Circadian Clock Proteins in Gingival Crevicular Fluid of Individuals With and Without Circadian Rhythm Disruption (Nursma-CIRC-HG)

May 11, 2026 updated by: Cuneyt Asim Aral, Inonu University

Investigation of Circadian Clock Gene/Proteins in Individuals With and Without Disrupted Circadian Rhythm: A Prospective Cross-Sectional Clinical Study

This prospective cross-sectional clinical study aims to investigate the relationship between circadian rhythm disruption and periodontal inflammation by evaluating circadian clock protein levels, inflammatory (IL-1beta) and anti-inflammatory (IL-10) cytokine levels in gingival crevicular fluid (GCF) of individuals with and without circadian rhythm disruption.

Participants aged 20-50 years will be classified into four groups based on their circadian rhythm status (disrupted/normal) and gingival health status (gingivitis/healthy). Clinical periodontal parameters including plaque index, gingival index, bleeding on probing, and probing depth will be assessed. Circadian rhythm status will be determined using validated questionnaires (Morningness-Eveningness Questionnaire and Munich Chronotype Questionnaire). Night-shift workers will represent the circadian rhythm disruption group.

GCF samples will be analyzed for circadian clock proteins (BMAL-1, CLOCK, PER-1, PER-2, PER-3, CRY-1, CRY-2, REV-ERB-beta, MTNR1B) and cytokines (IL-1beta, IL-10) using ELISA. Serum cortisol and melatonin levels will be measured for biochemical verification of circadian rhythm status.

Gingivitis groups will receive standard periodontal treatment and be re-evaluated at 2 weeks post-treatment. A total of 116 participants (29 per group) are planned for enrollment.

Study Overview

Status

Not yet recruiting

Conditions

Intervention / Treatment

Detailed Description

Gingival tissue plays a critical role in maintaining oral health as a fundamental component of periodontal tissues. Recent evidence suggests that circadian rhythm mechanisms, beyond microbial factors, may significantly influence periodontal disease pathogenesis through modulation of host immune responses.

The circadian rhythm is an endogenous mechanism regulating biological processes in approximately 24-hour physiological and behavioral cycles, controlled centrally by the suprachiasmatic nucleus in the hypothalamus. At the molecular level, core clock genes (CLOCK, BMAL1, PER1-3, CRY1-2) operate through the Transcription-Translation Feedback Loop (TTFL). CLOCK and BMAL1 proteins form heterodimers in the cell nucleus, bind to E-box regions on DNA, and initiate transcription of PER and CRY genes. Subsequently, PER and CRY proteins synthesized in the cytoplasm return to the nucleus and inhibit the CLOCK-BMAL1 complex, creating approximately 24-hour rhythmic gene expression cycles. Regulatory nuclear receptors such as REV-ERBa and ROR also contribute to the fine-tuning of this mechanism.

Recent studies have demonstrated the presence of functional peripheral clock mechanisms in periodontal tissues. CLOCK, BMAL1, PER1-3, and CRY1-2 gene expression has been identified in gingival fibroblasts and periodontal ligament fibroblasts. Experimental studies have shown that circadian rhythm disruption can increase inflammatory processes, enhance macrophage activation, elevate inflammatory cytokine production, and accelerate alveolar bone loss in periodontitis models.

Despite these findings, human studies evaluating the relationship between inflammatory cytokines and circadian rhythm proteins in gingival crevicular fluid (GCF) remain limited. This study addresses this gap by evaluating gingival health status using clinical periodontal parameters in individuals with and without circadian rhythm disruption.

Study Design:

Participants will be classified into four groups based on circadian rhythm status and periodontal health:

  • Group 1: Normal circadian rhythm / Periodontally healthy (control)
  • Group 2: Normal circadian rhythm / Gingivitis
  • Group 3: Circadian rhythm disruption / Periodontally healthy
  • Group 4: Circadian rhythm disruption / Gingivitis
  • Group 5: Group 2 at 2 weeks post-periodontal treatment
  • Group 6: Group 4 at 2 weeks post-periodontal treatment

Time Points:

T0 (Baseline): Clinical periodontal examination, GCF, saliva, and serum sampling in all groups.

