The Effect of Smoking on Peri-implantitis

Study Overview
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Brief Summary
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the primary aim of this project is to evaluate the microbiological and inflammatory effect of smoking status and smoking severity on periimplantitis lesions. The secondary aim is to compare the effect of smoking on periimplantitis and periodontal microbiota and inflammation in the same individuals. There will include 96 patients, equally divided into four groups: Smokers with peri-implantitis (n=24), non-smoker individuals with peri-implantitis (n=24), smokers with healthy peri-implant tissues (n=24), non-smoker individuals with healthy peri-implant tissues (n=24). Microbiological and biochemical analyses will be performed on the samples taken.

Detailed Description
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The cross-sectional study will be carried out in collaboration with the Departments of Periodontology in the Faculty of Dentistry at Bern University, Switzerland, Hatay Mustafa Kemal University, Dokuz Eylul University, Gazi University, and Ege University, Turkey. The first step of the study will be carried out in Turkey, and the second step in Switzerland. Since Hatay is one of the three provinces most affected by the 2023 earthquake in Turkey and Hatay Mustafa Kemal University Dental School was severely damaged, it is impossible to recruit samples from the patients in Hatay. Therefore, this project has been planned as a multicenter, and patient samples will be recruited from the Dokuz Eylül, Gazi University, and Ege University, Department of Periodontology, Faculty of Dentistry. The samples will be analyzed in the Laboratory of Oral Microbiology, Department of Periodontology, University of Bern, Switzerland. The study protocol was approved by the Ethics Committee for the Use of Human Subjects in Research of Dokuz Eylul University (Protocol No: 2023/GOA-8246). This study will include 96 patients, equally divided into four groups: Smokers with peri-implantitis Non-smoker individuals with peri-implantitis Smokers with healthy peri-implant tissues Non-smoker individuals with healthy peri-implant tissues Smoking status will be categorized into two groups: smoker and non-smoker (never smoker). The non-smoker group will include former smokers who stopped smoking at least 6 years ago. The cigarettes smoked per day, smoking index (self-reported average cigarettes per day x years of tobacco use), and smoking pocket year will be calculated (self-reported average cigarettes per day x years of tobacco use / the number of cigarettes in a pocket). Smoking status will be detected according to self-report. Smoking status will be later validated by saliva cotinine levels. Salivary cotinine concentration for smoking will be accepted at 5-7 ng/ml. Smoking severity will be determined by higher concentrations. Periodontal Parameters Clinical periodontal measurements will be assessed using the following for periodontal diagnosis. The measurements will performed using a Williams periodontal probe (Hu-Friedy, Chicago, IL, USA) and include probing pocket depth (PPD), clinical attachment level (CAL), plaque index (PI) 26, gingival index (GI) 27, and percentage bleeding on probing (BOP) 28 at six sites per tooth (mesio-buccal, buccal, disto-buccal, mesio-lingual, lingual and disto-lingual) on each tooth. Diagnosis of periodontal diseases and conditions will be made according to the radiographic and clinical diagnostic criteria proposed by the 2017 World Workshop on Classification of Periodontal and Peri-implant Diseases and Conditions.29 Peri-implant health will be assessed by the absence of visual signs of inflammation, bleeding on probing, and further bone loss following initial healing, which should not be ≥2 mm. The criteria for patients with periimplantitis will be PPD≥6 mm in conjunction with profuse bleeding and ≥3 mm bone loss. Patient demographics and clinical periodontal/peri-implant examination The following variables will be collected: Gender Age BMI Implant age Number and location of implants Surgical phase steps Complications related to implant treatment Implant malposition Implant Mobility and Discomfort (differences in Periotest values-PTVs) Time from loading of implants to current examination (time in function) Restorative design Prosthesis retention Radiographic bone level Kind and size of bone defect Presence of keratinized mucosa Frequency of dental brushing Interproximal flossing/brushing Frequency of dental appointment History of periodontitis Reason for tooth loss Use of alcohol Collection of Saliva Saliva samples will be obtained from patients as unstimulated. Samples will be obtained over 10 min when the participants will be seated. The