This observational study aims to investigate whether periodontal inflammation is associated with alterations in the expression of circadian clock-related genes and proteins in gingival tissues. Circadian rhythms regulate many biological processes, including immune responses and inflammation. Although experimental studies suggest a link between circadian disruption and periodontal disease, human data under controlled chronotype conditions are limited.

A total of 60 systemically healthy, non-smoking individuals aged 22-45 years with comparable sleep patterns (intermediate chronotype and 6-9 hours of sleep) were included. Participants were classified as periodontally healthy, gingivitis, or stage III grade B periodontitis according to established diagnostic criteria. Gingival tissue samples were collected during clinically indicated procedures within a standardized morning time window (09:00-11:00).

Gene expression levels of circadian clock components (CLOCK, BMAL1, PER1-3, CRY1-2, Rev-Erb-β, ROR-α) and inflammatory mediators (IL-1β, IL-6, TNF-α, NF-κB, IFN-γ, RANKL, OPG) were analyzed using RT-qPCR, Western blot, and ELISA techniques. Associations between molecular findings and clinical periodontal parameters were evaluated.

The study seeks to clarify whether periodontal disease itself may disrupt local circadian regulatory mechanisms in gingival tissues.

Circadian rhythms are generated through transcriptional-translational feedback loops involving core clock genes such as CLOCK, BMAL1, PER1-3, CRY1-2, ROR-α, and REV-ERB-β. These molecular oscillators regulate immune function, inflammatory signaling, and bone metabolism. Experimental evidence suggests that circadian dysregulation may aggravate periodontal inflammation and alveolar bone loss; however, comprehensive human data under controlled chronotype conditions remain limited.

This single-center, observational case-control study includes 60 systemically healthy, non-smoking individuals (30 males, 30 females) aged 22-45 years. Chronotype was determined using the Munich Chronotype Questionnaire, and only individuals with intermediate chronotype and self-reported sleep duration between 6 and 9 hours were included to minimize circadian variability.

Participants were classified into three groups (n=20 per group): periodontally healthy, gingivitis, and stage III grade B periodontitis according to the 2017 World Workshop criteria. Comprehensive periodontal examination included plaque index, gingival index, bleeding on probing, probing depth, and clinical attachment loss measurements. Gingival tissue biopsies were obtained during clinically indicated procedures and collected between 09:00 and 11:00 a.m. to standardize circadian timing. Samples were stored at -80°C until molecular analysis.

Total RNA was extracted from gingival tissues, and gene expression was quantified using RT-qPCR with normalization to β-actin and analysis via the 2^-ΔΔCT method. Protein expression of circadian clock components was assessed by Western blot, and inflammatory cytokine levels (IL-1β, IL-6) were quantified by ELISA. Correlation analyses were performed to evaluate associations between circadian gene expression, inflammatory mediators, and clinical periodontal parameters.

The primary objective is to determine whether periodontal inflammation is associated with disruption of gingival circadian clock gene and protein expression in individuals with comparable chronotype profiles.

Inclusion Criteria:

• Adults aged 18 years or older
• Systemically healthy individuals
• Presence of at least 20 natural teeth
• Individuals classified as periodontally healthy, gingivitis, or Stage III Grade B periodontitis according to the 2018 classification of periodontal diseases
• Willingness to provide written informed consent

Exclusion Criteria:

• Presence of any systemic disease affecting periodontal status (e.g., diabetes mellitus, autoimmune diseases, cardiovascular diseases)
• Use of antibiotics or anti-inflammatory medications within the previous 3 months
• Periodontal therapy within the last 6 months
• Current smokers or individuals who quit smoking within the past 5 years
• Pregnancy or lactation
• Use of medications known to influence immune or inflammatory responses
• History of systemic conditions that may affect wound healing

Systemically healthy adult individuals seeking care at the Department of Periodontology, Inonu University Faculty of Dentistry, were recruited. Participants were categorized into three groups based on periodontal status: periodontally healthy, gingivitis, and Stage III Grade B periodontitis according to the 2018 classification of periodontal diseases. All participants required clinically indicated periodontal procedures from which gingival tissue samples could be obtained.

De-identified individual participant data may be made available upon reasonable request to the corresponding investigator, subject to institutional approval and ethical considerations.