The human body constantly experiences mechanical forces, including gravity, tensile strains and compressive loads. It is now widely recognized that tissues grow and remodel in response to these environmental cues. The typical example consists in the morphological adaptations of the skeleton to mechanical changes. Physical loading positively influences bone density and stiffness whereas they are reduced under mechanical unloading. This skeletal adaptability actually reflects the intrinsic potential of bone cells to sense and respond to mechanical stress. Bones comprise three types of cells, namely bone-forming osteoblasts, bone-resorbing osteoclasts and regulatory osteocytes. All three cell types were shown to be sensitive and responsive to mechanical forces. Basically, physical stress induces the up-regulation of osteocytes and osteoblasts whereas it inhibits osteoclast activity. Nevertheless, the exact molecular mechanisms through which mechanical forces modulate bone cell physiology are poorly understood. Yet, it becomes a question of primordial importance due to the growing incidence of osteoporosis. Osteoporosis is the most common bone disorder which is characterized by bone loss, weakening and subsequent fracture. Nowadays, there are still no efficient preventive or therapeutic strategies. In addition to a hormonal cause, the onset of the disease correlates with reduced physical activity. Mechanical forces play thus an important role in maintaining bone tissue homeostasis. Therefore, understanding how bone cells perceive and react to mechanical (un)loading could lead to the development of new therapeutic drugs for osteoporosis. In this proposal, bone-cell mechano-biology will be thoroughly investigated at the single-cell level by means of emergent micro- and nanotechnologies. Mechano-biology is an interdisciplinary field which aims at unravelling how cells detect mechanical forces and transduce them into biochemical signals, which further elicit a cellular response. Practically, osteocytes, osteoblasts and osteoclasts will be grown as single-cell arrays and homo- and heterogeneous interconnecting cell networks on micropatterned substrates. These cell platforms will then be used for conducting 3 types of mechano-biological assays: (1) mechanical stimulation by AFM indentation, (2) mechanical loading through microfluidics and (3) mechanical unloading via simulated microgravity. The effects of mechanical (un)loading will be assessed through the monitoring of fluorescently-labelled mechano-signalling reporters by means of super-resolution fluorescence microscopy. Experiments on single-cell arrays will deepen the knowledge on the mechano-sensitivity and -responsiveness of each cell type. Additionally, the project places a great emphasis on the study of cell-cell communication between bone cells exposed to mechanical (un)loading. This will be achieved through the use of homo- and heterogeneous interconnecting cell networks. These results will shed light on the cell-cell communication under mechanical stress and thereby on the cooperative response of bones cells to mechanical stimuli. Altogether, the insights gained in this research will contribute to the overall understanding of bone-cell mechano-biology and may potentially identify new therapeutic targets. The developed assays may also be implemented in companies performing drug screening. The project will be realized at the NANO-VUB lab in close collaboration with the NANO-EPFL lab, under the supervision of Prof. Ronnie Willaert (promoter) and Prof. Giovanni Dietler (copromoter), respectively. These two labs compose the recently founded International Joint Research Group (IJRG) VUB-EPFL NanoBiotechnology & NanoMedicine (NANO). Each one in specialized in specific techniques that will be exploited in this project. The NANO-VUB is experienced in micropatterning and microfluidics whilst the NANO-EPFL has expertise in AFM indentation and simulated microgravity. Optimal completion of the project will thus require some mobility although the NANO-VUB lab will be the main place of research.