2.5. Rat Calvarial Defect Model

Thirty male Sprague-Dawley rats, aged eight weeks (250–300 g), were purchased from Bio-LASCO Co. Ltd. (Taipei, Taiwan). The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC-17-068) at the National Defense Medical Center (Taipei, Taiwan). Rats were randomly divided into three groups of treatment: Lesion control, SF scaffold, and hASCs-SF scaffold. Rats were anaesthetized with an intraperitoneal injection of xylazine (8 mg/kg) and ketamine (100 mg/kg), followed by a subcutaneous injection of enrofloxacin (0.05 mg/kg) as microbial prophylaxis. After being shaved and sterilized, the skin and underlying tissues including the temporal muscle were detached to expose the calvarial bone. Calvarial bone defects were generated with the aid of a dental bur and two defects of 5 mm in diameter were generated per cranium. The calvarial defects were either left empty or surgically filled by putting the tested materials inside the defect. All rats undergoing surgery were kept warm with an electric blanket during the operation and for 3 h afterwards. Rats were kept in individual cages under aseptic conditions and were sacrificed at six and twelve weeks after surgery. Then, bone specimens were harvested for further evaluation.