Amena Saïed

Change in porosity is the major determinant of the variation of cortical bone elasticity at the millimeter scale in aged women

At the mesoscale (i.e. over a few millimeters), cortical bone can be described as two-phase composite material consisting of pores and a dense mineralized matrix. The cortical porosity is known to influence the mesoscopic elasticity. Our objective was to determine whether the variations of porosity are sufficient to predict the variations of bone mesoscopic anisotropic elasticity or if change in bone matrix elasticity is an important factor to consider. We measured 21 cortical bone specimens prepared from the mid-diaphysis of 10 women donors (aged from 66 to 98 years). A 50-MHz scanning acoustic microscope (SAM) was used to evaluate the bone matrix elasticity (reflected in impedance values) and porosity. Porosity evaluation with SAM was validated against Synchrotron Radiation μCT measurements. A standard contact ultrasonic method was applied to determine the mesoscopic elastic coefficients. Only matrix impedance in the direction of the bone axis correlated to mesoscale elasticity (adjusted R(2)=[0.16-0.25], p<0.05). The mesoscopic elasticity was found to be highly correlated to the cortical porosity (adj-R(2)=[0.72-0.84], p<10(-5)). Multivariate analysis including both matrix impedance and porosity did not provide a better statistical model of mesoscopic elasticity variations. Our results indicate that, for the elderly population, the elastic properties of the mineralized matrix do not undergo large variations among different samples, as reflected in the low coefficients of variation of matrix impedance (less than 6%). This work suggests that change in the intracortical porosity accounts for most of the variations of mesoscopic elasticity, at least when the analyzed porosity range is large (3-27% in this study). The trend in the variation of mesoscale elasticity with porosity is consistent with the predictions of a micromechanical model consisting of an anisotropic matrix pervaded by cylindrical pores.

Site-matched assessment of structural and tissue properties of cortical bone using scanning acoustic microscopy and synchrotron radiation μCT

200 MHz scanning acoustic microscopy (SAM) and synchrotron radiation muCT (SR-muCT) were used to assess microstructural parameters and tissue properties in site-matched regions of interest in cortical bone. Anterior and postero-lateral regions of ten cross sections from human cortical radius were explored. Structural parameters, including diameter and number of Haversian canals per cortical area (Ca.Dm, N.Ca/Ar) and porosity Po were assessed with both methods using a custom-developed image fusion and analysis software. Acoustic impedance Z and degree of mineralization of bone DMB were extracted separately for osteonal and interstitial tissues from the fused images. Structural parameter estimations obtained from radiographic and acoustic images were almost identical. DMB and impedance values were in the range between 0.77 and 1.28 g cm(-3) and 5.13 and 12.1 Mrayl, respectively. Interindividual and regional variations were observed, whereas the strongest difference was found between osteonal and interstitial tissues (Z: 7.2 +/- 1.1 Mrayl versus 9.3 +/- 1.0 Mrayl, DMB: 1.06 +/- 0.07 g cm(-3) versus 1.16 +/- 0.05 g cm(-3), paired t-test, p < 0.05). Weak, but significant correlations between DMB and Z were obtained for the osteonal (R(2) = 0.174, p < 10(-4)) and for the pooled (osteonal and interstitial) data. The regression of the pooled osteonal and interstitial tissue data follows a second-order polynomial (R(2) = 0.39, p < 10(-4)). Both modalities fulfil the requirement for a simultaneous evaluation of cortical bone microstructure and material properties at the tissue level. While SAM inspection is limited to the evaluation of carefully prepared sample surfaces, SR-muCT provides volumetric information on the tissue without substantial preparation requirements. However, SAM provides a quantitative estimate of elastic properties at the tissue level that cannot be captured by SR-muCT.

High resolution ultrasound elastomicroscopy imaging of soft tissues : system development and feasibility

Research in elasticity imaging typically relies on 1-10 MHz ultrasound. Elasticity imaging at these frequencies can provide strain maps with a resolution in the order of millimetres, but this is not sufficient for applications to skin, articular cartilage or other fine structures. We developed a prototype high resolution elastomicroscopy system consisting of a 50 MHz ultrasound backscatter microscope system and a calibrated compression device using a load cell to measure the pressure applied to the specimen, which was installed between a rigidly fixed face-plate and a specimen platform. Radiofrequency data were acquired in a B-scan format (10 mm wide x 3 mm deep) in specimens of mouse skin and bovine patellar cartilage. The scanning resolution along the B-scan plane direction was 50 microm, and the ultrasound signals were digitized at 500 MHz to achieve a sensitivity better than 1 microm for the axial displacement measurement. Because of elevated attenuation of ultrasound at high frequencies, special consideration was necessary to design a face-plate permitting efficient ultrasound transmission into the specimen and relative uniformity of the compression. Best results were obtained using a thin plastic film to cover a specially shaped slit in the face-plate. Local tissue strain maps were constructed by applying a cross-correlation tracking method to signals obtained at the same site at different compression levels. The speed of sound in the tissue specimen (1589.8+/-7.8 m s(-1) for cartilage and 1532.4+/-4.4 m s(-1) for skin) was simultaneously measured during the compression test. Preliminary results demonstrated that this ultrasound elastomicroscopy technique was able to map deformations of the skin and articular cartilage specimens to high resolution, in the order of 50 microm. This system can also be potentially used for the assessment of other biological tissues, bioengineered tissues or biomaterials with fine structures.

