## 2 Materials and Methods  
### Study Design and Patient Selection  
The present pilot study was conducted in accordance with the ethical principles including the World Medical Association Declaration of Helsinki, the Belmont report, the Council for International Organizations of Medical Sciences (CIOMS) guidelines and the International Conference on Harmonization in Good Clinical Practice (ICH-GCP). The study was approved by the Bioethical Committee of \"Carol Davila\" University of Medicine and Pharmacy Romania, no. 176/2018. The procedure was explained, and written consent of each subject was obtained. Seven consecutive patients (Group 1) aged between 28 and 46 years, with at least twenty teeth, none of them with untreated periapical lesions visible on radiographs, without periodontal surgery in the last six months, and three healthy individuals between 25 and 44 years old, fully dentate, without dental caries or periodontal lesions (Group 0--reference), all of them being nonsmokers, were enrolled. Before enrollment, the periodontal status of all participants was clinically assessed, and only those with absence of visual signs of inflammation and bleeding on probing [20] were included. The mandatory criteria for the inclusion of patients in Group 1 were the following: the presence of at least one AlphaBio SPI (Alpha-Bio Tec, Petah Tikvah, Israel) dental implant inserted, loaded with a correctly executed dental prostheses for more than twelve months before enrolling in the present study; clinically healthy mucosa, no clinical or radiological signs of peri-implant/periodontal inflammation; without the presence of stomatitis or other pathological lesions; nonsmokers; good systemic health with no immunosuppressive, no medications that could affect salivary secretion nor the use of corticosteroids as medication; no antibiotic nor antifungal treatments in the past 3 months. All included implants were inserted and restored by a single experienced implantologist (S.D.) using a strict protocol, according to the manufacturer's instructions. In brief, each patient underwent preoperative cone beam computed tomography (CBCT) scanning prior to implant insertion. After local anesthesia, the full-thickness flap was elevated to obtain direct visual access to the residual bone and the osteotomies were performed under cold saline irrigation, using a freehand technique. After insertion, all implants were covered with cover screws and then the flap was sutured with nonabsorbable interrupted sutures, and patients received written hygiene recommendations, including Chlorhexidine 0.2% and a prescription for prophylactic antibiotics (amoxicillin and clavulanic acid 1 g/every 12 h), in addition to an anti-inflammatory (ibuprofen 600 mg/every 8 h) for 5 days. Implants were exposed after 4 months (Type 4C--Late Placement and Conventional Loading according to [PERSON] et al. 's classification [21]) and healing abutments were inserted. Two weeks after healing abutment insertion, the prosthetic procedure was initiated. Prefabricated titanium abutments and cemented metal-ceramic crowns were used to restore the inserted implants. Saliva collection was performed for Group 0 (n = 3, reference group) and Group 1 (n = 7) on the same morning, without external stimuli 90 min before collection (unsaturated sample), by a single investigator ([PERSON]), following the same protocol. From each patient, 3 mL of saliva was collected from the floor of the mouth using a 5 mL sterile syringe. The saliva was then placed in Falcon-type millimeter tubes (Merck KGaA, Darmstadt, Germany), coded and stored at a temperature of \(-18\,^{\circ}\)C for a maximum of four hours until being analyzed by an investigator ([PERSON]) who was unaware of the identity of the samples. On the same day, the patients in Group 1 underwent radiographic examination. Digital periapical radiographs of the inserted dental implants, using individualized holders to ensure repeatability, were performed.