A non-invasive in vitro technique for the three-dimensional quantification of microdamage in trabecular bone

Abstract

An accurate analysis and quantification of microdamage is critical to understand how microdamage affects the mechanics and biology of bone fragility. In this study we demonstrate the development and validation of a novel in vitro micro-computed tomography (microCT) method that employs lead-uranyl acetate as a radio-opaque contrast agent for automated quantification of microdamage in trabecular bone. Human trabecular bone cores were extracted from the femoral neck, scanned via microCT, loaded in unconfined compression to a range of apparent strains (0.5% to 2.25%), stained in lead-uranyl acetate, and subsequently re-scanned via microCT. An investigation of the regions containing microdamage using the backscatter mode of a scanning electron microscope (BSEM) showed that the lead-uranyl sulfide complex was an effective contrast agent for microdamage in bone. Damaged volume fraction (DV/BV), as determined by microCT, increased exponentially with respect to applied strains and proportionately to mechanically determined modulus reduction (p<0.001). Furthermore, the formation of microdamage was observed to occur before any apparent stiffness loss, suggesting that the localized tissue yielding occurs prior to the structural yielding of trabecular bone. This non-invasive in vitro technique for the detection of microdamage using microCT may serve as a valuable complement to existing morphometric analyses of bone.

Figure 1

MicroCT detection of increased DV/BV with increasing apparent strains. The left column shows a 3D reconstruction from a central volumetric cube of the cancellous bone cores loaded to respective strains. The right column shows a longitudinal cross-section along the axis of loading from the corresponding the respective volumes on the left. While DV/BV was computed based on the 4mm³ center cubes, the 3D views shown here are based on 1.5mm³ cubes for the ease of visualization. The darker shade of gray indicates regions stained by lead-uranyl sulfide as detected by microCT, while the lighter shade show unlabeled bone.

Figure 2

Back-scattered SEM image showing the presence of lead-uranyl acetate around the long crack (white arrow) and in the surrounding region of diffuse microdamage.

Figure 3

A 2mm cubic volume showing damaged regions which has been labeled with lead-uranyl sulfide. The darker shade of gray indicates regions stained by lead-uranyl sulfide as detected by microCT, while the lighter shade shows unlabelled bone.

Figure 4

MicroCT determined damage volume fraction (DV/BV) increased exponentially to the applied apparent strains in the cancellous bone cores (Pearson correlation; p<0.001).

Figure 5

Mechanically determined modulus reduction increased proportionally to the applied apparent strains in cancellous bone cores after the onset of yielding (r²=0.98).

Figure 6

Damage volume fraction (DV/BV) increased proportionately to the modulus reduction in the cancellous bone cores (r²=0.64). The applied apparent strain for each sample is shown beside the respective points.