Effects of insertion torque values on the marginal bone loss of dental implants installed in sheep mandibles

Implants characteristics

Eighty dental implants with Morse taper connection manufactured and marketed by Implacil De Bortoli (São Paulo, Brazil) with two distinct macrogeometries were used (n = 40 per model): Duo Cone (DC) implants, which feature trapezoidal threads and a conical design; and, Maestro (MAE) implants, which feature trapezoidal threads, conical design, and healing chambers distributed between the threads in the implant body. Both implant models used have the same type of surface treatment, that is, they are blasted with microparticles of titanium oxide (Ø100–150 µm) followed by conditioning by maleic acid. This surface presents a roughness pattern with Ra of 0.56 ± 0.10 µm³⁰. Implants with the same dimensions, 4 mm in diameter and 10 mm in length, were used. In addition, each implant after insertion into the bony site received a healing abutment 3.5 mm in diameter by 3.5 mm in length (n = 80). Figure 1 shows the image of the implants and the healing abutment used in this study.

Animal selection and care

The present study was reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Ten Santa Inês sheep of both genders from the department of surgery of the Faculty of Veterinary Medicine of the University of Rio Verde (Rio Verde, Brazil) weighing between 31 and 40 kg were used. The study was approved by the Animal Research Ethics Committee (UniRV nº07/2020) and followed the ethical principles of the National Council for the Control of Animal Experimentation (CONCEA), as well as the concern for the welfare of animals in accordance with the Law nº 11.794 of October 8, 2008 (Procedures for the Scientific Use of Animals).

Prior to surgical procedures, all animals were housed for a minimum period of 15 days to adapt to environmental conditions and human coexistence, as well as to detect possible illnesses. The animals were housed in two groups according to the time of sacrifice, that is, five animals per pen, provided with drinkers and feeders. The animals were fed industrialized chow and water ad libitum. These conditions were maintained throughout the study period.

Anesthetic, surgical procedure and proposed groups

Due to the possibility of regurgitation and aspiration of rumen content, which can lead to asphyxia or pneumonia, the animals were fasted on solid and liquid for 24 h prior to the procedure³¹.

The anesthetic medication was a mix of atropine sulfate at a dose of 0.02 mg/kg and morphine sulfate at a dose of 0.4 mg/kg, given via intramuscular injection. After 15 min, 0.1 mg/kg of xylazine and 8 mg/kg of ketamine, both in the same syringe, were given intramuscularly. The animals were intubated (orotracheal intubation) with a flexible tube and kept on oxygen. Intravenous fluid therapy was performed with a lactated ringer solution, through cannulation of the brachycephalic vein, with a 24G catheter, throughout the surgical procedure, in a 10 to 15 ml/kg/hour venous drip. Heart rate and oxygen saturation were monitored with the aid of a pulse oximeter.

For the surgical procedure to install the implants, antisepsis of the oral cavity was performed with 0.12% chlorhexidine and local anesthetic infiltration based on 2% mepivacaine and 1:100,000 epinephrine. After 5 min of anesthetic infiltration, an incision was made over the crest of the ridge in the diastema area, passing through the gingival sulcus of the first two posterior teeth, which were carefully extracted. Thus, two osteotomies were performed in the diastema area and another two in the alveoli of the mesial roots of the extracted teeth, as shown schematically in Fig. 2a. Then, four groups were formed as follows: group DCws, in which the implants were installed in healing bone (without a socket); group DCfs, in which the implants were installed in the post-extraction area (fresh sockets); group MAEws, in which the implants were installed in healing bone (without a socket); group MAEfs, in which the implants were installed in the post-extraction area (fresh sockets). The implants were installed on both sides of the mandible in each animal, being distributed as follows: DC, MAE, DC, MAE in the left hemimandible, and MAE, DC, MAE, DC in the right hemimandible. All implants were positioned 2 mm infra-bone, which was controlled by the marking on the drive used for the implant installation, as shown in Fig. 2b.

The osteotomies were performed with the same drill sequence (Fig. 3), according to the manufacturer’s recommendations, varying the drilling speed according to the implant model: for DC implants, pilot drill 2.0 mm, 3.5 mm conical drill and 4.0 mm conical drill, all at 1200 rpm; and, for MAE implants, a pilot drill 2.0 mm at 1200 rpm, a 3.5 mm conical drill at 600 rpm and a 4.0 mm conical drill at 600 rpm. A digital surgical motor (iChiropro Bien-Air) and contra-angle 20:1 Bien-Air (Bien-Air Surgery SA, Le Noirmont, Switzerland) was used. In sites of immediate implantation in fresh sockets, these were drilled with the same sequence of drills following the path of alveoli until reaching the planned depth. All osteotomies were performed under intense irrigation with a 0.9% sodium chloride solution. All implants were installed using the motor described above (iChiropro Bien-Air) with a rotation of 30 rpm, which registered and informed, through the software coDiagnostiXTM, the maximum torque of the implant during the insertion.

