Research design
This in vivo study had a comparative, randomized, prospective research design, and each group consisted of 10 male beagle dogs that were veterinarian-controlled, healthy, and of similar weight. Animal Research Reporting in Vivo Experiment (ARRIVE) guidelines were used, and surgical procedure was approved by the Local Animal Experiments Ethical Committee of Erciyes University. Adequate measures were taken to minimize the pain or discomfort in the animals. A total of 36 dental implants (tissue level, 3.3 × 10 mm, Straumann AG, Basel, Switzerland) were inserted in the animals according to the non-submerged healing protocol. Figure 1 presents the flowchart of the research design employed in the study.
Sedation, anesthesia, animal care, and sacrifice
All the interventions were performed under general anesthesia. Enteral nutrition was stopped 12 h before the surgical procedure. General anesthesia was achieved with 2 mg/kg xylazine hydrochloride (i.m.) (Rompun, Bayer, Istanbul, Turkey) and 5 mg/kg ketamine hydrochloride (i.m.) (Alfamyne, Egevet, Izmir, Turkey). After the surgery, a 3-day antibiotic therapy with Streptomycin 0.5 g/day (I.E. Ulagay, Istanbul, Turkey) was administered in each dog. Postoperative care included daily observations regarding appetite and the documentation of adverse events such as bleeding, pain, swelling, and discomfort. At the end of the experiment, all the animals were sacrificed with a large dose of pentobarbital (i.v.). The animals in groups 3 and 4 were sacrificed at month 6 and the animals in groups 1 and 2 were sacrificed at month 8 after the extraction surgery.
Surgical procedure
The surgical procedure was commenced by the extraction of the mandibular second, third, fourth pre-molars, and the first molar bilaterally. The pupillary reflex was controlled after the administration of anesthetic drugs. Peri- and intra-oral tissues were disinfected with 10% povidone-iodine solution, and the surgical area was covered with sterile covering. Infiltration anesthesia with 2% articaine (Ultracaine DS, Sanofi Aventis Drugs, Istanbul, Turkey) was applied to the premolar area for hemostasis and for post-operative pain control. A full-thickness vestibular flap was elevated gently, and surgical tooth extraction was performed using surgical burs with straight elevators. Surgical wounds were closed with 3/0 vicryl sutures and streptomycin 0.5 g/day was administered for 3 days postoperatively. After the extraction, a 3-month period was allowed for healing of the alveolar bone and soft tissue (Fig. 2).
After the 3 month healing period, a second surgery was performed for implant insertion. Pre-surgical procedures were the same as those described above. In addition, a horizontal incision was made along the edentulous premolar area. A mucoperiosteal flap was gently elevated to expose the recipient bone and the implant sockets were prepared using a commercially available surgical set (Straumann® instruments, Straumann AG, Waldenburg, Sweden) under sterile saline irrigation. All the implants had a sandblasted and acid-etched (SLA) surface and were of the same size and length (3.3 × 10 mm, tissue level). All implants were inserted to the level of the machined surface left below the bone. Non-submerged healing protocol was performed in all the groups and the flaps were closed with 3/0 vicryl sutures.
In group 1, after the insertion of the implants, 3/0 silk sutures (Doğsan, Trabzon, Turkey) were placed below the free gingival/mucosal margin around the implants and plaque control was terminated for 2 months (Fig. 3). To promote plaque retention and peri-implantitis, the animals were fed a soft diet. The implants were removed with reverse torque after the induction of peri-implantitis (Fig. 4). After the removal, all the implant surfaces were cleaned by air-flow with bi-carbonate granules for 1 min prior to the treatment with citric acid (pH: 1). Subsequently, the implant surfaces were rinsed with sterile saline solution and then all the implants were inserted in the contralateral side of the mandible of the same dog. After a 3-month osseointegration period, the animals were sacrificed with a high dose of pentobarbital (i.v.).
In group 2, the same procedures were applied as in group 1. However, unlike the implants in group 1, the implants in group 2 were sterilized by autoclave treatment at 121 °C for 30 min. Afterwards, the sterilized implants were inserted in the contralateral side of the mandible of the same dog from which the implants were retrieved. After a 3-month osseointegration period, the animals were sacrificed with a high dose of pentobarbital (i.v.).
In group 3, failed implants due to peri-implantitis were obtained from human subjects. The surface of the implants were cleaned and sterilized with autoclave and then the implants were inserted into the mandibles of the dogs. After a 3-month osseointegration period, the animals were sacrificed with a large dose of pentobarbital (i.v.).
In group 4 (control group), no implant insertion was performed and after a 3-month osseointegration period, the animals were sacrificed with a large dose of pentobarbital (i.v.). The preparation times, surgeries, and observation time points of all four groups were summarized on the time arrow (Fig. 5).
Experimental design
Resonance frequency analysis (RFA) measurements
Implant stability was measured using resonance frequency analysis (RFA) with an Osstell® device (Osstell AB, Goteborg, Sweden). All implants were placed with non-submerged healing protocol and the Osstell® sensor was positioned perpendicular to the long axis of the implant in accordance with the guidelines provided by the manufacturer. The results were calculated in the form of objective ISQ values (ranging from 1 to 100). The RFA measurements were performed from four different directions (mesial, distal, lingual, and vestibule), and the mean ISQ was recorded as the final value.
Removal and preparation of the implant-bone specimens
The implants with a neighboring bone were removed en bloc, and the adhesive soft tissues were dissected to investigate the healing status and the bone-implant contact (BIC) percentage. The specimens were fixed in 10% neutral buffered formalin for 48 h and dehydrated in subsequent concentrations of 70–99.9% ethanol. After dehydration, the specimens were embedded in methyl methacrylate (Technovit 7200 VLC, Heraeus Kulzer GmbH & Co. KG, Wehrheim, Germany) without decalcification. 200-μm-thick slides were cut from the blocks using a band saw (Exakt 300 CL, Exakt Apparatebau, Norderstad, Germany).
Histologic and histomorphometric analysis
The 50-μm-thick final histological slides were prepared by grinding with 320–4000 grit sandpapers (Hermes Schleifmittel GmbH & Co. KG, Hamburg, Germany). The final sections were mounted and stained with toluidine blue for histologic and histomorphometric analysis. In order to measure the BIC percentage, digital images of the sections were obtained by a digital camera (Olympus DP 70, Olympus, Tokyo, Japan) attached to a microscope (Olympus BX50). The obtained images were transferred to a computer and were histomorphometrically analyzed using ImageJ analysis software (ImageJ, National Institutes of Health, Bethesda, Maryland, USA) (Fig. 6). The BIC percentage was calculated by an experienced researcher blinded to the study protocol.
Statistical analysis
Statistical analyses were performed using SPSS v.20.0 (IBM, Chicago, IL, USA). The Shapiro-Wilks normality test was used to verify the normality of the data. All variables were normally distributed and thus parametric tests were used for intra-group (paired sample t test) and inter-group (one-way ANOVA/Tukey’s test) comparisons. A p value of < 0.05 was considered significant.