The primary objective of this study is to evaluate interleukin-1(IL-1) gene group polymorphisms in patients with peri-implantitis and to compare them with patients with peri-implant health (control group), taking into account smoking status, gender, age and history of periodontitis.The aim of this investigation is also to look at the levels of the inflammatory response markers IL-1 beta, prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF alpha) in the peri-implant crevicular fluid of patients with healthy oral implants in comparison with individuals with peri-implantitis, under consideration of the patients´ individual IL-1 genotype.

The main hypothesis is that individuals carrying the polymorphism in the IL-1 gene cluster are susceptible to develop peri-implantitis through altered IL-1 and IL-1 receptor antagonist (IL-1Ra) production. A second hypothesis is that both in healthy individuals and especially pronounced in patients having peri-implantitis, an IL-1-positive genotype will result in higher levels of inflammatory cytokines (IL-1 beta, PGE2 and TNF alpha) than an IL-1- negative genotype.

Patients included in the study will be recruited from the Dental Clinic Egas Moniz implant maintenance program and will only be used for this study. All possible candidates will receive a questionnaire and if the patient's medical history is in accordance with study inclusion criteria, and if they agree to participate, informed consent will be signed. All clinical data will be collected by the same examiner.

Genes IL-1A and IL-1B control the production of the proinflammatory proteins, IL-1 alpha and IL-1 beta, respectively. IL-1RN gene controls the synthesis of the IL-1Ra, which impedes the function of IL-1alpha and IL-1beta by competing for receptor binding. Polymorphisms of the following genes will be analyzed: IL-1A-889, IL-1B + 3954, IL-1B-511 and IL-1 RN from patients with peri-implantitis and peri-implant health. For the investigation of genetic polymorphisms, it will be collected a sample of cells from the jugal mucosa with the aid of a swab.

For the biochemical analysis of the inflammatory mediators IL-1 beta, TNF alpha and PGE2 a peri-implant crevicular fluid collection will be performed by inserting paper strips into peri-implant sulcus or pocket, in situations of peri-implantitis from the most affected location, while in situations of peri-implant health from the mesio-buccal location.

The study will be conducted according to the principles of the Helsinki Declaration (reviewed in 2013). Appropriate measures will be taken for the protection of patient data. The study was approved by Instituto Universitário Egas Moniz (IUEM) Scientific Council on 3 July 2019 and the approval of the Ethics Committee of Egas Moniz was received on 24 July 2019. Authorization was also obtained by the Clinical Direction of Egas Moniz Dental Clinic to carry out research work at the Clinic of the University.

The study will consist of two groups, the first consisting of patients with peri-implantitis (bleeding and / or suppuration on probing, probing depth (PD) equal or greater than 6mm and bone loss equal to or greater than 3mm) and the second group (control group) formed by patients with peri-implant health (peri implant mucosa without inflammatory signs and absence of peri-implant bone loss). The definition of peri-implantitis and peri-implant health will be in accordance with the new classification of periodontal and peri-implant diseases (American Academy of Periodontology (AAP) and European Federation of Periodontology (EFP) 2017)

A peri-implant probing examination (6 locations with a CP12 graduated periodontal probe) and evaluation of plaque accumulation and gingival indexes (4 locations) will be performed as usual at implant maintenance appointments. The presence of bleeding on probing or pus will also be evaluated. An implant control radiograph will be taken in cases where there is a probing depth more than 6mm. All clinical data will be collected by the same examiner. Prior to the study, a training and calibration exercise will be performed for PD measurements using patients not included in the study.

The periodontal condition of the patients will be also evaluated. To consider periodontal health, patients should have the following clinical characteristics: up to 10% of locations with bleeding on probing, absence of erythema and edema, absence of patient symptoms, absence of clinical attachment loss and bone loss and PD < 3mm (AAP and EFP 2017). A patient is a periodontitis case if: interdental clinical attachment loss is detectable at least 2 non-adjacent teeth, or buccal or oral clinical attachment loss >3mm with pocketing >3mm is detectable at least 2 teeth. The classification of periodontitis will be done on a multi-dimensional staging and grading system.

In the case of a peri-implant health situation, plaque control measures will be taken, while in cases of peri-implantitis the patient will be referred for a specific treatment appointment. In situations of patients with more than one implant affected by peri-implantitis, the one with the greatest bone loss will be chosen for the study.

The genetic analysis will be done at the IUEM Molecular Biology Laboratory with the DNA extraction technique followed by polymerase chain reaction followed by restriction fragment length polymorphism.

For the biochemical analysis the paper strips will be inserted into the peri-implant sulcus/ pocket until resistance is felt, waiting 30 seconds before removal. Visually contaminated samples (blood) will be discarded. Subsequently paper strips will be placed on Periotron® 8000 (Periotron Ide-interstate, NY, USA) for quantification of the collected peri-implant crevicular fluid volume. The strips of paper will then be placed in eppendorf tubes with filter and frozen at -80ºC until shipment to the Universidad Complutense de Madrid. At this university, the concentration of inflammatory mediators IL-1 beta and TNF alpha will be analyzed using multiplexed fluorescent sphere immunoassays using the Luminex® 100/200™ system and PGE2 by enzyme-linked immunosorbent assay (ELISA).

The prevalence of IL-1 gene group polymorphisms in patients with peri-implantitis and healthy ones will be compared by using appropriate inferential statistics methodologies (bivariate analysis/association tests), taking into account several sociodemographic and clinical variables: smoking status, gender, age and history of periodontitis. Based on a medium effect size (w=0.3), with 80% power and a 5% alpha level, the sample size for this study was calculated as n=88, thus, n= 44 for each group (periimplantitis and peri-implant health). Further, a multivariable regression logistic model will be constructed in order to evaluate the effect of those covariables and quantify the risk, through determination of the correspondent adjusted Odds Ratio (OR), towards the presence of peri-implantitis. Additionally, the levels of the inflammatory response markers IL-1 beta, PGE2 and TNF-alpha, present in the peri-implant crevicular fluid, will be compared between the group of patients with peri-implantitis and the patients with healthy oral implants, taking into account the above mentioned variables, by using appropriate inferential statistics methodologies (parametric/non parametric tests). All inferential analysis will be performed at a 5% significance level.

Inclusion Criteria:

• patients with Caucasian parents and grandparents
• patients with dental implants placed at the Dental Clinic Egas Moniz or out
• patients with peri-implantitis diagnosis
• implants in function for at least 1 year
• patients included in a periodontal / peri-implant support program
• patients who agree to participate in the study and sign informed consent

Exclusion Criteria:

• patients with any systemic condition that affects the immune system
• pregnant women
• patients who have taken chronic antibiotics or anti-inflammatory drugs in the last 6 months
• patients who have undergone peri-implant treatment in the area to be evaluated

Patients included in the study will be recruited from the Dental Clinic Egas Moniz implant maintenance program and will only be used for this study. All possible candidates will receive a questionnaire and if the patient's medical history is in accordance with study inclusion criteria, and if they agree to participate, informed consent will be signed.

The collections of DNA samples were collected only for the purpose of analyzing the polymorphisms of the IL-1 gene group. Participants authorized their collection only for this purpose. For ethical and confidentiality reasons, these samples may not be accessible to other researchers. DNA samples will be eliminated after conducting the study.