Cm h111

Four human OS cell lines, MNNG (Catalogue No.: HTX1631), HOS (Catalogue No.: HTX3087), U2OS (Catalogue No.: HTX1634), and 143B (Catalogue No.: HTX2774), were provided by Otwo Biotech (ShenZhen) Inc. (Shenzhen, China). And one osteoblast cell line named hFOB1.19 (Catalogue No.: CL-0353) that was used as standard control was provided by another company, Procell Life Science&Technology Co., Ltd. (Wuhan, China). All the OS cell lines were incubated with a complete growth medium consisting of Dulbecco's modified Eagle's medium (DMEM, Gibco, Shanghai, China), 10% of fetal bovine serum (FBS, Tianhang, Zhejiang, China), and 1% of dual antibiotics (penicillin and streptomycin) (Solarbio, Beijing, China) in an incubator with the atmosphere condition of humidity and the concentration of carbon dioxide (CO₂) at 5%; the temperature was set 37°C. At the same time, hFOB1.19 cells were cultured with a particular culture medium for human osteoblast (CM-H111, Procell Life Science & Technology Co., Ltd.) in a cell culture equipment with the atmosphere condition of humidity and the concentration of carbon dioxide (CO₂) at 5%; the temperature was set at 34°C. And they were passaged with the ratio of 1 : 3 after the cells reached 90% of confluence.

Osteoblasts (CP-H111, Procell, Wuhan, China) will be cultured in 24-well plates for 24 h with complete culture medium for human osteoblasts (CM-H111, Procell, Wuhan, China) in cell incubator (51032124, Thermo Fisher, MA, USA) under 5% CO₂ and 37°C, and then, these cells were collected and diluted with a PBS solution at a concentration of 1 × 10⁴ cells/mL. The above osteoblast diluents were divided into control group, nHA group, and NHA+RPR group. 1 mL osteoblast diluent and 2 mL special culture medium for human osteoblasts were added to each group, among which the control group was given no therapy, nHA sterilized by a high-pressure pot was added to the nHA group, and nHA and PRP were added to the nHA+RPR group. At last, the above cells were placed in the cell incubator and cultured for another 12 hours. Proliferation test will be performed by immunofluorescence. Anti-ki67 (ab15580, Abcam, USA) was used for labeling of living cells, and Alexa Fluor 488 (Cy2, FITC) was used as fluorescent secondary antibody. After incubation in dark at 26°C for 40 minutes, labeled cells were analyzed under a confocal microscopy (FV3000, OLYMPUS, Japan).