A review of in-situ mechanical testing combined with X-ray microfocus computed tomography: Application and current challenges for biological tissues

Biological tissues undergo physiological mechanical loading during their functioning in vivo. To properly respond to these mechanical signals, tissues have a highly complex microstructural organization. However, there is not yet sufficient knowledge about the link between their microstructural organization and their mechanical behaviour. Therefore, there is a need for methods to dynamically assess how the microstructure of biological tissues changes during mechanical loading. 4D-µCT is an imaging technique combining mechanical testing with X-ray microfocus computed tomography (µCT) imaging. It has been extensively used to visualize, at the micro-scale and in full 3D, the deformation of the microstructure of non-biological materials during mechanical loading. Additionally, postprocessing of the 4D-µCT datasets allowed 3D strain field calculations. This review aims to provide an overview of the current state of the art of the use of 4D-µCT specifically for the assessment of the mechanical behavior of biological tissue, and this both for mineralized and unmineralized tissues. We highlighted the advancements as well as the current limitations and challenges to overcome, such as the need for complex loading modes, the effect of X-rays on the mechanical behavior and the need to keep the samples hydrated during testing. We finally conclude with some future perspectives.