A correlative X-ray and electron microscopy framework for 3D bone imaging and its application to bone development and disease in human and animal tissue

Osteocytes are stellate cells which form a network within hard bone matrix and play a key role for bone adaptation during development, ageing and bone diseases including osteoporosis. The mechanisms by which osteocytes sense loading and transmit signals to regulate remodelling are not well understood. Detailed knowledge of the three-dimensional (3D) structure of the osteocyte network and the surrounding lacuno-canalicular network is essential to elucidate these mechanisms. 3D imaging on cellular and sub-cellular scales will allow quantitative hallmarks of health and disease to be derived and will lead to improved computational models of bone mechanotransduction. Until now the location of the cells within calcified bone matrix and the submicrometre dimensions of the networks have posed challenges for 3D imaging. Serial block-face scanning electron microscopy (SBF SEM) is a novel imaging technique which produces high resolution 3D data of the osteocyte and lacuno-canalicular networks (ON&LCN) simultaneously. The objective of this project is to develop a correlative X-ray and SBF SEM (CXEM) workflow for mammalian bone tissue, providing quantitative data, which will enable realistic computational modelling of bone mechanobiology. This project aims to develop protocols for SBF SEM sample preparation and imaging and to combine SBF SEM with X-ray micro computed tomography in a correlative workflow which allows derivation of established and novel quantitative measures of the ON&LCN across length scales in relevant tissue volumes. CXEM imaging and image analysis workflows are applied to juvenile and adult murine bone tissue and to bone tissue from osteoporotic (OP) and osteoarthritic (OA) human donors. CXEM applied to juvenile and adult mouse tibia has produced data on osteocyte density, porosity, and cell measures including volume and number of processes. Pericellular space volume and width were quantified in 3D for the first time. The results show that values for pericellular space volume used in previous studies have been overestimated and that the width of the pericellular space is more irregular than values derived from 2D imaging methods and used in computational models, possibly leading to the underestimation of peak strain sensed by osteocytes. Tissue from OP and OA donors was imaged and analysed using CXEM. Osteocyte number density is 37% of lacunar number density and osteocyte porosity is 30% of lacunar porosity in these samples. These findings illustrate that using lacunar number density and porosity measures to represent osteocyte measures in diseased human bone tissue is misleading. 93% of lacunae contained a cell showing ultrastructural changes indicative of deteriorating health or cell death. This indicates that the cell network, crucial for mechanobiology and bone homeostasis, is compromised in OA and OP tissue, although not in significantly different ways. CXEM enables imaging of the hard and soft components of bone simultaneously, at high resolution and in 3D, producing unique quantitative measures. CXEM assessment of the ON&LCN in health and disease paves the way for the study of mechanotransduction mechanisms by computational models based on accurate geometries. This will lead to the identification of relevant features of healthy and diseased bone at the cell level, which could serve as targets for the diagnosis and treatment of bone-related diseases, impacting significantly on public health