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). 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 cellsSummary
Introduction
Osteoporosis is a metabolic disease of the bone responsible for a loss of bone resistance and an increase in fracture risk. World Health Organization (WHO) estimations are about 6.3 millions of femoral neck fractures in the world by 2050. These estimations make osteoporosis a real problem in term of public health. Knowledge in biological tissues mechanical behaviour and its evolution with age are important for the design of diagnosis and therapeutic tools. From the mechanical aspect, bone resistance is dependent on bone density, bone architecture and bone tissue quality. If the importance of bone density and bone architecture has been well explored, the bone tissue quality still remains unstudied because of the lack of biomechanical tools suitable for testing bone at this microscopic dimension. Therefore the goal of this study is to estimate the osteoporotic cancellous bone tissue mechanical behaviour at its microscopic scale, using an approach coupling mechanical assays and digital reconstruction. The experimental study is based on cancellous bone tissue extracted from human femoral head. Forty 8mm diameters bone cylinders have been removed from femoral head explanted after a femoral neck fracture treated by arthroplasty. These cylinders have been submitted to a digitally controlled compressive trial. Before and after the trials, microscanner analyses with an 8 μm spatial resolution have been realized in order to determine the micro structural parameters. The cylinders have been rebuilt with the digital model-building in order to estimate the mechanical behaviour and the bone quality.Introduction
Materials and methods
