A total of 17 New Zealand white male rabbits (3–3.5 kg) underwent general anesthesia with Zoletil® and xylazine. This study was approved by the Institutional Animal Care and Use Committee at the Clinical Research Institute of Seoul National University Hospital Biomedical Research Institute (IACUC No. 13-0038). The hind limb was disinfected with betadine after hair removal. The anterior aspect of the tibia was exposed and two holes were generated by pre-drilling. The two holes were spaced no less than 15 mm apart in order to minimize the interaction between them. After the two holes for the implant fixture in the tibia were placed using a lance drill, they were enlarged to a diameter of 2 mm using a twist drill. Next, a straight drill was used to further enlarge each of the holes first to 2.8 mm, then to 3.3 mm, and finally to 3.8 mm; and then the holes were countersinked. The depth of each of the holes was 8.5 mm and dental implant fixtures, 4 mm in diameter, (Ø4, 8.5 mm length, MegaGen, Seoul, South Korea) were inserted. The insertion torque of the implant was 45 Ncm. Three groups were allocated as follows: the implant only group (implant group), the injected β-TCP-hydrogel power gel composite implant group (the hydrogel group), and the rhBMP-2 (200 μg) loaded powder gel β-TCP-hydrogel composite implant group (BMP-2 group). After two tibiae of a rabbit were exposed, two implants of the same group were inserted per each tibia. The block randomization was used to decide which implant to place into each tibia. After inserting a cover screw, and fascia layer, the wound was then sutured (Fig. 2). Immediately after the surgery, 300 mg cefazolin was injected intramuscularly and antibiotics were administered for 2 days. The experimental animals were euthanized 4 weeks later.
Fig. 2 Surgical procedures. a Tibial diaphysis in the tibial medial aspect was exposed and two, 4 mm diameter holes were generated; b A composite of rhBMP-2 loaded porous β-TCP microspheres and hyaluronic acid powder-gel was injected; c A dental implant fixture was inserted