Immunological-Clinical Evaluation of the Etiopathogenesis of Peri-Implantitis

The following research experiment project is part of the scientific section of a project funded under the Erasmus+ program (KA220-HED - Cooperation partnerships in higher education) entitled "Integrating Peri-Implantitis Research into Higher Education Curriculum - Developing and Integrating Evidence-Based Teaching Materials and Clinical Tools." According to the current state of knowledge, there are two main theories explaining the development of peri-implantitis. The first theory suggests that the primary etiological factor is the invasion of periopathogens along the gingival crevice surrounding the implant, resembling the pathogenic development seen in natural periodontal diseases such as gingivitis and periodontitis. In this scenario, bacterial invasion is facilitated by the distinct structure of the peri-implant soft tissues, which differ from natural gingival tissue in several ways: a more uniaxial, parallel orientation of collagen fibers relative to the implant's long axis, lower cellular density, and reduced mitotic potential of the surrounding soft tissue.

The second theory proposes that marginal bone loss around the implant is the result of an immuno-osteolytic reaction, with pathogenic bacteria appearing as a secondary phenomenon. According to this theory, the initiation of the pathological process is attributed to an immune response triggered by the presence of titanium ions and other metallic elements, such as vanadium, which is commonly found in titanium alloys. In vitro models have demonstrated that an increase in Ti concentration leads to macrophage activation and increased secretion of pro-inflammatory cytokines, particularly interleukin IL-1B and arachidonic acid derivatives. This macrophage activation is significantly higher in cases where prior bacterial lipopolysaccharide (LPS) exposure has occurred, suggesting an additive effect of bacterial factors in the overall disease process. Furthermore, vanadium appears to disrupt the balance of T/B lymphocytes, suppress the secretion of anti-inflammatory cytokines, and activate the innate immune response via Toll-like receptors and NF-κB signaling pathways.

Despite conceptual differences between these two theories, both agree on the fundamental premise that biological processes occurring on the implant surface and in its immediate vicinity play a critical role in the development of peri-implantitis. These processes are modulated by the physicochemical properties of the implant surface.

The immuno-osteolytic concept of peri-implantitis, also known as the foreign body reaction theory, is largely based on physicochemical changes that occur on the implant surface over time due to its exposure to the oral tissue environment. These changes are associated with gradual degradation, corrosion, and aging of titanium alloys.

In biomedical applications, biphasic titanium alloys are most commonly used, particularly the Ti-6Al-4V alloy, which contains 6% aluminum and 4% vanadium. Aluminum stabilizes the α-phase, improving mechanical strength and reducing weight, while vanadium stabilizes the β-phase, enhancing alloy plasticity.

All known metals and alloys undergo corrosion under specific conditions. Most of them are thermodynamically unstable, tending to transition from their metallic state to ionic form. The degree of thermodynamic stability of a metal is characterized by its standard electrode potential, which describes the free energy change when an ion transitions from the metal to a solution. The more negative the standard electrode potential, the lower the metal's resistance to corrosion. The standard ionization potential of titanium is theoretically established at 1.63V, indicating high electrochemical potential and corrosion susceptibility of pure titanium. However, in practice, titanium exhibits lower corrosion potential due to its tendency to undergo passivation, forming a stable oxide layer that acts as a barrier preventing direct contact between the metal and the external environment.

The stability of the titanium dioxide (TiO₂) passive layer in the oral cavity is compromised by salivary enzymes and bacterial metabolism, particularly acidic byproducts such as lactic, acetic, and propionic acids. These substances create localized pH shifts towards acidity, which are unfavorable for titanium alloys and accelerate corrosion. Another factor increasing titanium corrosion in dental applications is the presence of fluoride compounds, which are commonly used in toothpaste, mouthwashes, gels, and fluoride foams. In aqueous solutions, fluoride exists primarily as the fluoride ion (F-), which has the ability to dissolve the passive oxide layer on titanium, significantly increasing corrosion rates.

A separate concern is the fatigue-related degradation of titanium implants. These fatigue processes are associated with repeated mechanical stress, particularly non-linear loading patterns. The first observable sign of fatigue is the formation of microcracks, detectable via scanning electron microscopy (SEM). In advanced stages, these microcracks lead to wear-corrosion phenomena, further accelerating surface degradation of titanium alloys.

Objectives of the Medical Experiment:

Clinical assessment of oral health, with a particular focus on periodontal indicators and oral hygiene status in participants affected by peri-implantitis.

Radiological evaluation of bone structure affected by peri-implantitis. Hematological assessment of peri-implantitis markers and the immunological profile of participants diagnosed with peri-implantitis.

Measurement of Ti++ ion concentrations and other metallic elements present in titanium alloys in tissues surrounding implants affected by peri-implantitis.

Corrosion analysis and SEM-based fatigue analysis of implant surfaces associated with peri-implantitis.

Study design A total of 100 adult volunteers of both sexes will be enrolled in the project. The study will be conducted within an international consortium formed for the project, comprising the following institutions: Wroclaw Medical University (Poland), Sapienza University of Rome (Italy), University of Barcelona (Spain), University of Bern (Switzerland), and Egas Moniz University (Portugal).

Each international partner institution will recruit 20 volunteers as participants in the experiment. Each of the participating institutions (Sapienza University of Rome, University of Barcelona, University of Bern, and Egas Moniz University) will submit an application to their respective local bioethics committee for approval to conduct the medical experiment.

Eligibility Criteria To be eligible for the study, participants must have undergone previous implant treatment that has resulted in active, advanced peri-implantitis.

