Osteogenic Differentiation Potential And Underlying Molecular Mechanisms Of Mesenchymal Stem Cells Under Simulated Microgravity

Microgravity is known to affect proliferation and osteogenic differentiation of marrow mesenchymal stem cells that may be a cause of reduction of bone formation during spaceflight.The mechanisms for those effects may involve cell adhesion and cytoskeleton changes that interfere with cell growth and differentiation signal transmission.In this study,C57BL/6 mouse bone marrow-derived mesenchymal stem cells(BMSCs)were cultured using three-dimensional two-axis clinostat to simulate microgravity(SMG).The effects of SMG on BMSCs cytoskeleton,cell adhesion,osteogenic differentiation,apoptosis and key molecule expressions were detected.Alizarin red staining was used to detect the osteogenic differentiation of bone marrow mesenchymal stem cells.Reverse transcription PCR was used to detect the m RNA expression of osteocalcin,osteopontin and osteonectin.The direct immunofluorescence staining was used to stain microfilaments to detect the changes of cytoskeleton under SMG.The effects of cytoskeleton changes on nuclear structure and function were studied by indirect immunofluorescence staining for Lamin A.The same method was used to detect the changes of the vinculin(VCL).Annexin-V PI double staining was used to detect the occurrence of apoptosis in simulated microgravity and osteogenic differentiation.Western blotting was used to detect the expression levels of key signal molecules in MAPK,PI3 K / AKT and mTOR signaling pathway such as Erk,p-Erk,p85?,Akt,p-Akt,S6K1 and Rho A.The results show that osteogenic differentiation inducing medium can efficiently induce osteogenic differentiation of mesenchymal stem cells,while simulated microgravity inhibits osteogenic differentiation of bone marrow mesenchymal stem cells and MAPK,PI3 K / AKT and mTOR signaling pathways involve in this process.The structure of cytoskeleton under microgravity conditions changed significantly,F-actin becoming disorganized,shorter and abnormal distributed and the amount of Vinculin reduced.The expression of vinculin decreased at the same time.Simulated microgravity can promote the occurrence of late cell apoptosis.The above findings suggest that the inhibiting effects of SMG on BMSCs osteogenic differentiation are relevant with disrupted cytoskeleton,decreased cell adhesion that interfere with cell growth and osteogenic differentiation signal pathways.