Visual mapping of computational shear stresses implies mechanical control of cell proliferation and differentiation in bone tissue engineering cultures

The advantages of longitudinal monitoring techniques are getting more attention in various tissue engineering approaches. They provide consecutive information about one and the same sample over\u3cbr/\u3etime and as such may decrease sample numbers tremendously. These techniques also allow taking the actual environmental status of a tissue into account for predicting future development. Micro-computed\u3cbr/\u3etomography has been previously shown to be suitable to monitor mineralized extracellular matrix deposition in bone tissue engineering cultures. In this study, shear stresses (SS) acting on human mesenchymal stromal cells (hMSC) seeded on silk fibroin scaffolds in a flow perfusion bioreactor were calculated by computational fluid dynamics. Two different flow rates were investigated, mimicking expected loads on cells during early bone repair (0.001 m/s) and during bone remodeling (0.061 m/s), respectively. The threedimensional (3D) distribution of these stresses was then visually mapped to the distribution of the mineralized extracellular matrix deposited by the cells. SS values from 0.55–24 mPa were shown to promote osteogenic differentiation of hMSCs, whereas SS between 0.06 and 0.39 mPa were found to induce cell proliferation. Histological and biochemical analyses have confirmed these findings. In the future, these results may allow predicting the behavior of hMSC in 3D tissue culture. The non-destructive nature of this technique may even allow tight control and adaptation of the mechanical load during culture by taking the present status of the tissue into account

Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds:A combined experimental and computational approach

\u3cp\u3eMechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (v\u3csub\u3elow\u3c/sub\u3e = 0.001m/s and v\u3csub\u3ehigh\u3c/sub\u3e = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at v\u3csub\u3elow\u3c/sub\u3e compared to v\u3csub\u3ehigh\u3c/sub\u3e and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at v\u3csub\u3ehigh\u3c/sub\u3e whereas the amount of DNA was increased at v\u3csub\u3elow\u3c/sub\u3e. The biological response at v\u3csub\u3elow\u3c/sub\u3e might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (v\u3csub\u3elow\u3c/sub\u3e) or bone remodeling (v\u3csub\u3ehigh\u3c/sub\u3e). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.\u3c/p\u3

Micro-computed tomography based modeling of shear stresses in perfused regular and irregular scaffolds

Perfusion bioreactors are known to exert shear stresses on cultured cells, leading to cell differentiation and enhanced extracellular matrix deposition on scaffolds. The influence of the scaffold’s porous microstructure is investigated for a polycaprolactone (PCL) scaffold with a regular microarchitecture and a silk fibroin (SF) scaffold with an irregular network of interconnected pores. Their complex 3D geometries are imaged by micro-computed tomography and used in direct pore-level simulations of the entire scaffold–bioreactor system to numerically solve the governing mass and momentum conservation equations for fluid flow through porous media. The velocity field and wall shear stress distribution are determined for both scaffolds. The PCL scaffold exhibited an asymmetric distribution with peak and plateau, while the SF scaffold exhibited a homogenous distribution and conditioned the flow more efficiently than the PCL scaffold. The methodology guides the design and optimization of the scaffold geometry

Differences in fetal bovine serum affect the responsiveness of cells to mechanical loads

Nowadays, the end-point of a cell culture in bone tissue engineering\u3cbr/\u3e(BTE) is the acquisition of a well mineralized extracellular\u3cbr/\u3ematrix. The biological performance of BTE relies on evaluation of\u3cbr/\u3ethe cell capacity to proliferate and to produce extracellular matrix by\u3cbr/\u3equantification of gene expression and by histology or calcium\u3cbr/\u3equantification assays. Micro-computed tomography (micro-CT) allows\u3cbr/\u3emonitoring of BTE mineral constructs in a non-destructive\u3cbr/\u3emanner. Although fetal bovine serum (FBS) is commonly used as\u3cbr/\u3esupplement in cell cultures, its high composition variability between\u3cbr/\u3edifferent brands and batches leads to differences in the experimental\u3cbr/\u3eoutcomes. Nevertheless, only few studies have focused on a systematic\u3cbr/\u3einvestigation of the differences. While we have recently\u3cbr/\u3ereported the influence of FBS type on matrix mineralization under\u3cbr/\u3estatic culture conditions, it is still unknown how FBS affects cells in\u3cbr/\u3edynamic cultures. Different FBS types were used to differentiate\u3cbr/\u3ehuman mesenchymal stem cells down the osteogenic lineage under\u3cbr/\u3edynamic spinner-flask bioreactors. Opposite to static culture conditions,\u3cbr/\u3edifferences in FBS affected the responsiveness of cells to\u3cbr/\u3edifferentiate under mechanical loads. Although all FBS types upregulated\u3cbr/\u3ethe expression of bone-specific genes, differences in the\u3cbr/\u3eosteogenic differentiation stage were observed among the different\u3cbr/\u3eFBS. Accordingly, micro-CT analysis only showed mineral deposition\u3cbr/\u3efor cultures in an advanced differentiation stage.\u3cbr/\u3eThus the selection of the FBS type is crucial for the success in the\u3cbr/\u3eacquisition of BTE constructs. The combination of micro-CT with\u3cbr/\u3emolecular biology techniqueswill benefit efforts to optimize scaffolds\u3cbr/\u3edesign and cell culture conditions for scaling-up the BTE constructs