Simulated Microgravity Inhibits The Proliferation And Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Microgravity is known to affect proliferation and osteogenic differentiation of marrowmesenchymal stem cells that may be a cause of reduction of bone formation duringspaceflight. The mechanisms for those effects may involve cell adhesion and cytoskeletonchanges that interfere with cell growth and differentiation signal transmission.In this study, mouse bone marrow-derived mesenchymal stem cells (BMSCs) werecultured using random positioning machine (RPM) to simulate microgravity (SMG). Theeffects of SMG on BMSCs proliferation, cytoskeleton, nucleus, cell adhesion, osteogenicdifferentiation, and key molecule expressions were detected.The growth curve was made to observe the cell proliferation. The directimmunofluorescence staining was used to stain the cytoskeleton including α-tubulin andmicrofilaments. The effects of cytoskeleton changes on nuclear structure and function werestudied by indirect immunofluorescence staining for LaminA and SUN1. The same methodwas used to detect the changes of the vinculin (VCL). The mechanism of SMG affectedBMSCs proliferation was studied by detecting the important signaling molecules ERK,p-ERK, AKT, p-AKT, p85α and p85β protein content. The key molecule focal adhesionkinase (FAK) was dected by combining the immunofluorescence staining with western blot.Under SMG and1G，the BMSCs were cultured using the mouse bone mesenchymalstem osteogenic differentiation medium to induce BMSCs to differentiate into osteoblasts.After osteogenic differentiating culture, alizarin red stain analysis, alkaline phosphatase (ALP)assay and RT-PCR for the gene osteocalcin (OC) were identified. The celldifferentiation-related signaling pathways important signaling molecules ERK, p-ERK,AKT, p-AKT, p85β were detected using western blot.Under SMG, BMSCs proliferation and osteogenic differentiation were inhibited.Meanwhile, BMSCs microfilament disrupted or abnormally distributed that resulted in cellshape changed and cell adhesion reduced. The expression of Focal Adhesion Kinase (FAK)that is an important element of focal adhesion decreased. Akt and ERK1/2phosphorylationlevels, as well as p85β decreased under SMG, all of them are key elements in cell growthand differentiation signal pathways.The above findings suggest that the inhibiting effects of SMG on BMSCs proliferationand osteogenic differentiation are relevant with disrupted cytoskeleton, decreased celladhesion that interfere with cell growth and osteogenic differentiation signal pathways.