Alkaline phosphatase alp

Second-generation BMSCs were inoculated in six-well plates. When the cell confluence was approximately 60% at the bottom of the culture bottle, according to the instructions of the BMSC osteogenesis induction kit (Cyagen Biosciences, Santa Clara, CA, USA), the medium in the experimental group was replaced with BMSC osteogenesis induction medium, while the cells in the control group remained in complete L-DMEM. After two weeks of osteogenic induction, the cells were fixed, alkaline phosphatase (ALP) (Cyagen Biosciences) staining was used to detect ALP activity, and 0.1% alizarin red (Cyagen Biosciences) staining was used to identify calcium nodules.

BMSCs of the third passage were seeded into six-well plates. When the cell confluence reached 60%–70%, the medium in the experimental group was replaced by BMSC osteogenic induction medium according to the BMSC osteogenic induction kit (Cyagen Biosciences, USA), and the medium in the control group was unchanged. After 2 weeks of osteogenic induction, cells were fixed and ALP activity was determined by staining with alkaline phosphatase (ALP) (Cyagen Biosciences). Calcium nodules were identified by staining with 0.1% alizarin red (Cyagen Biosciences).

Passage 3 hMBSCs were seeded at a density of 1.5 × 10⁵ cells/well in a six-well plate. When the cells reached 100% confluence, the OriCell™ human mesenchymal stem cell adipogenic differentiation medium (Cyagen Biosciences, Shanghai, China) was added to wells according to the manufacturer's instruction. After 28 days of induction, Oil red O (Cyagen Biosciences) staining was performed to assess the differentiation potential of adipogenesis formation of intracellular lipid droplets. For osteogenic differentiation, hMBSCs were cultured with OriCell™ human mesenchymal stem cell osteogenic differentiation medium (Cyagen Biosciences) for 10 days and 21 days to analyze the middle and late stages of osteogenic differentiation. The differentiation potential for osteogenesis was assessed through alkaline phosphatase (ALP) (Cyagen Biosciences) staining at the middle stage and Alizarin Red (pH 4.2, 40 mM) (Cyagen Biosciences) staining at the late stage.