T1 (Day 14): Re-evaluation of clinical parameters and biological sampling in gingivitis groups after standard periodontal treatment.

Clinical Periodontal Assessment:

All periodontal measurements will be performed by a single examiner using a standard periodontal probe at six sites per tooth (mesiobuccal, buccal, distobuccal, mesiolingual, lingual, distolingual). Parameters include plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL).

GCF Sampling:

GCF samples will be collected using sterile Periopaper strips placed in the gingival sulcus for 30 seconds after supragingival plaque removal and isolation. GCF volumes will be quantified using a Periotron device. Samples contaminated with blood will be excluded.

Saliva Sampling:

Unstimulated saliva samples will be collected as an alternative biological material if circadian rhythm proteins cannot be detected at sufficient levels in GCF.

Serum Sampling:

Venous blood samples will be collected from the antecubital vein for biochemical evaluation of circadian rhythm status through cortisol and melatonin level analysis.

All biological samples will be collected during standardized morning hours (09:00-11:00) to minimize diurnal variation in biomarker levels.

Laboratory Analysis:

GCF samples will be analyzed using ELISA for circadian rhythm-related proteins (MTNR1B, BMAL-1, CLOCK, PER-1, PER-2, PER-3, CRY-1, CRY-2, REV-ERB-beta) and inflammatory biomarkers (IL-1beta and IL-10).

Statistical Analysis:

Data will be analyzed using SPSS. Normality will be assessed by Shapiro-Wilk test. Comparisons among independent groups will use one-way ANOVA or Kruskal-Wallis test, with Tukey or Dunn post-hoc tests. Pre- and post-treatment comparisons will use paired t-test or Wilcoxon signed-rank test. Correlations will be assessed by Pearson or Spearman analysis. Multivariate regression models will control for age, sex, BMI, and waist circumference as covariates. Significance level: p < 0.05.

This study was approved by the Inonu University Health Sciences Scientific Research Ethics Committee and will be conducted in accordance with the Declaration of Helsinki. Written informed consent will be obtained from all participants.

Study Type

Observational

Enrollment (Estimated)

116

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Contact Backup

Study Locations

  • Turkey (Türkiye)
    • Malatya
      • Malatya, Malatya, Turkey (Türkiye), 44210
        • Inönü University Faculty of Dentistry, Department of Periodontology

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult

Accepts Healthy Volunteers

Yes

Sampling Method

Non-Probability Sample

Study Population

Systemically healthy volunteers aged 20-50 years presenting to Inonu University Faculty of Dentistry, Department of Periodontology. The study population includes individuals with normal circadian rhythm and night-shift workers with circadian rhythm disruption. Participants are classified as periodontally healthy or with gingivitis based on clinical periodontal examination. Circadian rhythm status is verified using the Morningness-Eveningness Questionnaire (MEQ) and Munich Chronotype Questionnaire (MCTQ).

Description

Inclusion Criteria:

  • Aged between 20 and 50 years
  • Systemically healthy
  • Diagnosed as periodontally healthy or with gingivitis based on clinical periodontal examination
  • Having at least 20 natural teeth in the oral cavity
  • Willing to provide written informed consent
  • Agreeing to participate in circadian rhythm assessment and completing the Morningness-Eveningness Questionnaire (MEQ) and Munich Chronotype Questionnaire (MCTQ)
  • Sleep duration of 6-9 hours per day
  • For periodontally healthy: PD <=3 mm, BOP <10%, CAL=0, no radiographic bone loss
  • For gingivitis: PD <=3 mm, 10%<=BOP<=30%, CAL=0, no radiographic bone loss
  • For circadian rhythm disruption group: being a night-shift worker

Exclusion Criteria:

