α mem culture medium

Mouse bone marrow-derived macrophages (BMMs) and the mononuclear macrophage cell line Raw 264.7 cells were selected as representative cells to investigate the cytocompatibility and osteoclast differentiation of the metal materials. The mouse osteoblastic cell line MC3T3-E1 was selected as a representative cell line to evaluate the osteogenic differentiation of the metal materials. The MC3T3-E1 cells and BMMs were cultured in α-MEM culture medium (Gibco), which supplemented with 10 % FBS and 1 % penicillin/streptomycin (P/S, Gibco) at 37 °C with 5 % CO2.

Unfractured tibia of 20-month old mice was isolated and contents of medullary cavity (bone marrow) were flushed using αMEM culture medium (Gibco) on POD 21 days. Cell suspensions were passed through an 18G needle to dissociate cell aggregates, and cells were plated in 24-well plates with a density of 5 × 10⁵ cells/ml in plating medium (αMEM, 10% FBS, 1% Penicillin/Streptomycin) or 1 × 10⁶ cells/ml for seven days. To determine the colony forming units-fibroblastic after14 days with plating media; wells were then washed with PBS, fixed with 10% formalin, and stained with 0.05% crystal violet. Cells were cultured in osteogenic differentiation media (αMEM, 10% FBS, 1% Penicillin/Streptomycin, 30 uM ascorbic acid, 10^–8 M dexamethasone, 8 mM sodium phosphate) for next 14 days. Cultures were incubated for 21 days at 37 °C with 5% CO₂ and media were replenished every 2 days. Wells were washed with PBS, fixed using 10% formalin, and stained for alkaline phosphatase (ALP) using NBT/BCIP and mineralization using 2.5% silver nitrate solution (Von Kossa) on a light box and 2% Alizarin Red S (pH 4.3). Colonies were defined by containing at least 25 cells.

The Institutional Review Board (IRB) and Ethical Committee of Zhejiang University, Hangzhou, China, approved this study. Whole-bone marrow samples were collected from healthy donors at the First Affiliated Hospital, Zhejiang University. All donors provided the written informed consent. Bone marrow cells were isolated and purified from bone marrow according to the methods of Zhang et al. [3 (link)]. The harvested cells were seeded in a 10 cm dish with α-MEM culture medium (Gibco, Shanghai, China) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 5 ng/mL basic fibroblast growth factor (BFGF; Life Technologies, Shanghai, China) and cultured at 37°C, 5% CO₂, and 95% humidity. Cells at approximately 80% confluency were trypsinized and reseeded at a density of 1 × 10⁵ cells/mL as passage 1. Cells at passage 3 were used for this study. These cells were characterized by surface markers CD19−, CD34−, CD14−, CD45−, HLA-DR−, CD105+, CD90+, CD73+, and CD29+ and showed the potentials of osteogenesis and adipogenesis.

α‐MEM culture medium and trypsin were obtained from Gibco (Gibco). Foetal bovine serum (FBS) was the product of Biological Industries (Beit‐Haemek). Collagenase II was purchased from Solarbio Life Sciences. β‐glycerophosphate, dexamethasone and ASAP (ascorbic acid 2‐phosphate) were all from Sigma. Human recombinant BMP‐2 was purchased from Beijing Biosynthesis Biotechnology Co. All other chemicals were of analytical grade.