samples will be kept at -80°C until being transferred to the Department of Periodontology, School of Dental Medicine, University of Bern, in dry ice for the analyses. Collection of Plaque Samples Plaque samples will be taken from the periimplantitis and periodontitis subgingival area in the periimplantitis group, and the periodontal healthy and peri-implant health subgingival area in the peri-implant health groups. Subgingival implant and dental plaque samples will be collected from the two deepest periodontal pockets and peri-implant pockets within 200 μl RNAlater in Eppendorf tubes. The sampling sites will be isolated from saliva and slightly air dried. The sample will be collected using a sterile Gracey curette (HuFriedy, Rotterdam, Netherlands). The samples will be taken from the peri-implant health area by the same method in the periimplant health group. Eppendorf microcentrifuge tubes will be stored at -80 °C until being transferred to the Institute of Dentistry, Bern University, in dry ice for the analyses. Collection of Gingival Crevicular Fluid (GCF) and Peri-implant Sulcus Fluid (PISF) Samples The samples will be taken from two deepest peri-implant pockets and two deepest periodontal pockets from peri-implantitis patients. Saliva contamination will be avoided by isolating each area with cotton rolls and dehydrating slightly with air. Plaque will be gently removed using a periodontal curette. Using the orifice method, 39 the samples will be obtained within 30 seconds with standardized paper strips (Periopaper; Oraflow Inc., Plainview, NY). The volumes will be measured on a precalibrated electronic gingival fluid measuring device (Oraflow Inc., Plainview, NY). 40 The readings from the electronic device will be converted to an actual volume (μl) by reference to a standard curve. Samples contaminated with blood or saliva will be excluded. All six Periopaper strips will be pooled in plastic Eppendorf microcentrifuge tubes and stored at -80 °C until being transferred to the Institute of Dentistry, Bern University, in dry ice for the analyses. Laboratory Analyses Saliva and plaque samples will be transferred to the Laboratory of Oral Microbiology, Department of Periodontology, School of Dental Medicine, University of Bern and all laboratory analyses will be performed by Dr. Aysegul Sari at the Laboratory of Oral Microbiology, Department of Periodontology, School of Dental Medicine, University of Bern, Switzerland after the material transfer agreement is signed. Biochemical Analysis Patients' smoking status will be determined by cotinine analysis using an enzyme immunoassay kit in the saliva samples. Microbiological analysis The presence and abundance of Fusobacterium nucleatum, Campylobacter rectus, Prevotella intermedia, Aggregatinacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Saccharibacteria (TM7), Streptococcus aureus, and Actinomyces oris, will be analyzed in plaque samples. The targeted identification of these bacteria will be made by Real Time Q-PCR. 16sRNA sequencing will be used for an unbiased and untargeted microbiological analysis. Total genomic DNA will be extracted, and then amplification of the 16S rRNA will be performed. GCF analysis GCF analysis is planned as a second study. Luminex bead-based multiplex immunoassay will be used to carry out the analysis of GCF and saliva samples. GCF and saliva markers were broadly selected according to previous literature as oxidative stress markers 8-OHdG, PARK7/DJ-1, and NADPH oxidase inflammatory markers IL-1beta, IL-17A/E, osteoprotegerin (OPG); connective tissue degradation markers MMP-8, MMP-3; repair/regeneration markers bone morphogenic protein (BMP)-2, epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF). Statistical Analysis Since there is no similar study in the literature, the sample calculation was made on the effect size. The necessary sample size was 24 for each group to detect a significant difference at an effect size (large effect size) of 0.4 and a power level of 90% with a 95% confidence level between the four groups. Gpower package version 3.1 was used for sample size calculations. The normality of continuous variables will be evaluated by Shapiro-Wilk's test. Nonparametric statistical methods will be performed for values with skewed distribution. Kruskal-Wallis test will be performed to compare more than two non-normally distributed variables, and Dunn multiple comparison test for post hoc pairwise multiple comparison analyses. A oneway ANOVA test will be performed to compare more than two normally distributed groups, and the Tukey test will be performed for post hoc pairwise multiple comparison analyses. General linear models will be performed to determine the effect of smoking severity on periimplant and periodontal disease.

