Genomic and Proteomic Analyses of Apoptosis Mechanisms in Diseased Peri-implant Tissue

The study received ethical approval from the Clinical Research Committee of Sakarya University Faculty of Medicine (Approval number: E-16214662-050.01.04-85,162-217) and was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. The study protocol was thoroughly explained to all participants, and written informed consent was obtained prior to their inclusion. Recruitment took place between March 2021 and June 2022, involving patients who attended the clinics of the Department of Periodontology, Sakarya University. These patients were either in the maintenance phase following dental implant therapy, actively undergoing implant treatment, or presenting with complications related to dental implants placed at other centers. A total of 72 participants, encompassing 72 dental implants, were enrolled in the study. These participants were categorized as follows: 23 systemically healthy individuals with peri-implant mucositis, 25 individuals with peri-implantitis, and 24 healthy controls. Participant demographics, along with medical and dental histories, were collected during interviews. A comprehensive full-mouth periodontal examination was conducted, and the following clinical parameters were measured at six sites around each implant using a calibrated manual periodontal probe (Hu-Friedy, Chicago, IL, USA): keratinized mucosa width (KTW), modified bleeding index (mBI), probing depth (PD), modified plaque index (mPI), and modified gingival index (mGI). All assessments were performed by a single calibrated periodontist (DY, Kappa coefficient: 0.92). Radiographic evaluations of marginal alveolar bone levels and bone loss percentages were conducted using ImageJ software (ImageJ, Wisconsin, USA). Peri-implant tissue health was classified in accordance with the 2017 classification of Periodontal and Peri-Implant Diseases.

Clinical Procedures and Tissue Sampling Following the baseline assessments, all participants underwent full-mouth prophylaxis and oral hygiene motivation as recommended by EFP treatment guideline. Patients diagnosed with peri-implant mucositis or peri-implantitis were treated by a single periodontist (DY). Granulation tissue samples were collected during the treatment of peri-implant mucositis (non-surgical) and peri-implantitis (resective, reconstructive, or combined surgical approaches). All granulation and mucosal tissue samples were immediately placed into Eppendorf tubes and stored at -80°C. Subsequently, the samples were transported to the Institute of Dentistry, University of Turku and Helsinki University Hospital, University of Helsinki, under dry ice conditions for biochemical analyses. Maintenance therapy was provided to all patients with peri-implant diseases upon completion of their respective treatment protocols.

Analyses of Bcl-2 Family Proteins The thawed tissue samples were first weighed and subsequently sectioned into small pieces with disposable scalpels (#11 and #15, Feather Safety Razor Co., Ltd, Osaka, Japan). The processed tissue fragments were transferred to 2 mL tubes containing four ultrapure zirconium microspheres (3 mm diameter) and 400 μL of lysis buffer. Homogenization was performed using a high-speed tissue homogenizer (TissueLyzer LT, Qiagen, Hilden, Germany) set at 2000 rpm for four cycles of 60 seconds each, with 20 second intervals on ice to prevent overheating. After homogenization, the samples were centrifuged at 10000 rpm for 4 minutes, and the zirconium beads were removed. Ultrasonication (UP50H, Hielscher, Teltow, Germany) was performed at 0.5 cycle and 100% amplitude in three 5-second cycles, with 20-second intervals on ice between the cycles. The samples were then centrifuged again at 10000 rpm for 4 minutes, and the supernatants were carefully transferred to new tubes. Total protein concentrations in the samples were measured by the Bradford assay. The levels of Bcl-2 family proteins were quantified using a bead-based immunoassay system (Luminex xMAP, Luminex Corporation, Austin, TX, USA) with commercially available kits (Bio-Plex Pro RBM Apoptosis Assays, Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer's instructions.

Immunoblot analyses of P. gingivalis gingipain and T. denticola dentilisin Immunoblot analyses were performed by mixing the samples at room temperature with Laemmli's sample buffer and heating them at 100°C for 5 minutes prior to electrophoresis. Pre-stained low-range molecular weight SDS-PAGE standards (Bio-Rad) were used as molecular weight markers. For P. gingivalis, gingipain and T. denticola dentilisin served as positive controls. All samples and markers were loaded onto 8% SDS-PAGE gels and electrophoresed at 150 V for one hour. The gels were then transferred onto nitrocellulose membranes, soaked in blocking solution, and incubated overnight with the respective polyclonal anti-gingipain and anti-dentilisin antibodies. Following incubation, the membranes were rinsed four times with TRIS-buffered saline containing 1% Triton X-100, then incubated with antirabbit IgG Horse Radish Peroxidase (HRP) in 5 mL TBST as a secondary antibody and rinsed four times. Detection and development of the immunoblot films were performed in a dark room, with analytes visualized as dark bands on the films. The films were subsequently scanned, and band intensities were quantified using the GS-700 Imaging Densitometer Scanner and the Quantity One program (Bio-Rad).