The Ethics Committee for Animal Research of the Veterinary School of the University of Teramo (Teramo, Italy) approved the study protocol, which followed the guidelines established by the European Union Council Directive of February 2013 (R.D.53/2013).
Two female sheep, 4–5 years old, were included in the study. Clinical examination determined that all animals were in good general health. Exclusion criteria included general contraindications (pregnancy, systemic disease) to implant surgery and active infection, or severe inflammation in the area intended for implant placement.
The animals were given thiopental (Thiopental, Höchst, Austria) for induction of anesthesia as needed. After oro-tracheal intubation and ventilation, anesthesia was sustained with nitrous oxide oxygen with 0.5% halothane. Physiologic saline solution was administered during surgery for fluid replacement.
The edges of the iliac crests were exposed through a skin incision of 15 cm in length. The skin and facial layers were opened and closed separately.
After dissection of the soft tissues, the bone was exposed and five undersized osteotomic sites were prepared in each (left and right) side of the iliac crest. In the right side of each animal (test group), implant bone sites were prepared using only the pilot drill 1.8 mm in diameter. Ten Expander® 3.8 × 10 mm implants (NoDrill®, Milano, Italy) were inserted in the right side of both animals with a hand control wrench. Maximum insertion torque values were between 45 and 60 N/cm. In the left side of each animal (control group), implant bone sites were prepared using the following burs sequence: pilot drill 1.8 mm in diameter, twist drill 2.8 mm in diameter, and the final drill 3.2 mm in diameter. Ten 3.8 × 10 mm Dynamix® implants (Cortex, Shlomi, Israel) were inserted in the left sides of both animals. Maximum insertion torque values were between 30 and 45 N/cm. The implant drilling procedures were carried out under profuse saline irrigation (1000 rpm). Implants were inserted in cancellous type IV bone.
After implant insertion, cover screws were secured and the surgical wounds were closed by a resorbable periosteal-muscular inner suture, followed by an external cutaneous 2-0 silk suture.
Each animal underwent systemic antibiotic therapy for 5 days with 8 ml long-acting Clamoxil (Pfizer Limited, Sandwich, USA). After surgery, animals received appropriate veterinary care and were allowed free access to water and standard laboratory nutritional support throughout the trial period.
The sheep were sacrificed 2 months after implantation by an overdose of sodium thiopental (Thiopental, Höchst, Austria).
Micromotion analysis
Bone blocks containing the implants were retrieved from each side of the iliac crest. Each implant was fitted with a one-piece 11-mm straight abutment.
The bone blocks were fixed on a customized loading device to measure implant secondary stability according to a previously described technique [19]. A digital force gauge (Akku Force Cadet, Ametek, Largo, USA) and, on the opposite side, a digital micrometer (Mitutoyo Digimatic Micrometer, Kawasaki, Japan) were used to measure implant micromotion during load application. Horizontal forces of 25 N/cm were applied onto the abutment of the implant perpendicularly to the major axis, and the lateral displacement was measured by the digital micrometer 10 mm above the crest. This parameter represents the “value of the actual micromotion” (VAM) as previously published [20] and validated [21].
ISQ analysis
Resonance frequency analysis was assessed at the time of animal sacrifice (after 2 months of healing) with the latest Ostell device (Osstell AB, Göteborg, Sweden). The implant stability quotient (ISQ), which ranged between 0 and 100, was recorded.
Removal torque value (RTV) testing
Removal torque value (RTV) was measured at the time of animal sacrifice (2 months after implantation) after VAM measuring procedures. The RTV was evaluated and recorded for each implant using a digital hand-operated torque wrench (Tonichi STC400CN) by unscrewing the implants until interfacial failure occurred. The digital torque wrench automatically registered the peak removal torque value on the digital display. After the initial interface detachment, the implants were repositioned back in their initial position as accurately as possible and processed for histologic analysis. Although the interfacial detachment created an artifact at the interface, its analysis would still be reliable according to Sennerby et al. [22], who used a similar procedure to study the morphology of the bone-metal rupture.
Histomorphometric analysis
Specimens were immediately fixed in 10% neutral buffered formalin and processed for histologic analysis. After dehydration, samples were infiltrated with a methyl-methacrylate resin from a starting solution 50% ethanol/resin and subsequently 100% resin, with each step lasting 24 h. After polymerization, the blocks were sectioned and then ground down to about 40 μm. Toluidine blue staining was used to analyze the different ages and remodeling pattern of the bone. The histomorphometric analysis was performed by digitizing the images from the microscope via a JVC TK-C1380 Color Video Camera (JVC Victor Company, Yokohama, Japan) and a frame grabber. The images were acquired with a × 10 objective over the entire implant surface. Subsequently, the digitized images were analyzed by the image analysis software IAS 2000 (Delta Sistemi, Roma, Italy).
For each section, the two most central sections were analyzed and morphometrically measured. The histomorphometric parameters calculated were the % bone-to-implant contact (%BIC) and the bone volume (%BV).
Host bone density analysis
In both iliac crests of each animal, a bone sample was harvested close to the implant sites. The bone samples collected were analyzed in order to establish the bone volume percentage (basal %BV).
Statistical analysis
Biomechanical (VAM, RT, and ISQ) and histomorphometrical data (BIC% and BV%) of test and control groups were statistically compared by the T test using a dedicated software (GraphPad Prism 6 - www.graphpad.com).