This study was approved by the University Animal Ethics Committee-CEUA/UNICAMP-(Campinas, SP) (no.2730-1/12). Six adult male mini pigs (BR-1 mini pigs, São Paulo, Brazil) with ~36 months old and weighed ~55 kg were used in the experiment. The mini pigs were kept in the Experimental Center of the Veterinary Faculty (FESB-Bragança Paulista, SP) and were allowed to adapt to the environment 1 week prior to surgeries. At the beginning of the study, all animals underwent a physical examination by a veterinarian and were found to be healthy. During the study period, the mini pigs were weighed if abnormalities in food intake were observed [21]. The identification of the animals was enabled by marking earrings numbered. The mini pigs were kept separately in cemented stalls. Fresh water was available ad libitum. For 12 h, before surgery, the animals were fasting with water ad libitum. The animals were inspected after the first few postoperative days for signs of wound dehiscence or infection and weekly thereafter to assess general health.

The removal torque and the histological and immunohistochemical analysis of peri-implant bone were conducted in the mandible of the mini pigs.

The animals were premedicated to induce anesthesia with midazolam (0.2 mg/kg) (Medley, Sumaré, SP, Brasil) and chlorpromazina IM (0.1 mg/kg) (Cristália, Itapira, SP, Brasil). An endotracheal tube was used for intubation, and a mixture of isofluran (Baxter Healthcare Corporation, IL, USA) with oxygen in a ratio 1:1 (5–10 mL/kg/min) was used to maintain anesthesia during the experiment. Local anesthesia was performed with lidocaine 2% with epinephrine 12.5 μg/mL (Xylocain/Adrenalin®, Astra, Wedel, Germany). After surgery, veterinarian Pentabiotic Reinforced antibiotic 40.000 UI/kg (Eurofarma, Itapevi, SP) and anti-inflammatory dexamethasone 3 mg/pig (MSD, São Paulo, SP, Brasil) were administered IM application .

The same operator performed all the surgeries and radiographic. The P1, P2, P3, and M1 teeth were extracted bilaterally of each animal. The tooth extractions were difficult in every case because the roots were divergent and usually curved distally. It was necessary to odontosection before extracting them [22]. After 4 months of healing, three external hexagon implants (EH) (Dentifix. Santa Rita do Passa Quatro, SP, Brasil) with the same diameter and length (ø4.1 × 10 mm) were placed with the STA surface on one side of the mandible (Figs. 1 and 2). The side was chosen by lot. Six months later, this implant group received a prosthetic (Fig. 3) to improve the bone tension [23]. Three months later, the 3 implants with prosthetic were removed, totalizing 9 months (9M) and opposite side of the mandible three new implants were placed and immediately removed (IR). Each miniature pig received 6 implants, 3 on each side of the mandible. Thus, a total number of 36 implants were placed. Pigs were anesthetized as described above; all dental implants were carefully removed by a counterclockwise force (removal torque) with a torque driver (Retriever Maximus - Belo Horizonte, MG, Brasil); and the level of torque required to remove the implant from the bone was recorded by mark-10 universal torque series sensor STW, and removal torque were read by a force/torque indicator model BGI (JLW Instruments, Chicago, IL, USA). Afterwards, anesthetized pigs were euthanized with pentobarbital; their mandibles were cut and the respective peri-implant bone was removed in small blocks (10×10×6 mm).