Microfibril Orientation Dominates the Microelastic Properties of Human Bone Tissue at the Lamellar Length Scale

The elastic properties of bone tissue determine the biomechanical behavior of bone at the organ level. It is now widely accepted that the nanoscale structure of bone plays an important role to determine the elastic properties at the tissue level. Hence, in addition to the mineral density, the structure and organization of the mineral nanoparticles and of the collagen microfibrils appear as potential key factors governing the elasticity. Many studies exist on the role of the organization of collagen microfibril and mineral nanocrystals in strongly remodeled bone. However, there is no direct experimental proof to support the theoretical calculations. Here, we provide such evidence through a novel approach combining several high resolution imaging techniques: scanning acoustic microscopy, quantitative scanning small-Angle X-ray scattering imaging and synchrotron radiation computed microtomography. We find that the periodic modulations of elasticity across osteonal bone are essentially determined by the orientation of the mineral nanoparticles and to a lesser extent only by the particle size and density. Based on the strong correlation between the orientation of the mineral nanoparticles and the collagen molecules, we conclude that the microfibril orientation is the main determinant of the observed undulations of microelastic properties in regions of constant mineralization in osteonal lamellar bone. This multimodal approach could be applied to a much broader range of fibrous biological materials for the purpose of biomimetic technologies.

Assessment of Microelastic Properties of Bone Using Scanning Acoustic Microscopy: A Face-to-Face Comparison with Nanoindentation

The current work aimed at comparing, on site-matched cortical bone tissue, the micron-level elastic modulus Ea derived from 200 MHz-scanning acoustic microscopy (SAM) acoustic impedance (Z) combined with bone mineral density (assessed by synchrotron radiation microcomputed tomography, SR-µCT) to nanoindentation modulus En. A good correlation was observed between En and Z (R2=0.67, p<0.0001, root mean square error RMSE=1.9 GPa). The acoustical elastic modulus Ea derived from Z showed higher values of E compared to nanoindentation moduli. We assumed that the discrepancy between Ea and En values may likely be due to the fixed assumed value of Poissons ratio while values comprised between 0.15 and 0.45 have been reported in the literature. Despite these differences, a highly significant correlation between Ea and En was found (R2=0.66, p<0.001, RMSE=1.8 GPa) suggesting that SAM can reliably be used as a modality to quantitatively map the local variations of tissue-level bone elasticity.

Spatial distribution of anisotropic acoustic impedance assessed by time-resolved 50-MHz scanning acoustic microscopy and its relation to porosity in human cortical bone

We used quantitative scanning acoustic microscopy (SAM) to assess tissue acoustic impedance and microstructure of cortical bone of human radii with the aim to provide data on regional distribution of acoustic impedance along the circumferential and across the radial directions in the entire cross-section of the radius diaphysis as well as to determine the range of impedance values in transverse (perpendicular to bone axis) and longitudinal (parallel to bone axis) cross-sections. Several microstructural features related to cortical porosity were analyzed in order to determine whether these features differ in different parts of the cortex and to assess the relationship between the microstructure and tissue acoustic impedance. Fifteen fresh bone specimens (human radius) were investigated using a SAM (center frequency of 50 MHz and -6 dB lateral resolution of approximately 23 microm). The sample acoustic impedance was obtained by means of a calibration curve correlating the reflected signal amplitude of reference materials with their corresponding well-known acoustic impedance. Tissue acoustic impedance and microstructural features were derived from the morphometric analysis of the segmented impedance images. A higher porosity was found in the inner cortical layer (mean+/-SD=8.9+/-2.3%) compared to the peripheral layer (2.7+/-1.5%) (paired t-test, p<10(-5)). ANOVA showed that most of the variance can be explained by the regional effect across the radial direction with a minor contribution due to between-sample variability. Similar to porosity, the number and diameter of pores were greater in the inner layer. In contrast to porosity, ANOVA showed that impedance variability can mostly be explained by between-specimen variability. Two-way ANOVA revealed that after compensation for the between-sample variability the variation in acoustic impedance across the radial direction was much larger than that along the circumferential direction. In addition to the significant difference between the inner cortical layer (8.25+/-0.4 Mrayl) and peripheral layer (8.0+/-0.5 Mrayl) (unilateral paired t-test, p<10(-4)), the values in the anterior region (8.2+/-0.5 Mrayl) were found to be significantly higher than those of the posterior region (7.9+/-0.6 Mrayl). Impedance mean value of longitudinal sections was lower than mean value measured in transverse cross-sections, resulting in an impedance acoustic anisotropy ratio of 1.17+/-0.03 in the inner cortical layer and 1.19+/-0.02 in the peripheral layer. SAM is a valuable tool to provide data on the spatial distribution of microstructural and microelastic bone properties that is useful to improve our understanding of the impact of bone microstructure on tissue material properties.

