Effect of Smoking on Macrophage Polarization in Periodontitis

The Effect of Smoking on Macrophage Polarization in the Pathogenesis of Periodontitis

This observational case-control study investigates the effect of smoking on macrophage polarization in the pathogenesis of periodontitis. Adult participants who were either systemically healthy smokers or non-smokers, and who met the clinical criteria for periodontal health or periodontitis, were included. Gingival tissue samples were collected during periodontal procedures performed after completion of Phase I periodontal therapy, and the expression levels of macrophage polarization markers (iNOS and Arginase-1) were analyzed using ELISA. In addition, comprehensive clinical periodontal measurements-including probing pocket depth, clinical attachment level, bleeding on probing, Plaque Index, and Gingival Index-were recorded to evaluate the relationship between smoking status, inflammatory burden, and macrophage phenotype. The study aims to clarify how smoking modulates the balance between M1 and M2 macrophage responses in periodontal tissues.

Study Overview

• Status: Completed
• Conditions: Periodontitis
• Intervention / Treatment: Other: Smoking Exposure

Detailed Description

Periodontitis is a chronic inflammatory disease in which macrophage polarization plays a key regulatory role in the host immune response. Smoking is a well-established risk factor that alters inflammatory pathways and immune cell behavior. This study was designed to examine how smoking influences macrophage polarization-specifically the balance between M1 (iNOS-mediated) and M2 (Arginase-1-mediated) phenotypes-in periodontal tissues.

Systemically healthy adult participants were recruited and categorized into four groups based on smoking status and periodontal condition: (1) healthy non-smokers, (2) periodontitis non-smokers, (3) healthy smokers, and (4) periodontitis smokers. Gingival tissue samples (approximately 2×2 mm) were collected during periodontal surgical procedures performed after completion of Phase I periodontal therapy, immediately stored at -80 °C, and processed for biochemical analysis. Comprehensive clinical periodontal measurements-including probing pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), Plaque Index (PI), and Gingival Index (GI)-were recorded on the same day to evaluate periodontal status and inflammatory burden. ELISA was used to quantify iNOS and Arginase-1 levels in gingival tissues.

The primary objective of this study is to determine whether smoking shifts the macrophage phenotype toward a more pro-inflammatory M1 profile or suppresses M2-associated regulatory pathways in the context of periodontitis. The findings are expected to contribute to a better understanding of smoking-related alterations in periodontal immune mechanisms.

• Study Type: Observational
• Enrollment (Actual): 80

Contacts and Locations

Study Locations

• Turkey (Türkiye)
  • Isparta
    • Isparta, Isparta, Turkey (Türkiye), 32000
      • Süleyman Demirel University Faculty of Dentistry, Department of Periodontology

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: N/A

• Sampling Method: Non-Probability Sample

Study Population

The study population consisted of systemically healthy volunteer adults aged 18-65 years who presented to the Periodontology Clinic of Süleyman Demirel University Faculty of Dentistry with a need for periodontal treatment. Eligible participants were assigned to one of four groups based on smoking status and periodontal condition: healthy non-smokers, periodontitis non-smokers, healthy smokers, and periodontitis smokers. All participants were evaluated according to the study's predefined inclusion and exclusion criteria before enrollment.

Description

Inclusion Criteria:

• The study population consisted of volunteer participants aged 18-65 years who were referred to the Department of Periodontology at Süleyman Demirel University Faculty of Dentistry for periodontal treatment and who met the inclusion criteria without violating any exclusion criteria.

Exclusion Criteria:

• Individuals who did not consent to participate in the study
• History of any periodontal treatment within the past 6 months
• Use of systemic antibiotics within the past 3 months
• Pregnancy or lactation
• Psychological or physical conditions that may interfere with cooperation during clinical examination
• History of substance or alcohol abuse
• Presence of severe malocclusion or ongoing orthodontic treatment
• Acute dental pain due to caries, abscess, or other odontogenic infections
• Presence of systemic diseases with oral manifestations or any systemic condition known to affect periodontal tissues
• History of malignancy
• Use of medications that can influence periodontal parameters (e.g., anti-inflammatory drugs) within the past 3 months
• Participation in another scientific or clinical research study within the past 3 months

