Effects of icariin-loaded microsphere-three-dimensional scaffold on osteogenic differentiation of rabbit bone marrow mesenchymal stem cells

Jin Dongsheng; Zhao Zhanghong; Zhu Ziyin; Zhang Sen; Sun Zuyan; Deng Jiang

doi:10.12307/2026.591

Abstract：BACKGROUND:Previous studies have shown that icariin has good potential in promoting osteoblastic differentiation;however,it has drawbacks such as easy drug loss,degradation,and difficulty in sustaining its effects. OBJECTIVE:To prepare a three-dimensional scaffold loaded with icariin microspheres and further explore the optimal drug concentration. METHODS:Rabbit bone marrow mesenchymal stem cells were isolated and cultured,and identified by detecting cell phenotype and multidirectional differentiation ability.Icariin-loaded microspheres were prepared by double emulsion solvent evaporation method,added into silk fibroin/chitosan/nanohydroxyapatite mixed solution.Vacuum freeze-drying chemical cross-linking method was used to prepare drug-loaded scaffolds containing different concentrations(0,0.1,1,10,50,and 100 μmol/L)of icariin.The effects of sustained-release microsphere three-dimensional scaffolds containing different concentrations of icariin on the proliferation activity,migration,and osteogenic differentiation of bone marrow mesenchymal stem cells were detected by CCK-8 assay,live/dead cell staining,scratch wound assay,Alizarin red staining,alkaline phosphatase staining,and western blot analysis. RESULTS AND CONCLUSION:(1)The CCK-8 assay and live/dead cell staining experiments indicated that the icariin-loaded sustained-release three-dimensional scaffold promoted the proliferation of bone marrow mesenchymal stem cells and enhanced cell viability,with the 10 μmol/L group showing the strongest effect(P<0.05).(2)Alizarin red staining and alkaline phosphatase staining results suggested that the 10 μmol/L group exhibited higher in vitro osteogenic mineralization compared to the control group(P<0.05).(3)Western blot analysis demonstrated that icariin promoted the expression of type I collagen,osteocalcin,Runt-related transcription factor 2,and vascular endothelial growth factor protein,with the 10 μmol/L group showing significantly upregulated osteogenesis-related gene expression(P<0.05).These findings confirm that the optimal drug-loading sustained-release concentration of icariin is determined to be 10 μmol/L.