Kombinierte Rasterkraft-, Zugkraft und Fluoreszenzmikroskopie zur Analyse der Mechanotransduktion in Osteoblasten

Aim of this work was to analyse cellular processes within bone to gain knowledge about osteoporotic disease and thus the pathological loss of bone mass and material on a macroscopic scale. Investigation has been focused on the Mechanotransduction of osetoblastic-like cells using primarily atomic-force and traction-force microscopy as well as different methods of fluorescence microscopy. New aspects concerning the reception of mechanical loading signals on a cellular level have been found both, from a biological and a physical point of view. Beyond these finding the cellular mechanics under external loading conditions has been analysed and a new cellular model was postulated describing the mechanical behaviour of cells. To realise defined mechanical stimulation to single bone cells a three-dimensional force-microscope and a new illumination source for ratio imaging techniques using fluorescent dyes has been developed. The technical enhancement of the traction-force microscopy method revealed a deeper insight into the mechanical behaviour of cells under changing mechanical environments. Former investigations on the mechanotransduktion in osteoblasts have shown that specific intracellular signalling pathways become activated due to the application of fluid shear-stress and uniaxial stretch. Delayed intracellular calcium uptakes in stimulated cells have been observed through the activation of the Phospholipase C (PLC). However, in this study locally applied mechanical stress did not yield calcium signals and this observation was independent from stimulation parameters like frequency, amplitude and number of cycles. Neither the activation of PLC nor the activation of mechanosensitive ion-channels was observable. A special mechanosensor, responsible for intracellular calcium events, was hence not affected by local mechanical stimulation. But both, uniaxial stretch as well as local stimulation using the force-microscope provoked rapid changes of the cellular adhesion forces transmitted via focal adhesions to an elastic substrate. In addition to this locally applied forces did generate a slow increase or decrease of adhesion forces in few cases. Overall, these active cellular responses have been independent of the intracellular calcium concentration. These findings suggest that different biochemical signalling cascades are initiated by different kinds of mechanical loading and that potentially different mechanosensors become activated. Hence, the cellular reaction onto a mechanical stimulation depends on the type of mechanical loading. Furthermore the activation of signalling pathways is regulated by the frequency of the mechanical stimulation ? a phenomenon that has been observed during the stimulation of macroscopic bone fragments. To clarify the cellular behaviour upon different types of stimulation the transmission of force throughout the cells depth during local stimulations has been determined using differential traction-force microscopy. Whereas stretch events do influence the adhesion site of the cell globally, local forces do only have a local impact. This lets assume that the mechanosensor of the cell might be located somewhere near the cellular adhesion points or within the cytoskeleton that is connected to focal adhesions. This assumption is capable to explain the phenomenon of diverging cellular responses. Since the experimental results of this work are contradicting common cellular mechanical models a new combined mechanical model was postulated which is able to describe the observed cellular behaviour more properly. However, to carry out a deeper insight into the bone-remodeling process investigations on the mechanotransduction on a cellular level has to proceed. Hopefully the continuations of this interdisciplinary work will help to find new therapeutical treatments in order to decrease the speed of osteoporotic disease.