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Actin filament, one type of cytoskeletons, plays a central role in mediating cellular in responses to mechanical loading. Many mechanorgulation studies are restricted to 2D models using isolated cells or monolayer cultures, even though it is know that cells behave differently in term of cell morphology, cell matrix adhesion composition and matrix mediated force transmission when they are in 3D configuration. This current study investigates the temporal change of actin network of hMSCs entrapped in 3D collagen construct upon cyclic compression. Human bone marrow mesenchymal stem cells were encapsulated in cylindrical collagen construct. A micromanipulator based loading device coupled to fluorescent microscope was used to deliver compression loading to the construct with 10% strain at 1Hz for different period of time. Rhodamine phalloidin was used to stain for the actin filament network to hMSC in the construct at different time points postcompression. An optimized loading protocol with 5hrs of continuous loading was delivered. Actin network concentrated at the cell periphery of cells exhibiting round morphology was observed immediately while elongated and polarized actin network was found after 24 hours. Detailed characterization of actin filament organization and their association with cell-matrix interaction molecules are warrented before the mechanisms of compression-induced hMSC alignment can be delineated.