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Healthy Non-Smokers; Systemically healthy individuals who do not smoke and present with clinically healthy periodontal status. Gingival tissue samples were collected after completion of Phase I periodontal therapy during routine periodontal procedures.
Periodontitis Non-Smokers; Systemically healthy individuals diagnosed with periodontitis who do not smoke. Gingival tissue biopsies were obtained after completion of Phase I periodontal therapy during periodontal surgical procedures.
Healthy Smokers; Systemically healthy individuals who smoke (≥10 cigarettes/day) and exhibit clinically healthy periodontal status. Gingival tissue samples were collected after completion of Phase I periodontal therapy during periodontal procedures.	Other: Smoking Exposure; Exposure to cigarette smoking, defined as smoking ≥10 cigarettes per day. Participants were classified as smokers or non-smokers based on self-reported smoking status.
Periodontitis Smokers; Systemically healthy individuals who smoke (≥10 cigarettes/day) and are clinically diagnosed with periodontitis. Gingival tissue samples were collected after completion of Phase I periodontal therapy during periodontal surgical procedures.	Other: Smoking Exposure; Exposure to cigarette smoking, defined as smoking ≥10 cigarettes per day. Participants were classified as smokers or non-smokers based on self-reported smoking status.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Gingival iNOS Level	Concentration of inducible nitric oxide synthase (iNOS) in gingival tissue biopsies measured by ELISA (ng/mg tissue).	At the time of periodontal surgery following completion of Phase I periodontal therapy.
Gingival Arginase-1 Level	Concentration of Arginase-1 in gingival tissue biopsies measured by ELISA (ng/mg tissue).	At the time of periodontal surgery following completion of Phase I periodontal therapy.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Probing Pocket Depth (PPD)	Mean probing pocket depth (mm) recorded at six sites per tooth using a periodontal probe.	Immediately prior to periodontal surgery, following completion of Phase I periodontal therapy.
Clinical Attachment Level (CAL)	Clinical attachment level (mm) recorded at six sites per tooth using a periodontal probe.	Immediately prior to periodontal surgery, following completion of Phase I periodontal therapy.
Bleeding on Probing (BOP)	Bleeding on probing percentage calculated as the proportion of bleeding sites to total sites examined.	Immediately prior to periodontal surgery, following completion of Phase I periodontal therapy.
Plaque Index (PI)	Dental plaque accumulation assessed using the Silness and Löe Plaque Index. Plaque scores were recorded at four surfaces per tooth and scored on a scale from 0 to 3, where 0 indicates no plaque and 3 indicates abundant plaque accumulation. Higher scores indicate worse plaque status.	Immediately prior to periodontal surgery, following completion of Phase I periodontal therapy.
Gingival Index (GI)	Gingival inflammation assessed using the Löe and Silness Gingival Index. Gingival Index scores were recorded at four surfaces per tooth and scored on a scale from 0 to 3, where 0 indicates normal gingiva and 3 indicates severe gingival inflammation. Higher scores indicate worse gingival status.	Immediately prior to periodontal surgery, following completion of Phase I periodontal therapy.

Collaborators and Investigators

• Sponsor: Suleyman Demirel University
• Investigators: Principal Investigator: Zuhal YETKİN AY, Prof. Dr., Suleyman Demirel University, Faculty of Dentistry, Department of Periodontology

Study record dates

Study Major Dates

• Study Start (Actual): December 1, 2024
• Primary Completion (Actual): July 15, 2025
• Study Completion (Actual): November 4, 2025

Study Registration Dates

• First Submitted: December 4, 2025
• First Submitted That Met QC Criteria: December 28, 2025
• First Posted (Actual): January 7, 2026

Study Record Updates

• Last Update Posted (Actual): January 7, 2026
• Last Update Submitted That Met QC Criteria: December 28, 2025
• Last Verified: December 1, 2025

More Information

Terms related to this study

• Keywords: Smoking; Periodontal Disease; Macrophage Polarization; iNOS; Periodontal Inflammation; M1/M2 Macrophages; Arginase-1
• Additional Relevant MeSH Terms: Mouth Diseases; Stomatognathic Diseases; Behavior; Periodontitis; Periodontal Diseases; Smoking
• Other Study ID Numbers: SDU-Perio-iNOS-ARG1-01; TDK-2024-9512 (Other Identifier: SDU BAP)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: Individual participant data will not be shared because this study is a single-center thesis research project, and data sharing is not permitted under institutional and ethical regulations.

Drug and device information, study documents

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