Volume 9 Supplement 9
Dynamic cultivation of human stem cells under physiological conditions
© Egger et al. 2015
Published: 14 December 2015
The development of stable tissue-engineered autologous bone grafts in the field of regenerative medicine is still a challenge. Perfusion bioreactors not only provide continuous nutrition supply and waste removal, but are also suitable for the controlled application of mechanical forces like fluid shear stress. Mechanical loading is known to cause mechanotransductive effects like the induction of differentiation, resulting in enhanced deposition of extracellular matrix .
In our study, we determined the optimal flow rate for the osteogenic differentiation of human adipose-derived mesenchymal stem cells (MSC) by applying fluid shear stress that mimics the physiological environment normally experienced by bone progenitor cells in vivo. For this, we first analyzed the porosity of cell substrates with nanofocus-computed tomography as well as their specific permeability at different flow rates. To investigate the effect of controlled application of physiologic fluid shear stress a flow rate of 0.3 ml/min was used to cultivate MSC in a self-developed perfusion bioreactor. Cells were seeded on a three-dimensional macro-porous zirconium dioxide ceramic scaffold (0.3·106 cells/scaffold) and cultivated in standard growth medium (GM)or osteogenic differentiation medium (ODM) under normoxic (21% O2) or hypoxic (5% O2) conditions for a period of 21 days. After cultivation cell viability was examined using MTT assay. Furthermore DAPI staining was used to evaluate cell distribution. Glucose consumption and lactate production were monitored and histological stainings (calcein, alicarin red, Von Kossa) were used to evaluate osteogenic differentiation.
Physiologic fluid shear stress together with physiologic oxygen conditions (5% O2) lead to higher cell viability. Furthermore the glucose metabolism is elevated under perfusion due to enhanced mass transfer. The application of fluid shear stress results in a stronger differentiation regarding matrix deposition even in standard growth medium without any osteoinductive supplements. These results underline the positive effects of dynamic cultivation and physiologic oxygen concentration which together mimic in vivo conditions. Consequently other factors like medium composition should be adjusted to be more physiologic and taken into consideration during a tissue engineering process to ensure a physiologic tissue maturation.
Part of this work was performed with financial support of the FFG BRIDGE project "3D Tissue".
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