Thirty-two 16-week-old male Sprague–Dawley rats, weighing 400–500 g, were used to assess in vivo bone regeneration. Rats were allowed to acclimate to their diet, water, and housing for 1 week prior to surgery. Before the surgical procedure was carried out, rats were anesthetized with 4 MAC of isoflurane (Hana Pharm, Seoul, Korea) in the induction cage and maintained with 1.5 ~ 2 MAC via a face mask at an O2 flow rate of 1–2 L/min. Tramadol (12.5 mg/kg; Hanall Biopharma, Seoul, Korea) and enrofloxacin (Baytril®, 5 mg/kg; Bayer, Leverkusen, Germany) were injected subcutaneously as preoperative analgesic and antibiotic, respectively. The surgical site was clipped, disinfected with chlorhexidine–alcohol solution, and draped with a sterile drape. The skin was incised on the midline of the skull, and the subcutaneous tissue and periosteum were incised and retracted to expose the calvarium. Bilateral calvarial defects, 5 mm in diameter, were generated using a trephine burr with a dental unit and flushed with normal saline to avoid overheating. Each bone defect was filled with 20 mg ceramic beads. Only ceramic beads were implanted in the bead control group, BMP-2 (1 µg/defect) or quercetin (8 wt%)-loaded ceramic beads were implanted in the experimental groups, and the defects were left empty in the negative control group. Fibrin glue (Greenplast®; Green Cross, Seoul, Korea) was applied to the implanted beads to prevent their migration. The periosteum, muscle, and subcutaneous tissue were sutured using 4–0 absorbable suture material (Ethicon, Edinburgh, UK), and the skin was closed using a 4–0 nylon monofilament suture (AILEE Co., Busan, Korea). All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Seoul National University (SNU-200612-4).
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