ABSTRACT
Objective To investigate the synergic effects of substrate stiffness and topography on the morphology, proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs). Methods rBMSCs were cultured on polydimethylsiloxane (PDMS) substrates with different stiffness (3.5, 0.27 MPa) and ordered ridges and grooves (width 0.3 or 1.8 μm, depth 0.5 μm) or planar substrate. Inverted fluorescence microscope was used to observe the morphology of rBMSCs. CCK-8 reagent was used to detect the proliferation of rBMSCs. Alkaline phosphatase (ALP) kit was used to detect the ALP activity of rBMSCs. Immunofluorescence technique was used to detect the expression of osteocalcin (OCN) and collagen I (COL Ⅰ). QRT-PCR technique was used to detect the expression of Runx2 mRNA. Results rBMSCs cultured on substrate with 3.5 MPa stiffness, 0.3 μm ridge width, 0.3 μm groove width showed greater proliferation, spreading, cytoskeleton arrangement, and OCN and COL Ⅰ secretion, ALP activity, Runx2 mRNA expression were significantly increased as compared to cells cultured on other groups. Conclusions Substrate stiffness has an obvious influence on rBMSCs proliferation, while substrate stiffness and topography can synergistically promote the proliferation and osteogenic differentiation of rBMSCs. The research findings not only help to understand the biophysical factors in the pathogenesis of certain diseases (such as osteoporosis), but also provide a theoretical basis for developing new materials for bone tissue engineering.
ABSTRACT
Objective To study mechanical responses from mesenchymal stem cells (MSCs) under different mechanical stimulus duration, by measuring its elastic modulus and characterizing its stress fibers. Methods High resolution images of MSCs cytoskeleton in vitro were acquired by using atomic force microscope (AFM) and laser scanning confocal microscope (LSCM). AFM cantilever with micro-bead attached probe was used to perform force-distance curve experiment on MSCs at the approaching time of 0.1，0.5, 1, 5,10 s, respectively. The elastic modulus of MSCs at 300 nm indentation depth were measured and compared. Results The rat MSCs cytoskeleton presented an intensely organized network structure. The elastic modulus of rat MSCs varied obviously for different mechanical stimulus duration. The median and quartile (QR) of MSCs elastic modulus were 10.02 (QR=9.66),1.94 (QR=7.71),3.63 (QR=19.33),17.15(QR=35.13), 23.52 kPa(QR=34.87), with probe approaching time at 0.1，0.5, 1, 5,10 s, respectively. The MSCs elastic modulus showed the tendency of increasing with stimulus duration increasing, except for the extremely short stimulus (0.1 s). Conclusions Unlike inorganic elastomer, rat MSCs possess complete and flexible mechanical load-bearing structure and can respond actively to a relatively longer mechanical stimulation, with an increase of elastic modulus. These results may provide basic data for further tissue engineering researches on mechanical regulation of MSCs behavior.