Elucidating the Role of STAT3 in Epithelial-microbiome Interaction During Periodontitis and Peri-implantitis

Aim: To characterize STAT3 activation and immune defense function in gingival tissue during gingival health, periodontitis, and peri-implantitis.

Subject Selection:

This study will comply with the Declaration of Helsinki (October 2013) and Chilean legal and regulatory requirements. The study will be conducted at the Dental Clinic of the Faculty of Dentistry of the University of Chile.

Subjects attending the Dental Clinic of the Faculty of Dentistry of the University of Chile and diagnosed with gingival health, periodontitis, and peri-implantitis, according to the classification of periodontal and peri-implant diseases, will be recruited and examined.

All subjects must sign an informed consent form previously validated by an ethics committee and meet the following inclusion and exclusion criteria:

Inclusion criteria

Patients in the Gingival Health Group:

• Meet the case definition of gingival health.
• ≥18 years of age.
• Willingness to donate gingival tissue and collect microbiological samples for the study.

Patients in the Periodontitis Group:

• Meet the case definition of periodontitis.
• ≥18 years of age.
• Willingness to donate gingival tissue and collect microbiological samples for the study.

Patients in the Peri-Implantitis Group:

• Meet the case definition of periodontitis and peri-implantitis.
• ≥ 18 years of age.
• Willingness to donate gingival tissue and collect a microbiological sample for the study.

Exclusion criteria

• Using systemic antibiotics (intravenous, intramuscular, or oral) in the last six months.
• Having used corticosteroids or other immunosuppressants (e.g., cyclosporine), cytokine therapy, methotrexate or any other immunosuppressive chemical agent, or high-dose commercially available probiotics (≥ 108 colony-forming units per day) in tablet, capsule, or powder form in the last three months. The use of fermented foods/liquids, e.g., milk, yogurt, etc., does not apply to the exclusion of the individual.
• Being pregnant or breastfeeding.
• Subjects who cannot give consent.

Intraoral Examination: All subjects will undergo a full-mouth examination using a specially designed form (Appendix 3). General history (age, biological sex, occupation), habits, diseases, and medications will be recorded. A periodontogram will also be included to record periodontal measurements, probing depths, clinical attachment loss, bleeding on probing, etc.

Sample Collection: Representative gingival collar biopsies (2 mm wide) will be collected under local anesthesia and placed in 4% formalin for histological analysis, T-PER (Thermofisher Scientific) for Western blot analysis, and RNA later (Thermofisher Scientific) for RT-qPCR analysis. In healthy subjects, these samples will be collected as biological waste and may be obtained, for example, in the following cases: crown lengthening surgeries, third molar extractions, or teeth where there is no significant risk of removing gingival tissue, molar areas with opercula, or gingiva covering part of the dental crown where removing gingival tissue is not a risk. In subjects with periodontitis and peri-implantitis, these samples will be collected during procedures before subgingival instrumentation and/or periodontal or peri-implant surgery in the most affected area at probing depths.

• STAT3 activation in gingival tissue during gingival health, periodontitis, and peri-implantitis: For samples stored in T-PER, protein extraction will be performed, and total STAT3 and its activation (pSTAT3) will be determined using Western blot using antibodies that recognize STAT3 and pSTAT3 at Tyr705 (Cell Signaling Technology, USA). GAPDH will be used as a loading control, and the bands obtained will be quantified using ImageJ software (NIH, Bethesda, USA).
• Characterization of pSTAT3 in gingival tissue during gingival health, periodontitis, and peri-implantitis: For samples fixed in 4% formalin, the localization of pSTAT3 (epithelium and connective tissue) will be determined using immunofluorescence using antibodies that recognize CD45 for immune cells, Ck5 for epithelial cells, and pSTAT3 for the activated protein. The co-localization of pSTAT3-Ck5 and pSTAT3-CD45 will be determined by confocal microscopy at 40x.
• Analysis of the pSTAT3-mediated immune-defense response in gingival tissue during gingival health, periodontitis, and peri-implantitis: For samples stored in RNA later, total RNA will be extracted, and the following genes associated with the immune-defense response will be amplified using the RT-qPCR technique:

Inclusion criteria

Patients in the gingival health group:

• Meet the case definition of gingival health (Chapple et al. 2018).
• ≥ 18 years of age.
• Willingness to donate gingival tissue and collect microbiological samples for the study.

Patients in the periodontitis group:

• Meet the case definition of periodontitis (Papapanou et al. 2018).
• ≥ 18 years of age.
• Willingness to donate gingival tissue and collect microbiological samples for the study.

Patients in the peri-implantitis group:

• Meet the case definition of periodontitis and peri-implantitis (Berglundh et al., 2018).
• ≥ 18 years of age.
• Willingness to donate gingival tissue and collect a microbiological sample for the study.

Exclusion criteria

• 1. Having used systemic antibiotics (intravenous, intramuscular, or oral) in the last six months.

• Having used corticosteroids or other immunosuppressants (e.g., cyclosporine), cytokine therapy, methotrexate, or any other immunosuppressive chemical agent, or high-dose commercially available probiotics (≥ 108 colony-forming units per day) in tablet, capsule, or powder form within the last three months. The use of fermented foods/liquids, e.g., milk, yogurt, etc., does not apply to the exclusion of the individual.

• Being pregnant or breastfeeding. 4. Subjects who cannot give consent.