All procedures and protocols in this study were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (A2018-322A) (Fig. 1). Six healthy 1-year-old male beagle dogs were used in this experiment. All surgical procedures were performed under general and local anesthesia. Medetomidine hydrochloride (0.05 mL/kg, Domitor®; Orion Corporation, Espoo, Finland) was administered intramuscularly as premedication. Spontaneous respiration was maintained by intravenous injection of sodium thiopental (0.005 mL/kg, Ravonal®; Mitsubishi Tanabe Seiyaku Co., Osaka, Japan). Lidocaine hydrochloride (2%, 1:80,000 epinephrine, Xylocaine; Fujisawa Pharmaceutical Co., Osaka, Japan) was administered as local anesthesia.
Bilateral mandibular second, third, and fourth premolars in the beagles were extracted to provide sufficient space for dental implant placement. After 12 weeks of spontaneous healing, mucoperiosteal flaps extending from the first premolar to the first molar were elevated along the crest of the alveolar ridge. The alveolar crest was flattened by a periodontal chisel (Jovanovic; Hu-Friedy, Chicago, IL, USA) to obtain sufficient bone width for the experiment. The bone harvested during this process was collected for use as an AG. In the experimental group, standardized bone defects (2 mm buccal-lingual width × 3 mm mesio-distal width × 3 mm depth) were surgically created on the planned implant site. After the creation of these defects, bone-level implant fixtures (Straumann φ3.3 mmNC, SLA®8 mm, Roxolid®) were placed at the center of the defects (Fig. 2a). The intrabony defects were filled with either AG or DBBM (Bio-Oss®; Geistlich Pharma., Switzerland) (Fig. 2b). In the control group, implant fixtures were placed without creating an intrabony defect first. The experimental groups and the control group were designated randomly by random function (Microsoft Excel 2011; Microsoft Corporation, Redmond, WA, USA). After placing the cover screw (Straumann NC Closure cap φ2.8 mm, H 0 mm), an absorbable collagen membrane (Bio-Gide®; Geistlich Pharma., Switzerland) was placed in the experimental groups. The flaps were repositioned and immobilized using sutures (Gore-Tex® CV-6 Suture; W.L. Gore & Associates, Inc., Newark, DE, USA). After the surgical procedures, an antibiotic (Penicillin G; Meiji Seika Pharma Co., Ltd, Tokyo, Japan) and an analgesic agent (Vetorphale; Meiji Seika Pharma Co., Ltd, Tokyo, Japan) were administered intramuscularly. After 2 weeks of healing, the sutures were removed. The surgical site was rinsed with a 2% solution of chlorhexidine (HiBiTane® concentrate; Sumitomo Seiyaku Co., Ltd., Osaka, Japan) three times a week for 12 weeks.
Twelve weeks after implant placement, the position of each implant fixture was confirmed by bone sounding, and the gingiva over the cover screw was removed. The cover screw was replaced by the healing abutment (Straumann NC conical shape, φ4.8 3.5 mm) (Figs. 2c).
After 4 weeks of healing, 3–0 silk ligatures (Blade silk; Hashimoto Co., Ltd., Tokyo, Japan) were placed in the peri-implant sulcus (Fig. 2d). The remaining ligature thread was checked, and dental radiographs were taken once a week to confirm bone resorption around the implant. Four weeks after ligation, the beagles were euthanized by an overdose of intravenous thiopental. The mandibules containing all surgical sites were dissected into blocks and fixed in 10% neutral buffered formaldehyde (Mild form® 10 N; Wako Pure Chemical industries) for morphological and histological evaluation. One experienced surgeon (T.M.) performed all surgical procedures in this experiment.
Radiographic analysis
The height of the alveolar bone around the implant fixture at the time of ligature placement and 4 weeks after ligation was compared on radiographs. The difference in the height was defined as the length of bone loss. In all groups, the length of bone loss was represented the average of the mesial and distal measurements. The ratio of the length of bone loss to the length of the implant fixture was calculated.
Histological analysis
The tissue blocks containing the implant were fixed in 10% formaldehyde solution (Mildform® 10 N; Wako Pure Chemical Industries, Ltd.), followed by dehydration with ethanol solutions of different concentrations, which were replaced with acetone. After further treatment with methylmethacrylate (MMA) solution, the specimens were embedded in MMA resin, polished into 30–40 μm thick non-demineralized sections, and stained with toluidine blue.
Histological examination was done using an optical microscope (ECLIPSE Ni-U; Nikon Corporation, Tokyo, Japan). Histological measurements were performed using a computerized imaging system consisting of a high-definition color camera head (DS-Fi2; Nikon Corporation, Tokyo, Japan). The following parameters were measured by the same experienced and blinded examiner (T.A.). Intra-examiner reproducibility was ensured by the examiner reading 18 sections from all sites and repeating the same procedure 24 h later. Calibration was accepted at 90% level.
Bone-to-implant contact (BIC) was calculated as the percentage of implant-bone contact within the region of interest (ROI). An ROI was defined as the same area of the bone defect (3 mm mesio-distal width × 3 mm depth). The percentage of new bone and bone defect areas in the ROI were calculated. All reported parameters represented the average of the mesial and distal measurements. These items were measured using image analysis software (ImageJ v.1.43u; National Institutes of Health, Bethesda, MD, USA).
The following parameters were evaluated for all specimens:
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BIC (%): bone contact percentage of the implant body length in the ROI
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First BIC (fBIC, mm): distance from IS to first bone-to-implant contact
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Area of bone loss (mm2): the area from the implant shoulder (IS) to the base of the defect
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Area of new bone (mm2): amount of new bone formation calculated from the base of the defect.
Statistical analysis
Means and standard deviations for each parameter were calculated for each group, and statistical analysis for each group was performed using analysis of variance (Microsoft Excel 2011; Microsoft Corporation, Redmond, WA, USA). The level of significance was set at P < 0.05. The data are expressed as mean ± SD.
Difference in the area of new bone between the AG and DBBM groups was assessed with the paired t-test, and a p-value < 0.05 was considered statistically significant.