Most of our knowledge of bone cell physiology is derived from experiments carried out in vitro on polystyrene substrates. However, these traditional monolayer cell cultures do not reproduce the complex and dynamic 3-dimensional (3D) environment experienced by cells in vivo. Thus, there is a growing interest in the use of 3D culture systems as tools for understanding bone biology. These in vitro engineered systems, less complex than in vivo models, should ultimately recapitulate and control the main biophysical, biochemical and biomechanical cues that define the in vivo bone environment, while allowing their monitoring. This review focuses on state of the art and the current advances in the development of 3D culture systems for bone biology research. It describes more specifically advantages related to the use of such systems, and details main characteristics and challenges associated with its three main components, i.e. scaffold, cells and perfusion bioreactor systems. Finally, future challenges for non-invasive imaging technologies are addressed.