Crack Propagation in Bone Captured with In Situ Mechanical Testing During AFM
By O. Katsamenis, T. Boughen, P. J. Thurner, University of Southampton, UK
Bone, like all tissues, is built from structural elements starting at the nanometer scale. The generally complex and hierarchical arrangement of these basic elements into progressively larger structural features renders bone an anisotropic and anatomically distinct material adapted to specific loads and loading cases. Due to the hierarchical structure and complexity of bone, the uncovering of structure-function relationships, i.e. the origin of material properties such as strength, toughness, and fatigue resistance, is usually a non-trivial task. Atomic force microscopy (AFM) offers an approach to overcome some of these difficulties. Because AFM allows for imaging in ambient – even hydrated conditions – it is feasible to perform in situ micro-mechanical testing experiments while conducting imaging. Here we present first data obtained from a micro-tensile testing apparatus, demonstrating the power of this technique.
Materials and Methods
Ovine and bovine femora were sourced from a meat wholesaler. Cortical bone samples about 0.5mm x 0.5mm in cross section and 25mm in length were prepared using a butchers bandsaw and a low-speed saw with a diamond wafering blade. The long axis of these samples generally coincided with the long axis of the whole bones. The sample surfaces where then polished using increasing finer grades of silicon carbide paper as well as diamond suspension. Subsequently, the samples were notched in the vicinity of their center using the low-speed saw and a razor blade. Both sample ends were embedded into epoxy resin using a mold to create circular ends that fit the receptacles of the in situ mechanical testing device (NanoRack™ Stretching Stage). Samples were progressively loaded using the NanoRack device and images were acquired using an MFP-3D™ AFM in AC (tapping) and contact mode.
Results and Discussion
The stress concentration created by the sharp notch allowed for controlled crack initiation at its root. This made it feasible to successfully capture crack propagation over a number of loading steps in areas of up to 50µm x 50µm. Cracks were generally deflected from their initial path, towards the long beam axis (Figure 1). This is to be expected as the underlying bone structure consists of either plexiform bone lamellae or of osteonal lamellae, which are bonded through a presumably softer layer made to a large part of noncollagenous proteins.
In addition to crack deflection, we also detected cracks running along the lamellar boundaries eventually breaking though a lamella (Figure 2A).
The complex correlation of bone micro- and nanostructure is evident, especially after more elaborate polishing of the sample surfaces. Figure 2B shows a sample polished to a surface roughness of about 25nm. Here the lamellar organization is clearly visible and the propagation path of the primary stable crack, together with secondary micro- or nano-cracks, is uncovered.
The data acquired so far show the feasibility of the NanoRack device to image bone samples under load and during crack propagation. In an ongoing study, we are currently progressing further towards correlating bone nano- and microstructure as well as composition with crack propagation. So far it seems that distinct microstructural features and phases of the nanocomposite bone are controlling crack propagation and hence bone toughness. Further research is now being conducted to clarify this, which will undoubtedly aid in establishing improved micromechanical models of bone and further our understanding of bone as a hierarchical composite material.
MFP-3D and NanoRack are trademarks of Asylum Research.
Figure 1: AFM height image showing a crack propagating in cortical ovine bone deflecting left and right.
Figure 2: A) AFM height (left) and amplitude (right) images showing a crack in cortical bovine bone deflecting and eventually breaking through a lamella. B) Bone lamellae (dashed lines) can be clearly seen in a sample with improved surface finish. The stable primary crack (white arrows) runs through the lamellar structure in a zig-zag manner increasing energy dissipation. Toughness is further enhanced by the opening of secondary micro- and nano-cracks (black arrows).
The MFP-3D NanoRack Sample Stretching Stage is a high-strain, high-travel manual stretching stage that provides two-axis control of tensile loaded samples with a maximum load of 80N.