Paraformaldehyde (pfa)

Osteoblastogenesis induction and alizarin red staining were performed as described previously [65 (link)]. BMSCs with a density of 5 ​× ​10⁴ ​cells/cm² were seeded on the surfaces of sterilized Ti, TNrs, and Fe₃O₄-TNrs or into the wells without any sheets, treated with 1 ​mT 15 ​Hz SEMF for 1 ​h per day. BMSCs were cultured with the osteogenic differentiation induction medium (Cyagen Biosciences, CA, USA). After 21 days of culture, the cells were fixed with 4% paraformaldehyde for 20 ​min, and stained with alizarin red (Cyagen Biosciences, CA, USA) for 5 ​min, washed with PBS three times, then the cells could be placed under the microscope (EVOS FL Auto, Life Technologies, USA) to observe and photograph.

pTa and pTi6Al4V scaffolds were placed in a 12-well plate, and the third-generation SD rat BMSCs were inoculated on the scaffolds with a density of 5 × 10⁴/well. The cells were observed every day and cultured continuously for 21 days. The culture medium was absorbed carefully, fixed with 4% paraformaldehyde (Cyagen Biosciences Inc, Guangzhou, China) for 60 min and washed twice with PBS; 0.1% alizarin red dye solution was added and stained for 30 min, followed by washing twice with PBS, which was observed and photographed under a light microscope.