Osteogenesis assay in vivo

NOD/SCID mice (male; age, 8 weeks; 18-22 g; n=21) purchased from SLRC Laboratory. Mice were bred under specific pathogen-free conditions at a constant temperature (23±1°C) with 55±15% humidity and a 12-h light-dark cycle (09:00-21:00). All the mice had free access to food and water. For the in vivo osteogenic ectopic assay, a total of 2×10⁶ MC3T3-E1 were seeded on a β-tricalcium phosphate (β-TCP) scaffold (Shanghai Bio-lu Biomaterials Co., Ltd.). The implants loaded with cells were implanted into an intramuscular pocket of the right femur of mice (21). The mice were anesthetized with an injection of 1% pentobarbital sodium (70 mg/kg) into the abdominal cavity. The mice were randomly divided into seven groups containing three mice per group: i) β-TCP (vehicle); ii) β-TCP loading LV-ctr MC3T3-E1 cells (LV-ctr); iii) β-TCP loading LV-Kaiso MC3T3-E1 cells (LV-Kaiso); iv) β-TCP loading Sh-ctr MC3T3-E1 cells (Sh-ctr); v) β-TCP loading Sh-Kaiso MC3T3-E1 cells (Sh-Kaiso); vi) β-TCP loading Sh-ctr MC3T3-E1 cells (Sh-ctr); and vii) β-TCP loading Sh-Itga10 MC3T3-E1 cells (Sh-Itga10). At 8 weeks post-implantation, the mice were sacrificed via carbon dioxide asphyxia. Briefly, the CO₂ flow rate was started with 8 l/min in a 30-lchamber. The implants were harvested and fixed in 4% paraformaldehyde (room temperature for 24 h) for µ-computed tomography (µCT) analysis. The bone mineral density (BMD) of implants was then generated by skyscan 1176 (22). The implants were decalcified in 10% EDTA and embedded in paraffin. Then 5-µm sections were stained with hematoxylin and eosin (cat. no. C0105M; Beyotime Institute of Biotechnology) for 10 min at room temperature, and washed with distilled water for 10 min. Sections were observed and imaged using a light microscope (E200; Nikon Corporation) at a magnification of ×200. All animal studies were approved by the Animal Ethics Committee of Changhai Hospital.