In Vitro Cell Experiments

Rabbit bone marrow mesenchymal stem cells (RBMSCs) and mouse embryonic osteoblast precursor cells (MC3T3E1), purchased from the Cell Bank of the Chinese Academy of Sciences, were cultured in the complete culture mediums made of Dulbecco’s modified Eagle medium added with 10% fetal bovine serum and 1% penicillin. Cells were incubated in the complete medium (5% CO2; 37°C).

The composite samples of the mPLA/HA screw and PLGA/β-TCP screw were prepared into wafers by a dissolution-casting method, which was sterilized for cell culture. Then, the 2 groups of samples were placed in 96-well plates, and the RBMSCs and MC3T3E1 were respectively seeded on the surface with a proper density. Another group of RBMSCs and MC3T3E1 without any composite samples was also cultured as a control group. Next, cell proliferation was examined using Cell Counting Kit 8 (Dojindo Molecular Technology) following the manufacturer’s instructions at 1, 3, 5, and 7 days after incubation.

The expression of the osteogenesis-related mRNA (osteopontin [OPN], osteocalcin [OCN], collagen type 1 [Col-I], and alkaline phosphatase [ALP]) of MC3T3E1 osteoblasts was detected by real-time polymerase chain reaction after incubation for 3 and 7 days for comparison among the mPLA/HA group, PLGA/β-TCP group, and a control group (β-actin was used as a quantitative control for RNA levels). The sequences of the primers are listed in Supplementary Tables S1 and S2 (available separately). Also, alizarin red staining was performed on the culture medium of the 3 groups on days 14 and 21, and the distribution of calcium and phosphorus in the culture medium was observed.