The Effects of Substrate Stiffness and Intermittent Hydrostatic Pressure during Osteogenic Differentiation

Substrate stiffness and mechanical stimuli are known to play important roles in regulating differentiation of stem cells. However, most studies on this have adopted only one of those two factors while other factor is kept constant. In this study we have varied those two factors simultaneously to investigate the combinational effects focusing on osteogenic differentiation of menschymal stem cells (MSCs). For this, we developed a novel bioreactor which enables us to engage multi-patterned intermittent hydrostatic pressure (M-IHP). It can engage independent hydrostatic pressure to each of three chambers with different magnitudes and frequencies. To have different stiffness of substrate we fabricated soft (~50 kPa) and rigid (~6.8 MPa) polydimethylsiloxane (PDMS) membranes using different curing agent ratio. The MSCs were seeded on those substrates with osteogenic medium. Twenty-four hours after seeding, three different magnitudes of IHP (5 kPa, 50 kPa, 500 kPa) were engaged independently to each chamber: for 3 days, 4 hours/day. The frequency was set as 2 min and 15 min for pressuring and resting, respectively. To evaluate the effects of substrate stiffness and mechanical stimuli, we conducted various biological assays such as measurements of DNA contents and alkaline phosphatase (ALP) activities, von Kossa/ALP staining. As results, we found that the effect of substrate stiffness is more observable than those of IHP magnitude in MSCs proliferation. The same result was found in case of differentiation.