Implant preparation and characterization
Threaded commercial titanium alloy implants (3.3 mm diameter and 8 mm length) from Zimmer® were used as control implants. They consisted of Ti6Al4V screws blasted with hydroxyapatite and subsequent acid attack with HCl. Screws with these prior surface treatments were submitted to a thermal treatment. The oxidation treatment was performed at 700 °C for 1 h, in air. After oxidation, samples were removed from the furnace and cooled at room temperature. Commercial and heat-modified screws were gamma-ray sterilized prior to implantation.
Scanning electron microscope (SEM) was used to characterize the surface morphology of the Ti implants. The surfaces were analysed by using a JEOL-6500F microscope equipped with a field emission gun (FEG) coupled with an energy dispersive X-ray (EDX) spectrometer. The images were taken using secondary electrons.
Animals and treatment
The experimental protocol of this study and animal care conformed to the European Communities Council Directive of 24 November 1986 (86/609/EEC) [19] and has been independently reviewed and approved by the Madrid Community Ethics Committee for Regional Clinical Research (CEIC-R). Forty New Zealand female rabbits of approximately 5 kg and 11 months old were chosen as experimentation animals.
Animals were divided into healthy non-ovariectomised group and ovariectomised (OVX) group to carry out the tests. In order to induce osteoporosis, female rabbits underwent bilateral ovariectomy under general anaesthesia and were fed a hypocalcic diet (8.2 g kg−1 calcium and 6 g kg−1 phosphorous) for 8 weeks [20]. Diet mainly consisted of 74.2 % barley, 20 % wheat bran, 5 % soy and 0.3 % salt was supplied by PanLab SL (Harvard Bioscience Group, Barcelona, Spain). This procedure was selected because the treatment based only on the ovariectomy and hypocalcic diet provokes spontaneous fractures [21, 22].
Eight weeks post-surgery, rabbits were subjected to a densitometry to the vertebral column and tibia bone to verify the establishment of osteoporosis model [21]. For the densitometric analysis, a Norland XR-26 densitometer was used (Norland Co., Fort Atkinson, WI, USA) calibrated prior to the measurement. The exploration parameters were as follows: speed 40 mm s−1, resolution 1.0 × 1.0 mm, and measurement resolution 0.5 × 0.5 mm. Forty percent of the total length of each bone was analyzed, including metaphysis and diaphysis regions to verify differences between bone mass of healthy and osteoporotic groups. The value obtained was the bone mineral density (BMD) in grammes per centimetre.
Implant procedure. Surgery
The experimental design carried out with the experimental animals is summarized in Fig. 1. Rabbits were randomly divided into two groups: control (healthy rabbits) and OVX (osteoporotic rabbits). Both of them were subjected to surgery to insert commercial (no thermally treated Ti6Al4V) and modified (thermally treated Ti6Al4V) implants. The half of the healthy and osteoporotic rabbits was treated with 4 IU of recombinant human growth hormone (rhGH) as lyophilized powder (Genotonorm® Pfizer, NY, USA) directly located in the place of insertion. Healthy and osteoporotic rabbits without implants were also included as reference at both implantation times in the study.
Incision was performed in the inner side of the proximal epiphysis of each tibia, under intramuscular anaesthesia. Transcortical osteotomy followed by drilling to generate a bed of 3.1 mm diameter and 8 mm deep was made, where the implant was inserted until touching the opposite cortical bone (Fig. 2). In the experimental group with local GH, 4 IU of rhGH as powder was added into the bone hole just before the insertion of the Ti6Al4V implant. Commercial implant in the right tibia and the modified implant in the left tibia were inserted. Only one implant was inserted in each tibia.
The injury was sutured with absorbable material after the implantation. Antibiotics and anti-inflammatory agents were delivered in the postoperative period to prevent infection and pain. Rabbits were sacrificed after 15 and 30 days of implantation by intravenous injection of 0.4 mg sodium pentobarbital (Dolethal®, Vetoquinol, Cedex, Francia) diluted in serum (Fig. 1).
Tibiae were cleaned from soft tissues to determine the bone mineral density (BMD). Densitometries at 0.5 mm above and 0.5 mm below the Ti6Al4V implant were acquired to compare the BMD of control and OVX groups.
Histological study of each tibia (including the Ti6Al4V implant) for control and modified implants and with and without GH was carried out. Clean hard tissues were fixed in 10 % pH 7 buffered formaldehyde and dehydrated in grading alcohol concentrations. Tibiae were cut into blocks and histologically prepared according to the modified Donath and Breuner method [23]. The preparation of hard tissue consisted of embedding in light-polymerizing 2-hydroxietyl-metacrylate. These blocks were cutting in five sections of about 200 μm and then grinding until achieving cross-sections of about 80-μm final thicknesses with an EXAKT cutting and grinding equipment (EXAKT, Norderstedt, Germany).
Bone-implant interface sections were examined under the optical microscope (Zeiss, Oberkpchen, Germany) using histological laboratory stains such as toluidine blue with Weigert haematoxylin (Merck, Kenilworth, NJ, USA) that allows the differentiation between osteoblasts and osteoclasts [24].
The images were processed and cross-sections were compared by means of the MIP-4 image analyser software (Digital Image Systems, SL, Barcelona) in order to quantify the bone fraction. The MIP-4 software is able to perform area and volume measurements through a computerized system connected to the optical microscope and histological lens. Area and length measurements on the images captured from the microscope were attained. All images were processed with ×10 magnification objective. The bone-to-implant contact (BIC) is calculated as the ratio of the length of the implant in contact to bone tissue and the implant perimeter, i.e. the percentage of the implant surface in contact with bone.
The quantitative results were processed with the statistical package Statgraphics plus 5.1. The significance of the differences between the groups was studied according to Student’s t test and the one-way ANOVA test (analysis of variance). p value was 0.05.