Caractérisation du réseau lacuno-canaliculaire osseux par microscopie optique

Abstract : This thesis focuses on the study of bone lacuno-canalicular network (LCN) using different optical microscopy techniques. The LCN is the porosity network in the bone matrix where the cellular network lie. It is formed of dendritic cells: the osteocytes which are connected to each other. Although it plays a major role in the formation, remodeling and maintenance of biomechanical properties of bone, only little is known about this network as a whole. This can be explain by the difficult characterization of such a dense and complex network with sub-micron resolution and scales up to the entire organ. In this work we have sought to improve the characterization of the LCN using two approaches: the development of a method to analyse the network on large scale using confocal microscopy on one hand, and the assessment of the potential of non linear microscopy technique to study the LCN on the other hand.First, we have developed a protocol from sample preparation to image processing and data analysis to optimize confocal imaging of bone tissue in order to obtain a quantitative large scale analysis of the network. Preliminary results show a wide variation of network parameters at all scales revealing its complexity. This analysis was then used in order to assess changes in the LCN across an entire mice femur.Secondly, we study the potential of the non-linear optical microscopies especially the third harmonic generation (THG) microscopy for imaging and the study of the LCN. Initially, we demonstrated the ability to visualise the LCN without fluorescent labelling using THG microscopy. From this proof of concept we explained the origin of the different ThG microscopy contrasts observed in bone tissue: a signal from the porosities allowing to visualize the network and a structured background signal generated at the interfaces between collagen fibrils. We also assess the possibilities of combinations between different non-linear signals, mainly THG and SHG (second harmonic generation) that can simultaneously image the network and the collagen matrix respectively. A correlation between the network structure and collagen organization has been established using the visualization of these two signals over large scales. Finally quantitative parameters of the LCN were obtained from THG images and applied to study the effects of microgravity on the cellular network structure.
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Rachel Genthial. Caractérisation du réseau lacuno-canaliculaire osseux par microscopie optique. Biophysique [physics.bio-ph]. Université Grenoble Alpes, 2016. Français. ⟨NNT : 2016GREAY058⟩. ⟨tel-01589674⟩

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