Smoking Influence on Apoptosis in Periodontitis

Influence of Smoking on Fibroblast Apoptosis in Patients With Chronic and Aggressive Periodontitis

Apoptosis is an evolutionary form of physiological cell death. Studies suggest that apoptosis is involved in the pathogenesis of periodontal diseases. Human gingival fibroblasts (HGF) have an important role in the periodontal immune response. It is believed that HGF can be diminished and/or eliminated by means of apoptosis.

Smoking is one of the most common risk factor of periodontal disease. Studies indicated that smoking can increase the risk of periodontitis by enhancing the apoptosis of gingival fibroblast.

The purpose of this study is to determine and to investigate apoptosis of HGF in gingival biopsies collected from smokers and non smokers who are diagnosed with chronic periodontitis or aggressive periodontitis.

Eighty subjects will be invited to participate in this study. Patients will be allocated into four groups (20 patients each). Gingival biopsies will be obtained from the base of papillae during surgical treatment (open flap curettage) and will be examined by Immuno-histochemical analysis. Immune-staining will be done using p53 monoclonal mouse anti-human antibody.

Study Overview

• Status: Completed
• Conditions: Aggressive Periodontitis; Chronic Periodontitis

Detailed Description

Background:

The mechanisms responsible for gingival tissue damage are poorly understood, and both immune-mediated reactions and direct cytopathic effects of bacteria maybe involved. Based on direct effect of bacteria in cell cultures, it has been suggested that apoptosis might play an important role in periodontitis (Chen 1994).

Programmed cell death (apoptosis) is normal physiologic process that contributes to maintaining tissue homeostasis. The process of apoptosis can be modulated by various stimuli hormones, cytokines, growth factors, infections and immune-responses (Renehan et al. 2001).

Among other factors, the products of two genes that encode protein p53 and Bcl-2 have been shown to play fundamental regulatory role in apoptosis. P53 is the protein product of tumor-suppressor gene, and expression of P53 can induce apoptosis. This protein is also implicated in the regulation of tissue dynamics, and is specifically thought to induce apoptosis in terminally differentiated cells, including inflammatory cells (Bulut et al.2006).

Fibroblast is a type of cell that synthesizes the extracellular and collagen in connective tissue. It plays a critical role in wound healing. Fibroblasts are the most common cells in connective tissue. The main function of fibroblasts is to maintain the structural integrity of connective tissue by continuously secreting precursors of extracellular matrix. it was reported that apoptosis levels increased in gingivitis and periodontitis (Jarenberg et al. 2002).

Cigarette smoking is a risk factor for many diseases, and recent evidence indicates that smoking adversely influences periodontal health. A number of epidemiologic studies have shown strong association between smoking and prevalence and severity of periodontitis (Bostrom et al. 1998). Whereas, the pathogenesis of periodontitis in smokers is poorly understood there are data suggesting that smoking effects on the periodontal tissues include defects in neutrophil function, impaired serum antibody responses to periodontal pathogens, and potentially diminished gingival fibroblast function (Shivanaikar et al. 2001). Smokers are considered a high-risk group for periodontitis, and smoking history is a useful clinical predictor of future disease activity (Hanokia et al. 2000).

Aims of the study:

• Investigation of the effects of smoking on fibroblasts apoptosis in smokers with periodontitis compared to non-smokers with periodontitis using immuno-histochemical methods.
• Comparison of apoptosis levels of gingival fibroblasts between chronic and aggressive periodontitis.

Materials and methods:

A total of 80 subjects will be invited to participate in this study from the patients referred to the Department of Periodontology, Faculty of Dentistry, University of Damascus. The study will be approved by a specific Review Board. Subjects will be recruited according to specific inclusion criteria after completion of medical and dental history questionnaires. Patients will sign a consent form after being advised about the nature of the study.

