Sterilization and Characterization of Biomaterial Scaffolds

Each disc of PCL or HAp-PCL was sterilized by a fully automated ethylene oxide gas sterilizer (SA-N160, Iki Co., Otsu, Japan). The rBMSCs were collected and cultured from the tibia and femur of 6-week-old male F344 rats according to the protocol in the previous article [49 (link)], and cells with the passage numbers of 3 and 4 were used for in vitro cell-based studies. The rBMSCs were cultured with α-MEM (FUJIFILM Wako Co., Osaka, Japan) containing 20% fetal bovine serum (FBS, Cytiva, Tokyo, Japan) and 1% antibiotics and antimycotics (Gibco Invitrogen, Carlsbad, CA, USA). The cells (3 × 10⁴ cells) were seeded onto each disc (PCL or HAp-PCL), and the discs were placed in a 24-well plate. The DNA concentration of cells 6, 12, and 24 h after seeding were measured using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instruction. After seeding rBMSCs on each disc (PCL or HAp-PCL) for 24 h, viability was assessed using the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher scientific Inc., Waltham, MA, USA) according to the manufacturer’s instruction. The stained cells were observed by confocal laser fluorescence microscope (LSM-700, Zeiss Microscopy, Jena, Germany). Each experiment was performed in triplicate.