Effect of Metallic Particles on the Progression of Peri-Implantitis

The concentrations of titanium (Ti), aluminium (Al), vanadium (V), molybdenum (Mo), and chromium (Cr) will be quantified separately in healthy gingival tissue and peri-implant granulation tissue using inductively coupled plasma triple quadrupole mass spectrometry (ICP-TQ-MS). The concentration of each metallic element will be reported in parts per billion (ppb) and compared between the active control and peri-implantitis groups. Unit of Measure: Parts per billion (ppb).

Alpha diversity of the submucosal microbiome will be assessed in biofilm samples collected from the peri-implant site with the greatest probing depth in participants with peri-implantitis. Bacterial DNA will be extracted and the microbiome characterised by 16S ribosomal RNA gene sequencing. Alpha diversity will describe microbial richness, defined as the number of microbial taxa detected within each sample, and evenness, defined as how uniformly their relative abundances are distributed. Results will be reported as alpha-diversity index values.

Beta diversity of the submucosal microbiome will be assessed in biofilm samples collected from participants with peri-implantitis. Bacterial DNA will be extracted and the microbiome characterised by 16S ribosomal RNA gene sequencing. Beta diversity will quantify the degree of similarity or dissimilarity in the identity and relative abundance of microbial taxa between samples. These comparisons will be used to evaluate whether microbial community composition varies according to metallic particle concentration and the clinical characteristics of the sampled peri-implant sites. Results will be reported as beta-diversity distance values.

Titanium dioxide (TiO₂) nanoparticles will be evaluated in healthy gingival tissue from the active control group and peri-implant granulation tissue from the peri-implantitis group. High-resolution transmission electron microscopy (TEM) will be used to visualise the particles, and energy-dispersive X-ray spectroscopy (EDS/EDX) will confirm their elemental composition. Nanoparticles will be classified according to their observed shape and structural features. The percentage of identified TiO₂ nanoparticles in each morphological category will be reported for each sample. Unit of measure: %.

Titanium dioxide (TiO₂) nanoparticles will be evaluated in healthy gingival tissue from the active control group and peri-implant granulation tissue from the peri-implantitis group. High-resolution transmission electron microscopy (TEM) will be used to visualise the particles, and energy-dispersive X-ray spectroscopy (EDS/EDX) will confirm their elemental composition. Particle diameters will be measured using ImageJ image-analysis software, and the mean diameter of the identified TiO₂ nanoparticles in each sample will be reported in nanometres (Unit of Measure: nm).

The intracellular presence and localisation of titanium dioxide (TiO₂) nanoparticles will be evaluated in cells isolated from healthy gingival tissue and peri-implant granulation tissue. Ultrathin tissue sections will be examined using high-resolution transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS/EDX) will be used to confirm the elemental composition of the identified particles. The analysis will describe whether TiO₂ nanoparticles are located within cells and their distribution and interaction with intracellular structures.

In participants with peri-implantitis, peri-implant bone loss will be measured radiographically in millimetres at the time of biological sample collection. The estimated average annual rate of bone loss will be calculated by dividing the radiographic bone loss by the time elapsed, in years, from implant placement to sample collection. Higher values indicate a faster estimated rate of peri-implant bone loss. Results will be reported in millimetres per year (mm/year).

Macrophage polarisation will be assessed in healthy gingival tissue from the active control group and peri-implant granulation tissue from the peri-implantitis group. Paraffin-embedded tissue sections will undergo immunohistochemical analysis using established markers for pro-inflammatory M1 and anti-inflammatory and tissue-repair-associated M2 macrophage phenotypes. The relative distribution of both macrophage populations will be quantified and reported as the M1/M2 ratio. A higher ratio indicates a relative predominance of the pro-inflammatory M1 phenotype.

Titanium dioxide (TiO₂) nanoparticles will be evaluated in healthy gingival tissue from the active control group and peri-implant granulation tissue from the peri-implantitis group. High-resolution transmission electron microscopy (TEM) will be used to visualise the particles, and energy-dispersive X-ray spectroscopy (EDS/EDX) will confirm their elemental composition. The number of identified TiO₂ nanoparticles in each tissue sample will be quantified using ImageJ image-analysis software and reported as the number of nanoparticles per sample.

Plaque will be clinically assessed at six sites around the selected implant in participants with peri-implantitis. At each peri-implant site, plaque will be recorded dichotomously as present or absent. Results will be reported as the presence or absence of plaque at each of the six assessed sites.

Depth of peri-implant sulcus measured at six sites per implant using standardized probing force (20-25 g). Unit of Measure: millimeters (mm)

Presence of bleeding and/or suppuration elicited within 30 seconds after gentle probing at six sites per implant using a standardized periodontal probe. Unit of Measure: Categorical (Present/Absent)

Radiographic marginal bone level measured in millimeters as the distance from the implant reference point designed to be in intimate contact with the bone to the most coronal bone-to-implant contact on standardized periapical radiographs. Unit of Measure: Millimeters (mm)

Distance from the mucosal margin to the mucogingival junction at the buccal aspect of the implant, measured using a periodontal probe. Unit of Measure: Millimeters (mm)

Accessibility for oral hygiene will be clinically assessed at the selected implant in participants with peri-implantitis. The peri-implant area will be classified as accessible when an interdental brush can be used to access the area for plaque removal and as non-accessible when the prosthetic design or surrounding structures prevent such access. Unit of Measure: Categorical (Yes/No)

Accessibility for oral hygiene will be clinically assessed at the selected implant in participants with peri-implantitis. The peri-implant area will be classified as accessible when an interdental brush can be used to access the area for plaque removal and as non-accessible when the prosthetic design or surrounding structures prevent such access. Unit of Measure: Categorical (Yes, No)

A previous diagnosis of periodontitis will be determined by reviewing the participant's clinical history and dental records, based on the 2018 EFP/AAP classification criteria (interdental clinical attachment loss ≥1 mm at ≥2 non-adjacent teeth, or buccal/oral clinical attachment loss ≥3 mm with probing depth >3 mm at ≥2 teeth, and/or radiographic evidence of alveolar bone loss). In patients treated for periodontitis, the endpoints of periodontal treatment will be taken into account, allowing probing depths up to 5 mm in the absence of bleeding on probing (BoP) or up to 4 mm in its presence. Unit of Measure: Categorical (Present/Absent).

Type of implant surface according to the manufacturer's specifications. Unit of Measure: Categorical (Treated, Untreated)

Adherence to supportive periodontal maintenance therapy (≥2 professional maintenance visits per year) following periodontal treatment will be determined from the participant's clinical history and self-report. Unit of Measure: Categorical (Present/Absent)

The surface treatment of the dental implant placed will be recorded from manufacturer specifications and surgical records. Unit of Measure: Categorical (anodised; acid-etched; acid-etched and sandblasted).

The type of prosthetic restoration supported by the implant will be recorded from clinical and prosthetic records. Unit of Measure: Categorical (single crown; partial prosthesis; full-arch prosthesis; overdenture; screw-retained; cement-retained).

The material of the prosthetic framework will be recorded from clinical and laboratory records. Unit of Measure: Categorical (titanium; zirconia; cobalt-chromium alloy).

Participant age at baseline (T0), calculated as the interval between the date of birth and the enrollment date. Unit of Measure: Years, months, and days.

Self-reported sex of the participan. Unit of Measure: Categorical (Male, Female, Other, Prefer not to disclose)