Study design
Study protocol was approved by ethics committee of Tanta University's specifying conditions and constraints for conducting and publishing studies involving animal models (No. R-OMPDR-6-23-1) and followed the ARRIVE guideline [16].
Sample size calculation
The sample size was calculated using G-Power software version 3.1.9.2 [17] to detect difference in torque testing. Based on Cochran et al., [18], results, and adopting a power of 80% (b=0.20) to detect a standardized effect size in torque testing (primary outcome) of 0.515, and level of significance 5% (α error accepted =0.05), the minimum required sample size was found to be 12 specimens per group (number of groups=4) (Total sample size= 48 specimens)[19, 20].
Experimental animals and their housing and husbandry
In this study, four osteotomy sites were prepared in the femur of 12 adult male dogs (4 and 6 mm in diameter). One standardized implant size (4mm diameter, 10 mm long) was inserted in each osteotomy site. A fixation bar was used to stabilize the implant suffering from compromised primary stability, Fig. 1-a.
The animal-house veterinarian evaluated 12 mature male 2-year-old beagle dogs weighing between 10 and 12 kg to rule out disease and ensure that they were provided with a balanced diet of milk, broth, and meat during the study period. All animals were kept in individual stainless-steel cages with direct access to water, proper ventilation, as well as a 12-hour light/dark cycles. Each animal received two implants in the same femur.
Surgical procedure:
To prevent postoperative infection, each animal was given a prophylactic antibiotic (ampicillin 25 mg/kg body weight) right before the procedure. An expert surgeon (Y.H) performed all the surgeries in a sterile environment in a veterinary operating room. The dogs were given a subcutaneous injection of atropine (0.05 mg/kg; Kwang Myung Pharmaceutical) and after 10 minutes of premedication, anesthesia was induced by injecting a mixture of 2mg/kg Xylazine (Xyla-Ject; Adwia Pharmaceuticals) and 5.5mg/kg ketamine hydrochloride (KETAMAX-50; Troikaa Pharma) into the cephalic vein of the forelimb and maintaining it with inhalation anesthesia.
The skin over the medial side of the femoral bone was incised, reflected, and the superficial fasciae, muscle tissue, and deep fasciae were also incised bluntly. The periosteum was next incised to reveal the femur shaft, which was ready to receive the dental implants. One sequential osteotomy was started with a pilot drill followed by 2-, 3.5- and 4-mm drill to a depth of 10mm. A second osteotomy, 2 cm apart, was prepared to a size of 6 mm in diameter. Dental implants (B&B DURA-VIT -EV, Italy) of the required size (4 mm in diameter and 10 mm long) were inserted in the two prepared osteotomy sites. A micro plate fixation bar (1.2 System Micro, Plates BioMaterials, Korea) was secured over the compromised implant using the cover screw of the manufacturer and two short fixation pins, Fig 1-b&-c. Deep muscles and fascia were sutured using absorbable suture (4:0 cat gut suture, Trugut, India) followed by skin using non-absorbable sutures (1:0 black silk, Assut, Egypt), and the surgical site was covered with soft cloths to prevent infection, Digital periapical radiographs were taken immediately after surgery and at each subsequent follow-up, Fig 2.
Reverse torque test (RTT):
After 8 weeks of healing, the implants were uncovered and connected to a surgical handpiece connected to a calibrated step motor using a motor driven hex tool. The motor was activated in anticlockwise direction to reach a maximum torque of 50 N.cm over the course of 30 seconds. The test was stopped once the implant started to rotate and rotating torque was reported.
Histomorphometry:
The animals were sacrificed with an overdose of thiopental sodium after 8 weeks, and their femur blocks were dissected. The blocks were promptly fixed for one week in 4% buffered formaldehyde. The specimens were next dehydrated in rising ethanol concentrations (50, 70, 90, and 100%) using a dehydration machine (ASP 300S, Leica Biosystems) with agitation and vacuum. The blocks were embedded in clear chemically polymerized methyl methacrylate resin and cut in a coronal-apical plane using a precision-cutting machine (Metkon's Micracut150 precision cutter), then ground and polished with 800-grit silicon carbide paper. After staining, (Stevenes blue and Van Gieson picrofuchsin), the sections were examined using a light stereomicroscope (BX61; Olympus Corp) equipped with a high-resolution digital camera (E330; Olympus Corp). Bone implant contact (BIC) was calculated as the amount of new bone in direct contact with implant surface and was calculated as a percentage of implant perimeter calculated from the most central section.
Data were collected and entered to the computer using SPSS (Statistical Package for Social Science) program for statistical analysis (ver 25)[21]. Data were entered as numerical or categorical, as appropriate. Kolmogorov-Smirnov test of normality of the distribution of the variables was not statistically significant, so parametric statistics was adopted [22]. Data were described using minimum, maximum, mean, standard deviation, standard error of the mean, 95% Confidence Interval of the mean, 25th to 75th percentiles. Comparisons were carried out between two independent normally distributed variables using the independent (Student’s) t-test. An alpha level was set to 5% with a significance level of 95%.