**4. Conclusions**

The present paper demonstrates the use of an indentation rig within an X-ray CT scanner to visualise and characterise the cracking of bone material at an unprecedented level of detail for the first time. This has revealed a range of surface and sub-surface damage mechanisms in the bone structure, such as localised plastic deformation and the propagation of different types of cracking. The results

show that indentation coupled with X-ray CT has the potential to quantify the bone fracture mechanics associated with aging and disease process, such as osteoporosis and osteogenesis imperfecta.

The interactions of cracks with the existing pore networks and micro-cracks have been visualised in high resolution in 2D and 3D to understand the fracturing process of the bone structure. Further research is recommended on the 3D visualisation of these interactions to understand the fracture mechanics of bone. Time-lapse 3D imaging is a promising tool to better understand in-vivo indentation of bone to evaluate bone quality, the effects of normal development, and the impacts of disease processes and potential treatments.

The results illustrate that the 3D propagation of cracks and other deformation effects within the bone under the indentation load can be visualised with a resolution of ~2 μm by laboratory-based X-ray CT. This is sufficient to observe early crack formation while enabling us to see the microstructural features that might help prevent crack propagation. However, the current acquisition rate means that each scan took 52 h to acquire. This limited the number of frames that could be acquired in a time-lapse sequence. Furthermore, it introduces the risk of creep and relaxation affecting the interpretation of the results. Therefore, in-situ indentation tests under synchrotron X-ray CT imaging would be desirable to observe in-situ crack development as the indenter is progressively loaded. This would also allow the use of CT data to better understand the strain-strain features that occur during indentation in terms of the plastic hinging and crack propagation sub-surface. Such an approach will enable better interpretation and of indentation curves in terms of the effects of aging or disease. Nevertheless lab. X-ray CT is demonstrated to be a useful tool for characterising and quantifying sub-surface indentation damage for bone indentation testing.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1996-1944/11/12/2533/s1, Video S1: Animated sequences showing indentation damage in bone, Document S1: Materials and methods.

**Author Contributions:** Conceptualization, T.L., E.A., P.J.W. and W.S.; Funding Acquisition, P.J.W.; Investigation, T.L. and E.B.; Methodology, T.L. and E.B.; Project Administration, P.J.W.; Recourses, W.S.; Supervision, P.J.W.; Visualisation, T.L., E.B., and E.A.; Writing—Original Draft; E.A., W.S. and T.L.; Writing—Review & Editing; E.A., E.B., W.S., T.L., and P.J.W.

**Funding:** The X-ray kit was funded by the Higher education Funding Council for England UK (HEFCE). We would like to acknowledge the UK Research Partnership Investment Fund (UKRPIF) and the Engineering and Physical Sciences Research Council (ESPRC) for funding the Henry Moseley X-ray Imaging Facility through grants (EP/F007906/1, EP/F001452/1 and EP/M010619/1). P.J.W. also acknowledges support from the European Research Council (ERC) gran<sup>t</sup> No. 695638 CORREL-CT. E.B. would like to acknowledge the Canadian Natural Sciences and Engineering Research Council's support through the Postdoctoral Fellowship program.

**Acknowledgments:** The authors would like to thank Xun Zhang and Douglas Stauffer for advising on the use of the Hysitron indentation rig, and Andrew Chamberlain for providing the bone samples.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
