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.

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.