Determining Which Hydrostatic Pressure Regimes Promote Osteogenesis in Human Mesenchymal Stem Cells.

Abstract

Compressive loading of bone causes hydrostatic pressure changes which have been proposed as an osteogenic differentiation stimulus for mesenchymal stem cells (hMSCs). We hypothesised that hMSCs are adapted to differentiate only in response to cyclic hydrostatic pressures above critical thresholds of magnitude and frequency which correspond to physiological levels of anabolic bone loading. Using a pneumatic-hydrostatic bioreactor, we applied hydrostatic pressure regimes to human hMSCs in 3D collagen hydrogel cultures for 1h/day over 28days to determine which levels of pressure and frequency stimulated osteogenesis in vitro. Stimulation of the 3D cultures with 0-280kPa cyclic hydrostatic pressure at 1Hz resulted in up to 75% mineralisation in the hydrogel (without exogenous growth factors), whilst static culture or variations of the regime with either constant high pressure (280kPa, 0Hz), low-frequency (0.05Hz, 280kPa) or low-magnitude (70kPa, 1Hz) stimulation had no osteogenic effects (< 2% mineralisation). Nuclear translocation of YAP was observed following cyclic hydrostatic pressure in mature MLO-A5 osteoblasts but not in hMSCs, suggesting that cyclic hydrostatic pressure activates different mechanotransduction pathways in undifferentiated stem cells and committed osteoblasts. Hydrostatic pressure is a potent stimulus for differentiating MSC into highly active osteoblasts and may therefore be a versatile tool for translational cell engineering. We have demonstrated that there are minimum levels of force and frequency needed to trigger osteogenesis, i.e. a pressure 'switch', which corresponds to the physiological forces experienced by cells in their native mesenchymal niche. The mechanotransduction mechanisms underpinning these effects are the subject of further study.