The selection of patients will be made according of the criteria approved by the 1999 international world workshop for a classification system of periodontal diseases and conditions (Armitage 1999) using five clinical parameters and full mouth or panoramic radiographs for diagnosis.

Subjects will be allocated into four groups:

• Smoking Chronic Periodontitis group (ChP-S): comprise 20 patients who are smokers and aged > 45 years and have presence of ≥2 non-adjacent sites per quadrant that were not first molars or incisors, with probing depth (PD) ≥5 mm, which bleed on gentle probing. The demonstrated radiographic bone loss ≥30% of the root length, patient with poor oral hygiene, the amount of accumulated plaque commensurate with the amount of clinical attachment level (CAL)
• Non-smoking Chronic periodontitis group (ChP-NS): comprise 20 age-sex matched patients who are non-smokers and have chronic periodontitis.
• Smoking Aggressive Periodontitis group (AgP-S): comprise 20 patients who are smokers and aged < 35 years and diagnosed with rapid attachment loss with periodontal pocket depth (PD) > 4 mm around at least three teeth other than the first molars and incisors. Rapid bone destruction (>50%bone loss at diseased sites). Weak relationship between dental plaque and the severity of gingival inflammation.
• Non-smoking Aggressive periodontitis group (AgP-NS): comprise 20 age- and sex matched patients who are non-smokers and have aggressive periodontitis.

Clinical measurements:

A standard periodontal probe will be used for recording periodontal indices at six sites per tooth. The examined clinical parameters include bleeding on probing (BOP), plaque index (PI), clinical attachment loss (CAL) and periodontal pocket depth (PPD) and gingival index (GI).

Biopsy collection and analysis

• The biopsy samples will be obtained from patients during surgical treatment (open flap curettage) where biopsy is taken from the base area of papilla.
• Samples will be placed in 10% formalin.
• The samples will be then installed in paraffin wax.
• Samples will be analyzed in the by means of p53 monoclonal mouse anti-human antibody.

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

Contacts and Locations

Study Locations

• Syrian Arab Republic
  • Damascus, Syrian Arab Republic, DM20AM18
    • Department of Periodontics, University of Damascus Dental School
  • Damascus, Syrian Arab Republic, DM20AM18
    • Ali Abou Sulaiman

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 60 years (Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

A total of 80 subjects will be invited to participate in this study from the patients referred to the Department of Periodontology, Faculty of Dentistry, University of Damascus. Patients should have a diagnosis of periodontitis (either chronic or aggressive). Any candidate will be approached and informed about this clinical study with the relevant information sheet. Informed consent forms will be obtained.Using five clinical parameters and full mouth or panoramic radiographs, the participants will be allocated to four groups according to their condition as well as their smoking habit. The description of these groups has given before.

Description

Inclusion Criteria:

• Patients of Syrian descent
• Systemically healthy
• Have at least 20 teeth

Exclusion criteria:

• Periodontal treatment during the last three months
• History of major systemic diseases
• Consumption of antibiotics or anti-inflammatory drugs in the last three months
• Smoking
• Alcohol consumption
• Pregnant and lactating women.

Study Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Cross-Sectional

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort
Smoking Aggressive Periodontitis group; This group comprises 20 patients who are smokers and aged < 35 years and diagnosed with rapid attachment loss with periodontal pocket depth (PD) > 4 mm around at least three teeth other than the first molars and incisors. Rapid bone destruction (>50%bone loss at diseased sites). Weak relationship between dental plaque and the severity of gingival inflammation.
Smoking Chronic Periodontitis group; This group comprises 20 patients who are smokers and aged > 45 years and have presence of ≥2 non-adjacent sites per quadrant that were not first molars or incisors, with probing depth (PD) ≥5 mm, which bleed on gentle probing. The demonstrated radiographic bone loss ≥30% of the root length, patient with poor oral hygiene, the amount of accumulated plaque commensurate with the amount of clinical attachment level (CAL)
Non-smoking Aggressive Periodontitis grp; This group comprises 20 patients who are age- and sex- matched to the 'Smoking Aggressive Periodontitis group' and are non-smokers suffering from aggressive periodontitis.
Non-smoking Chronic periodontitis grp; This group comprises 20 patients who are age- and sex-matched to the 'Smoking Chronic Periodontitis group' and are non-smokers suffering from chronic periodontitis.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
P53 levels in gingival biopsy samples	This variable is going to be measured by an immunohistochemical analysis	The measurement will be performed at T0 (baseline measurement) once the sample has been recruited

