Abstract
Summary
Shear stress and hydrostatic effects on the hMSCs early mechano gene response were similar. For the same magnitude gene response, the hydrostatic compression (1.5×105 Pascal) is a 200000 times greater than the force exerted by shear stress (0.7 Pascal).
Introduction
In the lab, a perfusion bioreactor designed to automate the production of bone constructs was developed. The proof of concept was established in a large animal model of clinical relevance. The cells perfused in the bioreactor are likely to perceive 2 types of stresses: shear stress and hydrostatic pressure. Optimization of this bioreactor implies a better understanding of the effects of these forces on the cells in order to have better proliferation and differentiation. An understanding of the response of one cell layer submit to various strength is relevant. The primary objective of this study was to test the hypothesis that hMSCs have the fundamental ability to distinguish between different types of mechanical signals as evidenced by distinct gene expression. The effect of shear stress on one cell layer cultures of hMSCs will be evaluated using a commercially available system called Ibidi. For the hydrostatic pressure as there is no commercial device available, our group has developed a prototype capable of delivering a well-defined mechanical loading to cells in culture.
Validation of the techniques: In order to validate the systems (shear stress and cyclic pressure apparatus) used in this study, we have used an osteocytes-like cell line, MLO-Y4. When stimulated by a 30 minutes PFF at 7 dyn/cm2 or hydrostatic compression at 1.5 bar, cells responded by producing NO in the culture media
NO release after mechanical stimulation of hMSCs: hMSCs were subjected to increased PFF (7 to 42 dyn/cm2) for 30 minutes. This stimulation resulted in an increased release of NO in the media compared to non-stimulated cells (p<0.05). Interestingly the level of NO was maximal at 7 dyn/cm2 and decreased with higher flow rate. Similar observation was made after hMSCs stimulation by hydrostatic pressure for 30 minutes: a peak of NO release at 1.5 bar was observed
Early gene expression of known mechano-sensitive genes: Gene expression analysis immediately after stimulation (PFF or hydrostatic compression) was performed on a range of known mechano-sensitive genes: NOS2, PTGS2, PTGES, IER3 and EGR1. Immediately after stimulation by PFF at 7 dyn/cm2 or hydrostatic pressure at 1.5 bars, the expression of all the genes of interest appear to be up regulated in stimulated cells
Conclusions
In the present study, hMSCs cells responses to two different treatments, shear stress and hydrostatic pressure, were monitored in parallel to study the difference in sensitivity to both mechanical stresses. Both systems used ensure a stable and reproducible strain condition in a well-controlled environment. We demonstrated that the shear stress and the hydrostatic pressure effects on the hMSCs were similar. We note that, for the same magnitude response, force exerted by the hydrostatic compression (1.5×105 Pascal) on the cells is a 200000 times greater than the force exerted by shear stress (0.7 Pascal). This shows that the direct effect of hydrostatic compression on the hMSCs is negligible compared to the shear stress.
