagnosis with traumatic ulcers)
• Use of removable dental prostheses (to avoid misdiagnosis with traumatic ulcers)
• Presence of periodontal disease (CPI score of 3 or 4)
• Caries risk not classified as very low (DMFT ≥ 5)
• Presence of pathological oral mucosal lesions such as lichen planus, pemphigus vulgaris, or mucous membrane pemphigoid

The study population will consist of a total of 102 participants, including 51 patients clinically diagnosed with minor recurrent aphthous stomatitis (RAS) and 51 healthy controls. All participants will be aged 18 years or older. Patients must have experienced at least three episodes of minor RAS within the past year and present with at least one active aphthous ulcer at the time of saliva collection. Only systemically healthy, non-smoking individuals without periodontal disease or oral mucosal pathology will be included. Control group participants will be matched by age and gender to the patient group and must have no lifetime history of RAS.