2.3.2. Surgical Procedures

The present study used six mature male Japanese Suffolk sheep (approximately 20–21 months old and 33–54 kg in weight). Intramuscular administration of atropine (0.06 mg/kg), xylazine hydrochloride (0.22 mg/kg), and ketamine (11 mg/kg) were used for general anesthesia. The anesthesia was maintained with endotracheal inhalation of 2% isoflurane throughout the operation. Under proper aseptic measures, a skin incision was made above the right iliac crest, and the tissues were dissected to expose the surgical site. Standardized critical-size bone defects (15 mm × 10 mm × 9 mm) in iliac crests were harvested by an oscillating saw under cooling with sterile physiologic saline, and PR-DDMs were grafted in the bone defects (Figure 2). After irrigation, absorbable sutures (Polyglactin, Vicryl^®, Ethicon Inc., New Brunswick, NJ, USA) were used to secure the muscle flaps, and the skin incisions were closed with sterile 2-0 silk sutures. Animals were treated with intramuscular injections of penicillin G and dihydrostreptomycin (10 mg/kg) daily for 7 consecutive days. Adequate measures were taken to minimize the pain or discomfort to the experimental animals. At 2 months, three sheep and the remainder were humanely euthanized at 4 months post-operatively with intravenous injection of sodium pentobarbital-based euthanasia solution. The surgical graft sites were trimmed from the iliac crests after confirming the death of each animal and fixed in a 10% neutral phosphate-buffered formalin solution.

(a) Schematic illustration, and (b) 3D micro-CT images of grafted PR-DDM into critical-size sheep iliac defect.