2.1 Ethical statements
The study was designed according to Directive 2010/63/EU on the protection of animals used for scientific purposes, in accordance with the protocol approved by the Ethical Committee of the Rof Codina Foundation and the regional government (Xunta de Galicia, Xefatura Territorial da Consellería do Medio Rural. Ronda da Muralla, 70. Lugo, Spain). The protocol was approved on April 8, 2020, number 01/20/LU-001. The study was conducted in accordance with the ARRIVE guideline.
2.2 Study design
The present study had a prospective, randomized, split-mouth design. Each animal received four implants equipped with blades in the coronal aspect to cut the cortical bone. The blades had different diameters with respect to the implant neck to modulate lateral pressure on the cortical bone.
2.3 Experimental animals and sample size
The data from an earlier study in dogs on implant osseointegration were used for sample calculation. In that study, different insertion torques were applied, which resulted in different compression forces on the recipient bone [28]. With a calculated effect size of 2.5668, applying an α=0.05, a power of 0.9, a two-tail evaluation resulted in a sample size of 5 pairs of animals to reject the null hypothesis that the difference is zero (G*Power 3.1.9.4) [41,42]. The sample was increased to 6 for possible complications. Hence, 6 female beagle dogs, mean age 38 months and mean weight 15.5 kg were included in the experiment.
2.4 Randomization and allocation concealment
Randomization was performed electronically by an author (D.B.) who was not involved in the selection of animals or implant placement. The allocation treatment was maintained in sealed opaque envelopes opened before implant installation and after recipient site preparation. The histological slides did not report any indication of the type of implant, and their identification on the histology was practically impossible in most of the histological slides because of the geometry of the blades, which were not parallel to the cut of the implant.
2.5 Implant characteristics
The implants used (CortyBlade® Leader Medica s.r.l., via Giacinto Andrea Longhin 11, 35129 Padova, PD, Italy) were 3.75 mm in diameter and 8.5 mm in length with a tapered one-piece conformation with double acid-etched surface. The transmucosal collar was 1.8 mm high, with a convergent conformation. The CortyBlade implants were equipped with coronal blades. Implants with four different blade diameters were used in this study. The difference in diameter of the blades in relation to the neck of the implant was as follows: -175 µm (control site), 0 µm, +50 µm, or +200 µm (Figure 1).
2.6 Anesthetic procedures
Animals were premedicated with a combination of medetomidine 10mcg/Kg i.m. (Sededorm® 1 mg/ml, Vetpharma Animal Health, Barcelona, Spain) and morphine 0.3 mg/Kg i.m. (Morfina Braun 2%; B. Braun Medical, Barcelona, Spain) induced with 2 mg/kg of propofol IV (Propofol Lipuro ® 10 mg/ml, B Braun, Melsungen, Germany) and maintained with isoflurane 1-1.5% in O2 (Vetflurane® 1000 mg/g, Virbac SA, Carros, France). Animals were monitored during anesthesia (cardiac and respiratory frequency, blood pressure, and CO2 rate), and the values were recorded every 5 min at the beginning of the infusion and later every 15 min).
2.7 Surgical procedure
The first and second mandibular molars were extracted bilaterally. After 3 months, full-thickness flaps were elevated bilaterally in the edentulous region and two recipient sites were prepared on each side of the mandible. Implants were installed with the base of the convergent neck flush to the buccal bone crest. Healing abutments were screwed onto the implants and resorbable sutures were provided to allow non-submerged healing (Figure 2).
2.8 Animal maintenance
Buprenorphine (0.01 mg/kg/i. m./8 h, Bupaq, Richter Pharma AG, Wells, Austria) was injected to control post-surgical pain. Meloxicam (Loxicom, Norbrook Laboratories, Monaghan, Ireland) was first administered before surgery (0.2 mg/kg/i. v.) and for two days subsequently (0.1 mg/Kg/p.o. once a day). Cefazolin (20 mg/kg/i.v., Cefazolina Normon, Madrid, Spain) and sodium cefovecin (8 mg/kg/s.i.d/s.c., Convenia, Zoetis, Madrid, Spain) were administrated during each surgery.
2.9 Euthanasia
The dogs were sedated and euthanized with an overdose of sodium pentobarbital (200 mg/kg/I.V.; Dolethal, Vetoquinol, France).
2.10 Histological processing
After fixation in buffered 10% formaldehyde solution, all blocks were dehydrated in a graded series of ethanol solutions and embedded in a light-curing resin (Technovit 7200 VLC; Heraeus-Kulzer GMBH, Werheim, Germany). Using a band saw equipment (Exakt Apparatebau, Norderstedt, Germany), the implants contained in the blocks were cut at the buccal aspect of their long axis. The specimens obtained were then ground and polished to obtained samples of about 50 µm. The histological slides were stained according to the Levai–Laczkó method.
2.11 Histomorphometric evaluation
All histological slides were evaluated by a trained examiner (E.F.D.R.) after calibration with an expert (D.B.). New bone and soft tissues were evaluated. The following land-marks were adopted: IS, implant shoulder; M, coronal margin of the rough surface, corresponding to the base of the convergent neck; AB, apical extension of the blades; B, coronal extension of the osseointegration on the implant surface; C, top of the bone crest The following measurements were carried out at both the buccal and the lingual aspects: (i) the distance between IS and B; (ii) the distance between IS and C; (iii) the bone-to-implant contact percentage (BIC%) in the region included between B and AB. Bone changes were calculated by subtracting the convergent neck height (1.8 mm) from IS-B (Figure 3).
2.12 Experimental outcomes and statistical methods.
The primary variables were the distance IS-C and marginal bone loss (MBL). The secondary variables were level of osteointegration and bone-to-implant percentage (BIC%) in the area B-AB. The values obtained are expressed as the mean ± standard deviation. The Shapiro-Wilk test was used to determine the normality of data and, according to the results, the differences between the test and control sides were evaluated by a paired t-test or a Wilcoxon matched-pairs signed rank test. The Friedman test was applied for evaluated differences between groups. GraphPad Prism (version 10.0.2 for Windows, GraphPad Software, Boston, Massachusetts, USA,) was used for statistical analyses. The level of significance was 5%.