Investigation of the mechano-sensitivity of osteoblasts by combined atomic-force fluorescence microscopy and optical flow stress analysis

我们的目标是分析骨细胞(成骨细胞)对机械刺激的接受，这是理解骨质疏松症的关键过程。我们使用原子力仪器和荧光显微镜相结合的方法来研究细胞在明确定义的机械力作用下的反应。通过一种基板变形的有限元分析，分析了单元对基板施加的力，无论是自加的力还是由原子力显微镜传递通过基板的力。细胞在机械刺激下的任何反应都涉及细胞内游离钙浓度的变化。因此，我们通过荧光成像检测细胞内游离钙浓度的变化，作为细胞反应的明确指标，即细胞是否对特定刺激做出反应。我们的中心目标是确定最佳的刺激条件(如刺激频率)，并在细胞中定位机械传感器。我们将分析刺激所需的力和能量，并将它们与细胞的张力进行比较。此外，我们还将探讨刺激后引起细胞内游离钙升高的钙离子的来源，并分析相邻细胞之间的通讯，以帮助理解整个复杂的机械接受过程。我们将对不同的细胞系进行比较研究，以确定成骨细胞在接受机械刺激中的特定功能。

Our goal is to analyse the reception of mechanical stimuli by bone cells (osteoblasts) which is a key process for the understanding of osteoporosis. We use a combination of an atomic force apparatus with a fluorescence microscope to investigate the reaction of cells onto application of well defined mechanical forces. Forces applied by the cells to the substrate, either self applied or forces transmitted through the cell by the AFM, are analysed by a type of finite element analysis of the substrate distortion. Any reactions of the cell upon mechanical stimulation involve variations of the free intracellular calcium concentration. Therefore, we detect changes in the intracellular free calcium concentration by fluorescent imaging as an unambiguous indicator for a cellular reaction, i.e. whether a cell reacts to a particular stimulus or not. Our central goals are to determine the optimum stimulation conditions (e.g. stimulation frequency) and to locate the mechanosensor in the cells. We will analyse the forces and energies required for stimulation and compare them to the tension forces of the cell. Moreover, we will investigate the origin of the calcium ions causing the intracellular free calcium increase after stimulation and analyse the communication of adjacent cells in order to contribute to the understanding of the whole complex mechanoreception process. We will perform comparative studies on different cell lines to work out the specific function of osteoblasts in the reception of mechanical stimuli.