During the initial visit, patients will undergo a medical interview to document their systemic diseases and dental treatment history, with particular emphasis on:

The type and brand of implant The prosthetic loading type of the implant The location and time of implantation The onset and treatment history of peri-implantitis

Each participant will then undergo a dental examination, utilizing a UNC15 periodontal probe (DepplerSA, Roller, Switzerland). This assessment will include:

Oral hygiene indicators: Oral Hygiene Index (OHI), Aproximal Plaque Index (API) Periodontal disease indices: Community Index of Periodontal Treatment Needs (CPITN) Occlusal abnormalities and increased facial muscle tension, particularly in the mentalis muscle and oral floor muscles Implant-Specific Assessments

For the implant site, the following parameters will be evaluated:

Pocket Depth (PD) - measured on four surfaces around the implant Bleeding on Probing (BoP) Index Width and height of the attached gingiva (HKT, WKT) Possible implant mobility, assessed using the Mobility Index (MOB) Following the clinical examination, each participant will be referred for a Cone Beam Computed Tomography (CBCT) scan.

CBCT Examination

The CBCT scan will be routinely evaluated, focusing on the implant region, with measurements including:

Bone loss percentage, calculated as the ratio of bone defect depth from the implant platform to the base of the defect, relative to the total implant length, assessed separately for each of the four implant surfaces (mesial, distal, buccal, and lingual/palatal).

Bone density, measured in Hounsfield units (HU)

A peri-implantitis process will be classified as active if:

PD at the implant site exceeds 6mm, with a positive BoP index Advanced peri-implantitis is diagnosed if bone loss exceeds 66% on at least one implant surface Treatment Eligibility Criteria According to established literature, in cases of active, advanced peri-implantitis, where bone loss exceeds 50% of the implant length, implant removal is considered the appropriate therapeutic approach. Only patients meeting this criterion will be enrolled in the project.

Exclusion Criteria:

General and local contraindications for surgical procedures Pregnancy Use of bisphosphonates or other antiresorptive medications in medical history Patients who do not meet the implant removal criteria and are not eligible for the study will be directed toward non-surgical or conservative-surgical treatment options.

Laboratory Analysis Procedures

During a subsequent procedural visit, a 20ml venous blood sample will be collected from the antecubital vein of each participant. The blood samples will be preserved in EDTA tubes and sent to a laboratory for further analysis, including:

Hematological Inflammation Indices: Systemic Immune-Inflammation Index (SII), Aggregate Index of Systemic Inflammation (AISI), Complete blood count with quantitative and percentage analysis, Indicator of Peri-Implantitis and Periodontal Disease Development: morphology by quantitative and percentage separation, Lipid profile: (CHOL , HDL, LDL, TG, APOA1, APOB);

• Inflammatory markers: CRP, ESR, ALB, TP, TF, FIB;
• Cytokine profile: TNF, IL- 1β, IL-6, IL-8, IL-10;
• HOMA2 Parameters: Glucose, Insulin, C-Peptide, HBA1;
• Thyroid profile:- TSH, FT3, FT4, T3, T4, rT3;
• Vitamin D level - 25(OH)D, 1.25(OH)2 D3 Laboratory and Surgical Procedures Each partner institution will conduct laboratory tests and clinical procedures at their respective clinical locations, in collaboration with a certified analytical laboratory.

Next, each participant will undergo implant explantation surgery, during which tissue samples will be collected for further analysis, followed by immediate bone defect regeneration at the implantation site. The procedure will be preceded by removal of the prosthetic restoration.

Following oral disinfection with a 0.2% alcohol-based chlorhexidine solution, local infiltration anesthesia will be administered using 2% articaine with epinephrine 1:200,000. A flap incision will be performed to access the peri-implant region.

A trephine drill with the smallest possible diameter will then be selected to encompass the implant collar. Using the following drilling parameters-800 rpm, 20 Ncm torque, and 0.9% NaCl irrigation-the drill will be lowered to the apical depth of the implant. The implant will be extracted along with any bone tissue tightly adhered to its surface.

The implant and bone tissue will be preserved in a 5% formalin solution. The surgical site will be sutured using non-resorbable 5-0 monofilament sutures.

Both specimens will be sent to research centers in Poland. The implant and tissue samples will be transferred to the Department of Inorganic Chemistry, Analytical Chemistry, and Electrochemistry at the Silesian University of Technology in Gliwice, Poland.

Laboratory Analysis

In the laboratory, biological material will be separated from the samples. The following analyses will be performed:

Titanium concentration measurement and detection of other metals present in the titanium alloy.

Implant surface will be analysed using: Light microscopy, Scanning electron microscopy (SEM), corrosion testing. The microscopic examination will assess the implant surface, identifying areas with fatigue-related material degradation, such as microcracks.

Post-Explantation Follow-Up

One week after the explantation procedure, a follow-up visit will take place. During this visit:

Surgical sutures will be removed. The healing process of the surgical wound will be assessed. If no complications are detected, the next follow-up visit will be scheduled four weeks later. During the four-week follow-up visit, the following evaluations will be performed:

Clinical assessment of the implantation site, including HKT and WKT parameters A second CBCT scan: to assess bone structure in the post-implantation region, with measurements including: Percentage of bone loss, calculated as the ratio of the smallest vertical dimension of the alveolar ridge in the post-implantation area to the height of the alveolar ridge in the immediate vicinity

Blood Sample Collection:

20ml of venous blood will be collected from the antecubital vein of each participant.

The blood sample will be preserved in EDTA tubes and sent to an analytical laboratory for further testing, including same parameters as mentioned previously.