Mc3t3 e1

Mouse osteoblast-like cells (MC3T3-E1) (Procell Life Science & Technology Co., Ltd.; Wuhan, China) were used for in vitro biocompatibility measurements, and they were cultured in DMEM medium with 10% fetal bovine serum, 1% streptomycin and penicillin at 37 °C. In this research, the sample extracts were used to study the biocompatibility. Each sample was immersed in complete culture medium for 72 h with a ratio of 3 mL/cm² in accordance with ISO10993−12 [42 ] to obtain the extracts. The extracts were preserved at 4 °C prior to the experiments.

The mouse macrophage line Raw 264.7, mouse preosteoblast cell line MC3T3-E1, and BMSCs were obtained from Procell Life Science & Technology (Wuhan, China). MC3T3-E1 cells were cultured in α-MEM supplemented with 10% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin. RAW264.7 cells were cultured in DMEM supplemented with 10% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin. Mouse BMSCs were cultured in mouse MSC Medium (Cyagen) according to the manufacturer’s protocol (Nuwacell, Hefei, China). The cell cultures were maintained in an incubator at 37 °C in a humidified atmosphere with 5% CO2.
C57BL/6J female mice of specific pathogen-free (SPF) quality were purchased from the Model Animal Research Center of Tongji Medical College of Huazhong University of Science and Technology (HUST, Wuhan, China). All mice were housed under controlled conditions, which included room temperature of 25 °C, humidity maintained at 60 ± 10%, and a natural light–dark cycle throughout the study. All animals were treated according to the regulations of Chinese law and the local Ethics Committee. All animal experiments were reviewed and approved by the IACUC of HUST (IACUC Number: 3469).

MC3T3-E1 and RAW 264.7 cell lines were used as models for osteoblast and osteoclast, respectively. Both cell lines were purchased from Wuhan Procell Life Science & Technology Co., Ltd. Construction of each stable-transfer cell line included 5 steps: (1) cell recovery and culture: α-MEM+10%FBS+1% (Penicillin-Streptomycin Solution) and DMEM+10%FBS+1% (Penicillin-Streptomycin Solution) were suitable culture medium for MC3T3-E1 and RAW 264.7 cell lines, separately; (2) cell passaging; (3) determine the optimal screening concentration of puromycin; (4) lentiviral infection; and (5) cell screening.