Smoking Influence on Apoptosis in 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.

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.

Eligibility Criteria:
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.