Bone microhardness has been successfully correlated with important functional parameters such as mineralisation and stiffness. It provides a means of examining the mechanical competence of bone at a micron scale, averaging the effect of osteonal lamellae but sensitive to variation in mineral content within a bone, and, with careful selection of indentation site, able to obtain material characteristics separate from any effects of porosity. However, the effect of bone’s viscoelasticity on such measurements has been largely ignored. This preliminary study investigates the post-indentation size change of Vickers indentations on wet bone. 4 axial slices of bovine femur were harvested from the same shaft, and polished. Each sample was subjected to 4 sets of 10 Vickers indentations with a load of 50 g and holding period of 15 s. The indentation size was measured immediately after the load was removed, and then again at intervals for a period up to 24 hours after the indentation was made. To avoid dehydration, the bone stood in water during the indentation testing and during measurement, and between each measurement period it was fully immersed in water. Measured hardness significantly decreased with time, by approximately 30% in total. The rate of post-indentation recovery is difficult to analyse since the driving force of residual strain decreases as recovery takes place. However a simple exponential fit to the variation of HV with time in the form of H = H(final).(1−exp(−kt)) + H(initial) suggests that the size of the indentation tends towards a constant size between 5 and 24 hours after indentation. Thus we conclude that care should be taken when making “early” measurements given the rapid rate of change in indentation size. Caution should also be employed when interpreting such data.

Cortical porosity is a useful evaluator of bone since it is sensitive to changes in bone turnover. The aim of this study was to evaluate cortical bone porosity of human vertebrae samples using Scanning Acoustic Microscopy (SAM). Currently the common techniques used to determine bone porosity are histomorphometry or scanning electronmicrosopy images. Both methods require extensive preparation of the bone samples. SAM represents a new technique with the great advantage of minimal sample interference since the bone is imaged in water, or saturated, and requires just one flat surface which is scanned (but not contacted) by the transducer. 46 specimens between the ages of 64–90 years were randomly selected and ground before SAM imaging of was carried out using a 400 MHz transducer. For each sample posterior and anterior sections of the cortical bone were scanned several times, and the porosity measured using Scion image software to process the images. It was possible to image the entire anterior or posterior cortex in a single image with 4 mm spatial resolution. Measured porosity was in the region 5 % – 21 %, and showed a significant increase with age for the female specimens but no age dependence in the male specimens. At low porosity (< 6 %) vertebral compressive strength was uncorrelated with porosity. However, at higher porosities strength was highly correlated with porosity. (As would be expected, strength decreased with increasing porosity). High frequency SAM has potential for future bone characterisation, particularly where it is desirable to correlate local measurements of material properties such as nanohardness or microhardness, with microstructure.

Intervertebral disc function and dysfunction is governed by its structural architecture of concentric layers of highly ordered collagen fibres. This architecture is important at the mm scale for overall mechanical performance of the disc; and at the micron scale for mechano-transduction signalling pathways of the disc cells that are responsible for matrix maintenance and therefore disc health. To understand such mechanical behaviour 3-dimensional collagen fibre architecture must be quantified in intact intervertebral discs. Conventional imaging modalities lack either the spatial resolution (e.g. x-ray diffraction) or penetration (e.g. optical, electron or confocal laser microscopy) to yield mechanically important information. Preliminary studies of scanning acoustic microscopy (SAM) at 50 MHz visualises alternating layers of fibre texture, however exactly what is being imaged requires both explanation and validation. Three-dimensional SAM data sets obtained from intact discs were compared to polarised-light and scanning electron micrographs of individual layers of fibres, peeled by micro-dissection from discs. The dimensions of the structural features were measured and recorded. Optical and electron microscopy revealed that each layer consisted of highly oriented collagen fibres of diameter 5 μm with regularly spaced splits between fibres with a spacing of approximately 20–30 μm. The SAM data sets showed layers with a uniform highly oriented fibre texture that reversed between adjacent layers. Resolution of the texture was limited by the acoustic system to approximately 30 μm. It is clear that SAM at 50 MHz cannot resolve and therefore image individual collagen fibres. However, the regular defects in the fibre layers can be visualised and convey complete information about local collagen fibre architecture. SAM therefore provides an effective way of quantifying the fibrous structure of intact, hydrated, unfixed intervertebral discs.