Animals
Experiments were carried out on 56 white laboratory female rats aged 6 months, with an average body weight of 300g ± 25. Rats were randomly assigned to 1 of 2 groups: Sham-operated control group (SH) or ovariectomy group (OVX). The study followed the requirements of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes. The study design was approved by the institution Bioethics Committee (Protocol No. 175 dated 26.02.2018).
Implants
Four commercially available porous implants were used in this study. The first, TANTALUM – porous Tantalum Trabecular Metal (Zimmer, Warsaw, IN, USA). The other three were made from titanium alloy Ti6Al4V using additive technologies: TTM (AK Medical, Beijing, China), CONCELOC (Smith & Nephew, Memphis, TN, USA), ATLANT (TITAN-MED, Kyiv, Ukraine). All materials were comparable in porosity – 80% or upper. TANTALUM has an elastic modulus of 3 GPa which is between that of cortical and cancellous bone, and similar to subchondral bone [29]. The elastic modulus of the other three materials was 12.9 GPa for TTM [30], 4.3 GPa for CONCELOC [31], and 113 GPa for ATLANT.
Surgical procedures
All surgeries were performed under general intramuscular anesthesia – ketamine 50 mg/kg. Bilateral ovariectomies were performed in 28 animals in the OVX group according to previously described methodology [28]. For SH group, the ovaries were not removed. At 3 months after ovariectomy, when osteoporotic bone changes had developed in OVX group [28], surgery was performed in both groups (OVX & SH) of rats. The skin of the lower extremities was shaved and treated with Betadine® solution. From the lateral access transcortical defects (diameter 2.5 mm, depth 3.0 mm) were created into the distal metaphysis of the left femur using a dental burr (Figure 1 A-B). The implants (TANTALUM, TTM, CONCELOC or ATLANT) were placed into the prepared defects (Figure 1 C) by using a press-fit technique. Wounds were treated with antibiotic powder and sutured in layers.
The euthanasia of rats was performed 45 days after surgery by administering a lethal dose of anesthetic (sodium thiopental, 90 mg/kg intramuscularly). This observation period was chosen for the reason that other researchers conducted similar experimental studies on rats in the same period [32], as well as the results of previous study performed by us [28]. Since for this period (45 days) the site of defect is substituted by new formed mature bone.
Histological study
After extraction the implanted left femurs (Figure 1 D) were fixed in a solution of 10% formalin, and decalcified in a 10% solution of formic acid. After decalcification, the implants were carefully removed, and the distal metaphysis of the femurs was dehydrated in alcohols of increasing concentration and a mixture of paraffin and xylene (1:1), and embedded into paraffin. Longitudinal histological sections of 5-6 µm (7 of each sample) thick were stained with hematoxylin and eosin (H&E). The bone structure was analyzed under light microscope BX63 (Olympus, Japan) and imaged with a digital camera DP73 (Olympus).
Histomorphometry
Three sections were obtained from each femoral distal metaphysis. The percentage of peri-implant bone area (BA%) around the implant at a distance of 500μm in the cancellous area was measured as described previously [26, 33]. The BA% allows us to evaluate all bone areas, not only along the perimeter of the implant, but also at a greater distance from the implant. This is especially important in osteoporosis, when overall bone area/mass is reduced. The measurements were made using CellSens Dimension 1.8.1 software (Olympus, 2013) for the Olympus BX-63 microscope (at a magnification rate of ×4).
Statistical Analysis
Measurement data were presented as mean ± standard deviation (SD). Comparison of the values in the groups of OVX rats and SH rats was performed by using the one-way ANOVA analysis with Bonferroni post-test. To compare values between OXV and SH groups we used unpaired t-test. A critical level of significance was accepted as 0.05. The analysis was performed using the IBM SPSS Statistics 19.0 software.