Nanoemulsion-Eicosapentaenoic Acid Enhanced Alkaline Phosphatase, Calcium Contents, and Surface Molecules Expression During Osteogenesis Using Mouse Multipotent Bone Marrow Stromal Cells

Abstract

Long-chain eicosapentaenoic acid (EPA) is found mainly in marine oils, and has wide ranging biological effects. In this study, we evaluated the effect of nanoemulsion-EPA on the osteogenic differentiation of mouse mesenchymal stem cells (D1 cells). D1 cells were cultured in osteogenic differentiation medium (ODM) for 6 days and treated with nanoemulsion-EPA for 2 days and then subjected to various tests; MTT assays, alizarin red S staining, alkaline phosphatase (ALP) activity determinations, calcium contents analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), confocal microscopy, and flow cytometric analysis. D1 cells were found to differentiate into osteoblasts in the presence of osteogenic differentiation medium, as determined by positive alizarin red S staining, increased alkaline phosphatase activity and calcium contents, osteocalcin, osteopontin and Runx2 mRNA expressions, and by positive immunofluorescence staining against CD44 or CD51. Furthermore, osteogenic differentiation was enhanced when cells were treated with nanoemulsion-EPA, as determined by alizarin red S staining, elevated ALP activity, calcium contents and osteocalcin and Runx2 mRNA expression, and immunofluorescence staining and flow cytometric analysis for CD44. Furthermore, pretreatment with p38 inhibitor prior to nanoemulsion-EPA decreased alizarin red S staining and ALP activity. These findings suggest that nanoemulsion-EPA enhances osteogenic differentiation via the p38 pathway when administered to mouse mesenchymal stem cells in osteogenic differentiation medium.