Official Title
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Evaluation of the Role of Smoking in the Peri-implantitis Pathogenesis

Conditions
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Periimplantitis, Microbial Colonization, Cytokines

Intervention / Treatment
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* Other: Recruiting samples


Participation Criteria
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Eligibility Criteria
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The inclusion criteria for study groups: 18 years old Body mass index (BMI) £30 kg/m Presence of min 1 implant and 1 tooth for the implant groups At least 1 year of implant loading for implant groups for the implant groups Exclusion criteria for study groups: Systemic diseases such as diabetes mellitus, hepatic or renal disease, or other medical conditions or transmittable diseases (ASA score III, IV, V) Pregnancy Having undergone antibiotics in the previous 6 months Periodontal treatment in the previous 6 months

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 70 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Smokers with peri-implantitis<br>24 patients with smokers with peri-implantitis were included in this group. | Other: Recruiting samples<br>* The GCF, plaque, and saliva samples will recruited from participants.<br>|
| Non-smoker individuals with peri-implantitis<br>24 patients with non-smokers with peri-implantitis were included in this group. | Other: Recruiting samples<br>* The GCF, plaque, and saliva samples will recruited from participants.<br>|
| Smokers with healthy peri-implant tissues<br>24 patients with smokers with healthy peri-implant tissues were included in this group. | Other: Recruiting samples<br>* The GCF, plaque, and saliva samples will recruited from participants.<br>|
| Non-smoker individuals with healthy peri-implant tissues<br>24 patients with non-smokers with healthy peri-implant tissues were included in this group. | Other: Recruiting samples<br>* The GCF, plaque, and saliva samples will recruited from participants.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The evaluation of the abundance of Fusobacterium nucleatum | The presence and abundance of Fusobacterium nucleatum will be analysed by real-time Q-PCR in plaque samples | 2 months | 
| The evaluation of the abundance of Campylobacter rectus in plaque samples | The presence and abundance of Campylobacter rectus will be analysed by real-time Q-PCR in plaque samples | 2 months | 
| The evaluation of the abundance of Prevotella intermedia in plaque samples | The presence and abundance of Prevotella intermedia will be analysed by real-time Q-PCR in plaque samplse | 2 months | 
| The evaluation of the abundance of Aggregatinacter actinomycetemcomitans in plaque samples | The presence and abundance of Aggregatinacter actinomycetemcomitans will be analysed by real-time Q-PCR in plaque samples | 2 months | 
| The evaluation of the abundance of Porphyromonas gingivalis in plaque samples | The presence and abundance of Porphyromonas gingivalis will be analysed by real-time Q-PCR in plaque samples | 2 months | 
| The evaluation of the abundance of Tannerella forsythia in plaque samples | The presence and abundance of Tannerella forsythia will be analysed by real-time Q-PCR in plaque samples | 2 months | 
| The evaluation of the abundance of Treponema denticola in plaque samples | The presence and abundance of Treponema denticola will be analysed by real-time Q-PCR in plaque samples | 2 months | 
| The evaluation of the abundance of Saccharibacteria (TM7) in plaque samples | The presence and abundance of Saccharibacteria (TM7) will be analyzed by real-time Q-PCR in plaque samplse | 2 months | 
| The evaluation of the abundance of Streptococcus aureus in plaque samples | The presence and abundance of Streptococcus aureus will be analysed by real-time Q-PCR in plaque samples | 2 months | 
| The evaluation of the abundance of Actinomyces oris in plaque samples | The presence and abundance of Actinomyces oris will be analysed by real-time Q-PCR in plaque samples | 2 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The evaluation of 8-OHdG, PARK7/DJ-1, and NADPH oxidase levels in GCF and saliva samples | The concentrations of 8-OHdG will be analysed by multiplex bead immunoassay method in GCF and saliva | 2 months | 
| The evaluation of IL-1beta, IL-17A/E, and osteoprotegerin (OPG) levels in GCF and saliva samples | The concentrations of IL-1beta, IL-17A/E, osteoprotegerin (OPG) levels will be analysed by multiplex bead immunoassay method | 2 months | 
| The evaluation of MMP-8 and MMP-3 levels in GCF and saliva samples | The concentrations of MMP-8, MMP-3 will be analysed by multiplex bead immunoassay method | 2 months | 
| The evaluation of bone morphogenic protein (BMP)-2, epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) levels in GCF and saliva samples | The concentrations of bone morphogenic protein (BMP)-2, epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) will be analysed by multiplex bead immunoassay method | 2 months |