  • Use of antioxidant or melatonin supplements
  • Presence of any systemic disease
  • Use of antibiotics, anti-inflammatory drugs, or immunosuppressants within the past 3 months
  • Having received periodontal treatment within the past 6 months
  • Pregnancy or lactation
  • Use of tobacco or tobacco products
  • Presence of acute oral infection, abscess, or advanced periodontal disease
  • Currently undergoing orthodontic treatment or having fixed orthodontic appliances
  • Inability to comply with study procedures or refusal to provide informed consent
  • Self-reported sleep duration less than 6 hours or greater than 9 hours per day

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

Cohorts and Interventions

Group / Cohort
Intervention / Treatment
Normal Circadian / Periodontally Healthy
Individuals with normal circadian rhythm and periodontal health (control group). Inclusion: PD <=3 mm, BOP <10%, CAL=0, no radiographic bone loss. n=29
Normal Circadian / Gingivitis
Individuals with normal circadian rhythm and gingivitis. Inclusion: PD <=3 mm, 10%<=BOP<=30%, CAL=0, no radiographic bone loss. These participants will receive standard periodontal treatment and be re-evaluated at 14 days post-treatment. n=29
Other: Standard Periodontal Treatment
Standard periodontal treatment including detailed oral hygiene instruction and professional dental surface cleaning (scaling and polishing). Applied to gingivitis groups (Group 2 and Group 4) at baseline, with clinical and biochemical re-evaluation at 14 days post-treatment.
Disrupted Circadian / Periodontally Healthy
Night-shift workers with circadian rhythm disruption and periodontal health. Circadian rhythm status verified by MEQ and MCTQ questionnaires. Inclusion: PD <=3 mm, BOP <10%, CAL=0, no radiographic bone loss. n=29
Disrupted Circadian / Gingivitis
Night-shift workers with circadian rhythm disruption and gingivitis. Circadian rhythm status verified by MEQ and MCTQ questionnaires. Inclusion: PD <=3 mm, 10%<=BOP<=30%, CAL=0, no radiographic bone loss. These participants will receive standard periodontal treatment and be re-evaluated at 14 days post-treatment. n=29
Other: Standard Periodontal Treatment
Standard periodontal treatment including detailed oral hygiene instruction and professional dental surface cleaning (scaling and polishing). Applied to gingivitis groups (Group 2 and Group 4) at baseline, with clinical and biochemical re-evaluation at 14 days post-treatment.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
GCF Circadian Rhythm Protein Levels
Time Frame: Baseline (T0) and 14 days post-treatment (T1) for gingivitis groups
Concentrations of circadian clock proteins (BMAL-1, CLOCK, PER-1, PER-2, PER-3, CRY-1, CRY-2, REV-ERB-beta, MTNR1B) measured in gingival crevicular fluid (GCF) using ELISA. GCF collected with Periopaper strips placed in the gingival sulcus for 30 seconds. Volumes quantified using a Periotron device.
Baseline (T0) and 14 days post-treatment (T1) for gingivitis groups
GCF Inflammatory Cytokine Levels
Time Frame: Baseline (T0) and 14 days post-treatment (T1) for gingivitis groups
Interleukin-1 beta (IL-1beta, pro-inflammatory) and Interleukin-10 (IL-10, anti-inflammatory) cytokine concentrations in gingival crevicular fluid measured by ELISA.
Baseline (T0) and 14 days post-treatment (T1) for gingivitis groups

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Inonu University

Collaborators

University of Turku
University of Minnesota
Harvard University

Investigators

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

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Estimated)

August 1, 2026

Primary Completion (Estimated)

January 1, 2027

Study Completion (Estimated)

March 1, 2027

Study Registration Dates

First Submitted

May 11, 2026

First Submitted That Met QC Criteria

May 11, 2026

First Posted (Actual)

May 18, 2026

Study Record Updates

Last Update Posted (Actual)

May 18, 2026

Last Update Submitted That Met QC Criteria

May 11, 2026

Last Verified

May 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • IUCREC.2026/9750
  • Will be applied (Other Grant/Funding Number: Inönü University Scientific Research Projects Coordination Office)

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

UNDECIDED

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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