After installing the 4 implants and placing the healing abutments, the mucosa was sutured with simple stitches and interrupted with 5.0 Nylon suture (Johnson & Johnson Health Care Systems Inc, Piscataway, USA). The same surgeon with extensive experience in dental implant procedures and previously calibrated performed the installation of all implants.

Postoperative care and euthanasia

In the immediate postoperative period, the animals were monitored until full re-establishment of consciousness and then sent to their individual boxes. To control pain and inflammation, an anti-inflammatory (meloxicam 0.5 mg/kg) was administered subcutaneously, once a day for three days, and an analgesic (tramadol hydrochloride 5 mg/kg) was administered subcutaneously, three times a day for three days. In addition, a single dose of 20% oxytetracycline antibiotic (0.1 mg/kg) was administered intramuscularly. The animals were clinically evaluated once a day for physiological parameters (heart rate, respiratory rate, defecation, and urination), behavior and return to feeding.

After 30 and 90 days after surgery (n = 5 animals per time), the animals were euthanized using pentobarbital (90 mg/kg at 3%, via intravenous), according to the CONCEA guidelines for the care and use of animals for scientific and educational purposes³².

Measurement of insertion torque and implant stability quotient (ISQ)

The insertion torque (IT) was measured during implant installation with the aid of the digital surgical motor, as described above, considering the maximum torque value obtained during insertion into the bone bed. The stability measured by magnetic frequency, in this case the implant stability quotient (ISQ), was performed with the aid of Osstell® (Osstell AB, Gothenburg, Sweden), and a SmatPeg sensor (type 49) was installed for each implant. For each implant, two measurements were acquired, one in the buccolingual direction and the other in the mesiodistal direction, with an overall average being made for each implant. These ISQ measurements were taken at three moments: immediately after implant placement (m1), in samples taken from animals sacrificed at 30 days (m2), and in samples taken from animals sacrificed at 90 days (m3).

Sample preparation and histological analysis

All samples were immediately immersed in 10% buffered formalin and kept in this solution for 7 days. Then, they were dehydrated in an ethanol solution sequence (50–100%) and embedded in a historesin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). The cuts were performed using an IsoMet 1000 machine (Buehler, Lake Bluff, USA). The slides were stained using the picrosirius–hematoxylin technique³³. Images were obtained using a Nikon E200 light microscope (Tokyo, Japan). For histological measurements, ImageJ for Windows™ software was used (National Institute of Health, Bethesda, USA). The percentage of contact between bone and implant (BIC%) was measured along the entire length of the implant surface.

Marginal bone level (MBL) measurements

Since all implants were installed 2 mm infra-bone in relation to the implant platform, measurements were made using the histological sections for the buccal and lingual marginal bone positions (MBL1), as shown in Fig. 4a. The periapical radiographs were used to measure the mesial and distal marginal bone level (MBL2), as shown in Fig. 4b. To take the radiographic images, a digital radiography system with a portable IriX-ray DX 3000 device (Dexcowin, Seoul, Korea) was positioned at 10 cm from the sample using a digital film RVG First intraoral system (Trophy, Toulouse, France). All images were taken observing the parallelism of the equipment with the implants. The exposure time to obtain each radiograph was adjusted to 0.35 s. The images were transmitted directly to the computer using the Trophy imaging software (Toulouse, France). All measurements were taken using the ImageJ software, which was calibrated against the implant diameter as a reference.

Sample size calculation and statistical analysis

The software SigmaStat 4.0 (Systat Software Inc, San Jose, USA) was used to calculate the sample size, based on a power of 85% to obtain a p-value of 0.05. Using the data (differences between the means and standard deviations), the calculated minimum sample size for each group at the 2 proposed time resulted in 8 samples. However, 10 samples were used to improve the sampling condition.

The normal distribution was tested using the D’Agostino–Pearson omnibus normality test. As the normality was confirmed, the generalized parametric linear model for repeated measures with a significance level of 5% was applied. The one-way ANOVA statistical test was used to determine the difference between the four groups in the same measured time for each parameter analyzed. The t-test was used to evaluate statistical differences in each parameter analyzed inside of each group among the two proposed times. The Bonferroni multiple comparison test was used to detect differences between the groups for each parameter and each time. Pearson’s correlation test was used to evaluate the correlation between the IT values and BIC%, IT values and MBL1, and IT and MBL2. For all statistical tests, we used the GraphPad Prism software version 5.01 (GraphPad Software, San Diego, USA), considering the result significant when p < 0.05.

Ethical approval

The present study was approved by the Animal Experimentation Committee (UniRV nº07/2020), University of Rio Verde (Rio Verde, Brazil). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Informed consent

For this type of study, formal consent is not required.