Subjects and implant data
The present study included 4 patients, 3 female and 1 male (mean age of 66 ± 5 years), who attended the Implant Dentistry Department at Federal University of Santa Catarina, complaining of pain, discomfort, suppuration, and/or bleeding at implant sites. All patients revealed a history of periodontal disease, were non-smokers (100%), and reported no additional systemic diseases. The patients exhibited a total of 5 machined titanium implants with a mean loading time of 12 ± 6 years, supporting single screw-retained prostheses and were diagnosed with peri-implantitis.
Peri-implantitis was defined as the combination of bleeding on gentle probing (BOP) with or without suppuration, probing depths (PD) ≥ 6 mm, and radiographic marginal bone loss (MBL) (i.e., interproximal bone levels ≥ 3 mm apical of the most coronal portion of the intraosseous part of the implant) [2]. All implant sites were associated with an advanced bone loss (> 6 mm/> 50% of the implant length) [14] and were clinically stable (i.e., no manually detected mobility) but were scheduled for explantation due to the advanced disease progression. The study protocol no. 3437751 was approved by the Human Research Committee at Federal University of Santa Catarina-Brazil, 2016–2021, and all patients signed an informed consent according to the university ethics regulations.
Clinical evaluation
The following clinical measurements were recorded using a color-coded plastic periodontal probe (PCV12PT Hu-Friedy Inc., Chicago, IL, USA): (1) BOP, evaluated as present if bleeding was evident within 30 s after probing, or absent, if no bleeding was noticed within 30 s after probing; (2) PD measured from the mucosal margin to the bottom of the pocket; (3) suppuration, evaluated as present if it was evident after probing and/or peri-implant palpation. All measurements were performed at 6 aspects per implant: mesio-vestibular (mb), mid-vestibular (b), disto-vestibular (db), mesio-oral (mo), mid-oral (o), and disto-oral (do) by one calibrated investigator (M.B.).
Radiological evaluation
Cone beam computed tomographic (CBCT) scans were obtained from each patient for the surgical planning of implant removal. Linear measurements of the defect length (DL) at affected implants were made by drawing a vertical line, following the long axis of the implant, from the implant shoulder (IS) to the bottom of the defect (BD) at buccal and oral aspects in the CBCT data sets (Implant viewer, version 1.84, USA). Radiological evaluations were performed by one experienced and calibrated examiner (M.G.).
Surgical procedure and retrieval of specimens
Implant removal in all patients was performed by the same clinician (M.B.) following a standardized surgical procedure. Patients were prescribed 500 mg of oral amoxicillin, 3 times a day during 1 week, starting 1 day before surgery. Patients were locally anesthetized (2% lidocaine, 1:100,000 epinephrine). The prostheses of the implants were removed. Surgical incision was performed, and full-thickness mucoperiosteal lingual and buccal flaps were raised; bone-implant biopsies were obtained using a trephine bur under copious irrigation with sterile saline. The implants were removed with the minimal required trephine diameter and without compromising the soft-tissue component for ethical reasons. Specimens were immersed immediately in 10% neutral buffered formalin. Following the removal of granulation tissue from the defect area, mucoperiosteal buccal and lingual flaps were repositioned and fixed with single sutures.
Histological processing
Specimens were dehydrated in ascending series of alcohol and embedded in light-cured resin (Technovit 7200, VLC+BPO, Heraeus, Kulzer Co., Wehrheim Germany). Undecalcified thin ground sections were prepared along the longitudinal axis (bucco-lingual direction) with a high precision diamond-coated band saw and precision grinding equipment (Exakt, Apparatebau, Norderstedt, Germany) [18]. Histological sections were stained with Levai-Laczko dye and scanned with an Olympus BX61VS microscope using a digital virtual light microscopy system (dotSlide 2.4; Olympus, Tokyo, Japan) at a resolution of 0.321 μm/pixel.
Histomorphometrical analysis
Histomorphometrical analysis and microscopic observations were performed by one calibrated examiner (M.G.). Digitalized histological sections (bucco-lingual direction) were analyzed using a morphometric software (Definiens Developer XD 2.0, Definiens AG, Munich Germany). The following landmarks were identified in the stained sections (buccal aspect) (Fig. 1): implant shoulder (IS), level of the alveolar bone crest (BC), bottom of the defect (BD), and the most apical extension of the residual bone tissue (A). Linear measurements were performed by drawing a vertical line following the long axis of the implant: defect length (DL), as measured from IS to BD (mm), residual bone (RB) as measured from BC to A, residual BIC (i.e., the length proportion of the implant surface that was in direct contact with mineralized tissue) as measured as a percentage of the distance from BD to A. The density of the RB was measured as a percentage by dividing the bone area (B.Ar) by the total area of tissues (T.Ar) surrounding the implant.
RB was divided into 500-μm-wide horizontal zones (from BD to A) following the long axis of the implant. For each of these individual zones, BIC and bone density (B.Ar/T.Ar) were determined. Osteocyte and empty osteocytic lacunae were counted along the region of interest (ROI) (i.e., the area encompassing the intra-thread regions along the buccal RB extension) (Fig. 1) using a software program (Image J, software, 1.52a, Maryland, USA). Osteocyte density and empty lacunae density were determined by calculating the ratio of the number of osteocytes or empty lacunae in the region of interest.
Statistical analysis
The statistical analysis was performed using a commercially available software program (SPSS, 19.0, Chicago, IL, USA). Mean values, standard deviations, and confidence intervals for each variable were calculated at the implant level.