Gracey curette

PD was defined as PPD ≥ 5 mm in ≥10 teeth, and BOP in ≥30% of sites. Periodontally healthy controls had PPD ≤ 4 mm and BOP in ≤30% sites. Periodontal assessment was carried out by two calibrated examiners (M.S.R. and A.M.F). RNA isolated from PanDCs and PBMCs then qrt-PCR was performed to detect molecular microbial signatures in each population. In addition, quantifications of Sg, Fn and Pg were carried out in the oral biofilm of healthy and PD patients. For each subject, subgingival biofilm samples were collected from the four deepest sites (one site per quadrant) that did not exhibit suppuration as observed by prior clinical examination. Before sampling, the teeth were isolated with cotton rolls and the supragingival plaque was removed. A mini-five Gracey curette (Hu-Friedy®, Chicago, IL, USA), was gently inserted into the pocket in the most apical portion and the subgingival biofilm was collected with a single stroke. The samples were placed in polypropylene tubes with 1.5 ml of a solution containing 100 ml buffer solution (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6) and stored at −80 °C until use.

Crevicular fluid and subgingival biofilm were obtained from the most severe site in each oral quadrant at the base of the periodontal pocket. The crevicular fluid was obtained by introducing filter paper for 60 s. Subgingival biofilm was obtained with a sterile Gracey curette (Hu-Friedy). For Candida spp. identification, crevicular fluid, and subgingival biofilm were collected in thioglycolate medium (BD) tubes. Non-stimulated saliva samples were collected in a clean 2 mL amber tubes and stored at −70°C. All samples were collected before noon after periodontal evaluation.

The subgingival microbial dental plaque samples were obtained at the first and third months. The clinician gently removed the subgingival microbial dental plaque from the deepest periodontal pockets of the patients using a sterile Gracey curette (Hu-Friedy, Chicago, USA). Subsequently, the levels of T. forsythia, P. gingivalis, and T. denticola were determined through DNA sequencing. DNA was isolated from the samples using the kit (GeneMATRIX, EurXTM, Gdansk, Poland) in line with the manufacturer’s instructions. Prior to DNA sequencing readout, the 16S V3 and V4 regions in each sample were amplified using PCR during amplicon library preparation. For the DNA purification, primer dimers and free primers were removed using magnetic beads (AMPure XP, Beckman Coulter, California, USA). This method was used to determine the genes by searching the sequence in terms of similarities and protein-coding potential and mutations in the genes, and also by comparing the sequences of the identified bacteria with those registered in Genbank (NCBI, GenBank, Bethesda, USA).

Peri-apical radiographs were obtained using the parallel technique, and the peri-implant bone levels were evaluated. Patients received a full mouth examination one week after supragingival scaling. Periodontal parameters, including pocket probing depth (PPD), bleeding index (BI) (Mazza et al., 1981 (link)), and plaque index (PLI) (Silness and Loe, 1964 (link)), were recorded at all six sites (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual) for each tooth. All clinical examinations were performed by the same clinician, and the treatment procedure was performed by another clinician.
Before treatment, oral hygiene instructions were provided to improve the plaque control. Under appropriate anesthesia, debridement with titanium curettes (Hu-Friedy, Chicago, IL, USA) and irrigation with 0.12% chlorhexidine were performed for the diseased peri-implant sites. For periodontitis sites, scaling and root planing using an ultrasonic device and a metal curette (Gracey curette, Hu-Friedy, Chicago, IL, USA) were performed before peri-implant mechanical debridement. A re-examination was performed eight weeks after the treatment.

At the initial visit, periodontal measurements of all participants were recorded, and their baseline GCF samples were collected at least two days later. Patients with severe periodontitis underwent oral hygiene instructions, full-mouth supragingival debridement, and quadrant-based SRP using both ultrasonic scalers (MiniPiezon, EMS Dental, Nyon, Switzerland) and hand instruments (Gracey Curette; Hu-Friedy, Chicago, IL, USA) under local anesthesia within one month. Follow-up examinations and GCF sample collections were conducted at 1, 3, and 6 months after treatment, with clinical measurements taken at each subsequent follow-up appointment. In addition, supportive periodontal therapy including oral hygiene instructions and supragingival debridement was provided at each subsequent visit.

The periosteum from four different skeletal sites (F-frontal bone, H-hyoid bone, P-parietal bone, T-tibia), each representing a unique signature of embryonic Hox code (positive/negative) and embryonic origin (neural crest (NC)/mesoderm) were analyzed. After the soft tissues were carefully removed, the periosteum was collected with a Gracey curette (Hu Friedy, Chicago, IL) and stored in RNAlater (Qiagen, Hilden, Germany). The tissues were homogenized in Qiazol (Qiagen) with a mortar and pestle complemented with Qiashredder columns (Qiagen) then purified with the miRNeasy kit (Qiagen) following the manufacturer’s instructions. The RNA purity was verified with Nanodrop (Thermo Fisher Scientific, Waltham, MA) and the integrity was evaluated with Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only samples with 260/230 ratios superior to 2.0 and RIN superior to 8 were further analyzed.