This protocol enables the observation of the microstructure of the whole human femur as it deforms under load and fracture. By imaging the whole femur with microstructural resolution, this protocol makes it possible to study how the different trabecular cortical compartment of the bone, synergically determine its capacity to withstand the load. Advancing our understanding of the bone fracture mechanism can inform advanced diagnostic methods for osteoporosis.
This protocol has been developed at the Australian Synchrotron, adapted for use in a commercial, large volume micro-CT scanner at University and applied to study different anatomical region, including the knee and shoulder. In principle, it can be extended to materials of similar size to whole human bones and joints. This study necessitates expertise in several disciplines, including imaging, solid mechanics, and computational modeling in biomechanics.
To begin, using a clinical CT scanner, scan the femur specimen with a slice thickness and an in-plane pixel size of approximately 0.5 to 0.7 millimeters. Alongside the specimen, scan a CT densitometry calibration phantom, with five known concentrations of dye potassium hydrogen phosphate. Next, segment the bone geometry from the clinical CT images, calibrate gray levels in the images to corresponding bone density values, and Young's modulus, using the density to elastic modulus relationship.
After creating a mesh of the segmented geometry, import the mesh into the finite element software. Fully constrained three to six millimeter deep, the distal end of the model, then replicate a single-leg stance loading configuration by applying a nominal force of 1000 Newton, abducted by eight degrees from the femoral shaft axis in the coronal plane and passing through the center of the femoral head. Solve the finite element model using the built-in PCG solver.
Then by executing the indicated commands, generate an element table containing the first and third principle strain components at the element's centroid. Next, execute the indicated command to calculate the strain ratio between the first and third principle strain components in the model and the bone yield strain, intention and compression. Scale the nominal force by the peak strain ratio, intention and compression, and discard the biggest of the two to estimate the fracture load.
Position a testing rig on the micro-CT rotation stage with the specimen in the reference unloaded condition and start the micro-CT scan. Repeat imaging twice for the unloaded condition and unwind the cable after scanning. Apply the force increment by manually actuating the screw jack mechanism at a constant rate of approximately one second per round, and perform the micro-CT imaging.
Repeat the force increment until the specimen fractures, as indicated by a sudden drop in the reaction force. Perform micro-CT imaging of the fractured specimen. Then visualize the sequence of projection images at various load steps.
Subsample the micro-CT images by four, to reduce the computation time. Rigidly co-register, in space, the images of the specimen under load, with those in the unloaded reference condition, using the distal diaphysis as the target of the co-registration. Create surface three dimensional models for visualization after binarizing the micro-CT images.
Determine the displacement in the images over a grid 50 pixels in size, using bone DVC. Then determine the strain tensor, by converting the grid into a finite element model. Apply the calculated displacement at the nodes and solve the model.
Next, using cubic interpolation, with the inter P three function in Mac-Lab, re-sample the displacement and strain volumes to match the size of the original size of micro-CT images. Visualize displacements, strain and microstructural images for large volume visualization and animation. For bone deformation analysis, display the permanent deformation of the bone by overlaying the images obtained in unloaded conditions and after the fracture.
Then display the progressive micro structural deformation of the bone by overlaying the three dimensional models in unloaded conditions, at increasing load levels, and post fracture. Display the strain of the bone at the fracture location. Finally, using descriptive statistics and regression methods, analyze the deformation energy, stiffness, and displacement.
Micro-CT imaging and concomitant mechanical tests allow for observing femoral neck fractures. An animation showed that the femoral head progressively rotated medially, while moving distally up to fracture. The head curvature flattened under the socket, where local cortical instability, but no instability of the underlying trabecular volume, was observed.
Fracture onset occurs by bending the cortex, either progressing along the main compressive trabecular group, or by shear at about 45 degrees from the main principle compressive, trabecular axis. Strain exceeded bone yield, once the four succeeded 50%of the expected fracture load, reaching eight to 16%compression before fracture. Permanent deformation was observed in the head region under peak compression.
Failure occurred under a complex strain state, showing compression, tension and shear strain. The deformation energy was a linear function of the displacement up to fracture, showing a stable fracture behavior. A critical aspect for replicating the protocol is obtaining the load step increment, which is important for controlling the number of load steps required to cause a fracture to displacement and planning of the experiment.
Obtaining good quality images is also important for a meaningful analysis of the data. The belief that elastic instability that demands the steep increase of fracture incidents above 60 years of age focused the case of research in fragility prevention on cortical thickness. The elastically stable fracture behavior demonstrated by this protocol, in lightly osteoporotic bones, shifts the current focus to cortical and trabecular interactions.
This procedure can advance considerative models of bone mechanics, inform the diagnosis of fragility and the design of implantable devices.
