Circadian Clock Gene Expression in Periodontal Disease (PERIOCLOCK)

Periodontal Inflammation Is Associated With Disruption of Gingival Circadian Clock Gene and Protein Expression in Individuals With Comparable Chronotype Profiles

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.

Study Overview

• Status: Completed
• Conditions: Periodontitis; Gingivitis
• Intervention / Treatment: Procedure: Gingival Tissue Biopsy

Detailed Description

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.

• Study Type: Observational
• Enrollment (Actual): 60

Contacts and Locations

Study Locations

• Turkey (Türkiye)
  • Malatya, Turkey (Türkiye), 44210
    • Inonu University Faculty of Dentistry

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

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.

Description

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

Study Plan

How is the study designed?

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Periodontally Healthy; Systemically healthy individuals with clinically healthy periodontal tissues, no attachment loss, and no radiographic bone loss.	Procedure: Gingival Tissue Biopsy; Collection of gingival tissue samples from interproximal sites during clinically indicated periodontal procedures for molecular and protein expression analyses.
Gingivitis; Systemically healthy individuals presenting with gingival inflammation without clinical attachment loss or radiographic bone loss.	Procedure: Gingival Tissue Biopsy; Collection of gingival tissue samples from interproximal sites during clinically indicated periodontal procedures for molecular and protein expression analyses.
Stage III Grade B Periodontitis; Systemically healthy individuals diagnosed with Stage III Grade B periodontitis according to the 2018 classification of periodontal diseases.	Procedure: Gingival Tissue Biopsy; Collection of gingival tissue samples from interproximal sites during clinically indicated periodontal procedures for molecular and protein expression analyses.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Relative mRNA Expression Levels of Circadian Clock Genes in Gingival Tissue	Quantitative assessment of BMAL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, REV-ERBβ, and ROR-α mRNA expression levels in gingival tissue samples using real-time quantitative polymerase chain reaction (RT-qPCR). Expression levels will be calculated as relative fold changes normalized to housekeeping genes and compared among periodontally healthy, gingivitis, and Stage III Grade B periodontitis groups.	At the time of gingival tissue collection (single time point)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Pro-inflammatory Cytokine Expression Levels	Relative mRNA expression levels of IL-1β, IL-6, TNF-α, and IFN-γ in gingival tissue samples assessed by RT-qPCR and compared among study groups.	At the time of tissue collection
NF-κB Expression Level	Relative expression level of NF-κB in gingival tissue samples determined by molecular analysis and compared among study groups.	At the time of tissue collection
Bone Metabolism Markers	Relative mRNA expression levels of RANKL and OPG in gingival tissue samples and evaluation of the RANKL/OPG ratio among study groups.	At the time of tissue collection

Collaborators and Investigators

• Sponsor: Inonu University
• Investigators: Study Chair: Cüneyt A Aral, Professor, DDS, PhD, Inonu University

Publications and helpful links

General Publications

• Czeisler CA, Duffy JF, Shanahan TL, Brown EN, Mitchell JF, Rimmer DW, Ronda JM, Silva EJ, Allan JS, Emens JS, Dijk DJ, Kronauer RE. Stability, precision, and near-24-hour period of the human circadian pacemaker. Science. 1999 Jun 25;284(5423):2177-81. doi: 10.1126/science.284.5423.2177.
• Kapila YL. Oral health's inextricable connection to systemic health: Special populations bring to bear multimodal relationships and factors connecting periodontal disease to systemic diseases and conditions. Periodontol 2000. 2021 Oct;87(1):11-16. doi: 10.1111/prd.12398.
• Chapple ILC, Mealey BL, Van Dyke TE, Bartold PM, Dommisch H, Eickholz P, Geisinger ML, Genco RJ, Glogauer M, Goldstein M, Griffin TJ, Holmstrup P, Johnson GK, Kapila Y, Lang NP, Meyle J, Murakami S, Plemons J, Romito GA, Shapira L, Tatakis DN, Teughels W, Trombelli L, Walter C, Wimmer G, Xenoudi P, Yoshie H. Periodontal health and gingival diseases and conditions on an intact and a reduced periodontium: Consensus report of workgroup 1 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Periodontol. 2018 Jun;89 Suppl 1:S74-S84. doi: 10.1002/JPER.17-0719.
• Dibner C, Schibler U, Albrecht U. The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol. 2010;72:517-49. doi: 10.1146/annurev-physiol-021909-135821.
• Hergenhan S, Holtkamp S, Scheiermann C. Molecular Interactions Between Components of the Circadian Clock and the Immune System. J Mol Biol. 2020 May 29;432(12):3700-3713. doi: 10.1016/j.jmb.2019.12.044. Epub 2020 Jan 10.
• Hastings M, O'Neill JS, Maywood ES. Circadian clocks: regulators of endocrine and metabolic rhythms. J Endocrinol. 2007 Nov;195(2):187-98. doi: 10.1677/JOE-07-0378.

Study record dates

Study Major Dates

• Study Start (Actual): January 20, 2024
• Primary Completion (Actual): March 2, 2025
• Study Completion (Actual): December 1, 2025

Study Registration Dates

• First Submitted: March 1, 2026
• First Submitted That Met QC Criteria: March 1, 2026
• First Posted (Actual): March 5, 2026

Study Record Updates

• Last Update Posted (Actual): March 6, 2026
• Last Update Submitted That Met QC Criteria: March 5, 2026
• Last Verified: March 1, 2026

More Information

Terms related to this study

• Keywords: Inflammation; Periodontitis; Circadian Rhythm; IL-6; OPG; RANKL; TNF-α; NF-κB; IL-1β; IFN-γ; BMAL1; CLOCK; Per1; Per2; Per3; CRY1; CRY2; ROR-α; Rev-Erb-β
• Additional Relevant MeSH Terms: Periodontal Diseases; Mouth Diseases; Stomatognathic Diseases; Pathologic Processes; Infections; Gingival Diseases; Pathological Conditions, Signs and Symptoms; Periodontitis; Inflammation; Gingivitis
• Other Study ID Numbers: IUCREC.2024/05.13; TDH-2024-3463 (Other Grant/Funding Number: Inonu University Scientific Research Projects Coordination Office)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED
• IPD Plan Description: De-identified individual participant data may be made available upon reasonable request to the corresponding investigator, subject to institutional approval and ethical considerations.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No