Single Cell Microfluidic Imaging for Spatial Mapping and Quantification of Gene Expression in an in vivo Model of Bone Adaptation
Mechanical loading is perhaps the most important physiologic factor regulating bone mass and shape. Age related bone loss and consequent osteoporosis have been attributed, at least in part, to the decrease in mechanical usage of the skeleton. Conversely it has been demonstrated that mechanical overloading results in enhanced bone formation, thus a detailed understanding of the biochemical processes governing load regulated bone formation could lead to the identification of molecular targets for the prevention and treatment of diseases such as osteoporosis. Osteocytes, embedded deeply within the mineralized bone matrix have been shown to be the key mediators of load induced bone remodeling, however, owing to their inaccessible location the molecular mechanisms by which mechanical forces are translated into anabolic signals remain poorly understood. In vivo studies have attempted to identify load regulated genes expressed by osteocytes, however, their interpretation is limited by the fact that they report the average effect of tens of thousands of cells. Furthermore, depending on their location within the diverse micro-architectures of bone these cells are exposed to varying magnitudes of mechanical strain. Therefore the immediate goal of the proposed research program is to develop a novel experimental approach to map the location of individual osteocytes within bone, quantify their micro-mechanical environment and analyze their individual patterns of gene expression exploiting microfluidic technology. Once established this single cell-resolution microfluidic imaging approach will be used to spatially map the expression of selected genes in loaded mouse bone. The availability of this approach will provide a framework, which can be extended to quantify, in a high throughput manner, the individual behavior of tens of thousands of cells. This will provide a generally applicable methodology, which will enable cells and their gene expression to be mapped in three dimensions thus making it possible to investigate how cellular processes are choreographed in space and time in whole organs.
Keywords: Mechanical Systems Biology, Bone Remodeling, Single Cell Gene Expression, Imaging, Microfluidicsback