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
presence or absence of fibroblasts' apoptosis in the gingival tissues	this variable is going to be measured by an immunohistochemical analysis	The measurement will be performed at T0 (baseline measurement) once the sample has been recruited

Collaborators and Investigators

• Sponsor: Damascus University
• Investigators: Principal Investigator: Ali Abou Sulaiman, DDS MSc PhD, University of Damascus Dental School, Department of Periodontology; Study Director: Sharif Barakat, DDS MSc Phd, University of Damascus Dental School, Department of Periodontology

Publications and helpful links

General Publications

• Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol. 1999 Dec;4(1):1-6. doi: 10.1902/annals.1999.4.1.1.
• Bostrom L, Linder LE, Bergstrom J. Clinical expression of TNF-alpha in smoking-associated periodontal disease. J Clin Periodontol. 1998 Oct;25(10):767-73. doi: 10.1111/j.1600-051x.1998.tb02368.x.
• Bulut S, Uslu H, Ozdemir BH, Bulut OE. Expression of caspase-3, p53 and Bcl-2 in generalized aggressive periodontitis. Head Face Med. 2006 Jun 20;2:17. doi: 10.1186/1746-160X-2-17.
• Chen Y, Zychlinsky A. Apoptosis induced by bacterial pathogens. Microb Pathog. 1994 Oct;17(4):203-12. doi: 10.1006/mpat.1994.1066.
• Jarnbring F, Somogyi E, Dalton J, Gustafsson A, Klinge B. Quantitative assessment of apoptotic and proliferative gingival keratinocytes in oral and sulcular epithelium in patients with gingivitis and periodontitis. J Clin Periodontol. 2002 Dec;29(12):1065-71. doi: 10.1034/j.1600-051x.2002.291203.x.
• Hanioka T, Tanaka M, Ojima M, Takaya K, Matsumori Y, Shizukuishi S. Oxygen sufficiency in the gingiva of smokers and non-smokers with periodontal disease. J Periodontol. 2000 Dec;71(12):1846-51. doi: 10.1902/jop.2000.71.12.1846.
• Renehan AG, Booth C, Potten CS. What is apoptosis, and why is it important? BMJ. 2001 Jun 23;322(7301):1536-8. doi: 10.1136/bmj.322.7301.1536. No abstract available.
• Shivanaikar S, Faizuddin M and Bhatt K. Influence of smoking on fibroblast apoptosis in chronic periodontitis. RGUHS J Dent Sciences. 2001; 3: 9-14

Study record dates

Study Major Dates

• Study Start: April 1, 2015
• Primary Completion (Actual): December 1, 2015
• Study Completion (Actual): March 1, 2016

Study Registration Dates

• First Submitted: March 14, 2014
• First Submitted That Met QC Criteria: April 7, 2014
• First Posted (Estimate): April 10, 2014

Study Record Updates

• Last Update Posted (Estimate): March 16, 2016
• Last Update Submitted That Met QC Criteria: March 15, 2016
• Last Verified: March 1, 2016

More Information

Terms related to this study

• Keywords: smoking; periodontal disease; apoptosis
• Additional Relevant MeSH Terms: Behavioral Symptoms; Stomatognathic Diseases; Periodontal Diseases; Mouth Diseases; Aggression; Periodontitis; Aggressive Periodontitis; Chronic Periodontitis
• Other Study ID Numbers: UDDS-Perio-01-2014