Milestones on the road to higher resolution, quantitative, and functional ultrasonic imaging

Ultrasonography is evolving rapidly with important recent advances in high-density transducer arrays, one-and-one-half-dimensional transducers, broad-band transducers, increased scanner bandwidth, and more sophisticated image-formation routines. Technical advances have clearly improved accuracy of image readings, heightened contrast and resolution, reduced noise, and reduced image slice thickness. It is within this fertile environment that very high-frequency ultrasound, harmonic imaging, and ultrasound contrast agents have emerged. Clinical applications of ultrasonography have also been extended to new fields, such as skeletal status assessment, which have long been considered beyond ultrasounds reach, and over the past 15 years, quantitative ultrasound bone densitometry has become an important part of the armamentarium for osteoporosis diagnosis. The state of these innovations, their contributions to diagnostic and monitoring capabilities, as well as the new applications they bring into reach will be discussed. We will explore several applications currently under development including ultrasound biomicroscopy (eye, skin, small animals), quantitative perfusion assessment, and pathology evaluation. Thus, ongoing research has not only significantly added to diagnostic ultrasounds existing capabilities, but also promises to further broaden the range of its clinical and biological applications.

Relative contributions of porosity and mineralized matrix properties to the bulk axial ultrasonic wave velocity in human cortical bone

Velocity of ultrasound waves has proved to be a useful indicator of bone biomechanical competence. A detailed understanding of the dependence of ultrasound parameters such as velocity on bone characteristics is a key to the development of bone quantitative ultrasound (QUS). The objective of this study is to investigate the relative contributions of porosity and mineralized matrix properties to the bulk compressional wave velocity (BCV) along the long bone axis. Cross-sectional slabs from the diaphysis of four human femurs were included in the study. Seven regions of interest (ROIs) were selected in each slab. BCV was measured in through-transmission at 5 MHz. Impedance of the mineralized matrix (Z(m)) and porosity (Por) were obtained from 50 MHz scanning acoustic microscopy. Por and Z(m) had comparable effects on BCV along the bone axis (R=-0.57 and R=0.72, respectively).

In vivo gene transfer into the ocular ciliary muscle mediated by ultrasound and microbubbles.

This study aimed to assess application of ultrasound (US) combined with microbubbles (MB) to transfect the ciliary muscle of rat eyes. Reporter DNA plasmids encoding for Gaussia luciferase, β-galactosidase or the green fluorescent protein (GFP), alone or mixed with 50% Artison MB, were injected into the ciliary muscle, with or without US exposure (US set at 1 MHz, 2 W/cm(2), 50% duty cycle for 2 min). Luciferase activity was measured in ocular fluids at 7 and 30 days after sonoporation. At 1 week, the US+MB treatment showed a significant increase in luminescence compared with control eyes, injected with plasmid only, with or without MB (×2.6), and, reporter proteins were localized in the ciliary muscle by histochemical analysis. At 1 month, a significant decrease in luciferase activity was observed in all groups. A rise in lens and ciliary muscle temperature was measured during the procedure but did not result in any observable or microscopic damages at 1 and 8 days. The feasibility to transfer gene into the ciliary muscle by US and MB suggests that sonoporation may allow intraocular production of proteins for the treatment of inflammatory, angiogenic and/or degenerative retinal diseases.

To what extent can cortical bone millimeter-scale elasticity be predicted by a two-phase composite model with variable porosity?

An evidence gap exists in fully understanding and reliably modeling the variations in elastic anisotropy that are observed at the millimeter scale in human cortical bone. The porosity (pore volume fraction) is known to account for a large part, but not all, of the elasticity variations. This effect may be modeled by a two-phase micromechanical model consisting of a homogeneous matrix pervaded by cylindrical pores. Although this model has been widely used, it lacks experimental validation. The aim of the present work is to revisit experimental data (elastic coefficients, porosity) previously obtained from 21 cortical bone specimens from the femoral mid-diaphysis of 10 donors and test the validity of the model by proposing a detailed discussion of its hypotheses. This includes investigating to what extent the experimental uncertainties, pore network modeling, and matrix elastic properties influence the models predictions. The results support the validity of the two-phase model of cortical bone which assumes that the essential source of variations of elastic properties at the millimeter-scale is the volume fraction of vascular porosity. We propose that the bulk of the remaining discrepancies between predicted stiffness coefficients and experimental data (RMSE between 6% and 9%) is in part due to experimental errors and part due to small variations of the extravascular matrix properties. More significantly, although most of the models that have been proposed for cortical bone were based on several homogenization steps and a large number of variable parameters, we show that a model with a single parameter, namely the volume fraction of vascular porosity, is a suitable representation for cortical bone. The results could provide a guide to build specimen-specific cortical bone models. This will be of interest to analyze the structure-function relationship in bone and to design bone-mimicking materials.