不同来源的小鼠骨源性间充质干细胞的分离培养及成骨能力比较OA

Objective To compare the osteogenic potential in mouse bone derived mesenchymal stem cells from different sources and provide an efficient osteogenic induction technical system for bone tissue engineering research.Methods Bone-derived mesenchymal stem cells were isolated from the leg bones of BALB/c mice through three sources:bone marrow(bone marrow,BM),cortical bone fragments digestion(bone digestion,BD),and cortical bone fragment culture(bone culture,BC).The proliferative capacity of the cells were compared via passaging and cell counting kit-8(CCK-8)assays,while flow cytometry was used to assess the stem cell proportions.Following osteogenic induction,osteogenic potential was evaluated using alkaline phosphatase(ALP)and Alizarin red staining,Western blotting,and real-time quantitative PCR,and the optimal cell type was determined.After inducing the selected cells in 3D culture on β-tricalcium phosphate(β-TCP)for 2 weeks,their osteogenic potential was assessed through Western blotting and real-time quantitative PCR experiments.The three types of cells were subcutaneously transplanted into the dorsal region of BALB/c mice.The samples were collected after 8 weeks,the osteogenesis was evaluated with histological staining.Results The MSC-BD and MSC-BC exhibited stronger proliferative capacity than MSC-BM.Compared with MSC-BD,MSC-BC showed significantly upregulated mRNA and protein expression levels of osteogenic markers(Runx2,Sp7),as well as enhanced ALP and Alizarin Red staining intensity.The HE staining of graft sections from MSC-BM and MSC-BD showed only fibrous connective tissue formation,while new bone formation was found with MSC-BC.The immunohistochemical staining results showed that MSC-BC had more osteocalcin-positive cells when compared with that in MSC-BD,while no significant positive cells were observed in MSC-BM.Conclusion The MSC-BC demonstrates superior proliferative capacity,higher stem cell proportions,and stronger osteogenic potential.