Comparative investigation of elastic properties in a trabecula using micro-Brillouin scattering and scanning acoustic microscopy.

Micro-Brillouin scattering (µ-BR) and a 200 MHz scanning acoustic microscope (SAM) with similar spatial resolutions were applied to evaluate tissue elastic properties in two directions in a trabecula. Acoustic impedance measured by SAM was in the range of 5-9 Mrayl. Wave velocities determined by µ-BR were in the range of (4.75-5.11) × 10(3) m/s. Both exhibited a similar trend of variation across the trabecula and were significantly correlated (R(2) = 0.63-0.67, p < 0.01). µ-BR is useful for the evaluation of tissue stiffness within a trabecula. Combined with SAM or nanoindentation, it can provide additional information to assess elastic anisotropy at the micro-scale.

Variations of microstructure, mineral density and tissue elasticity in B6/C3H mice.

200-MHz scanning acoustic microscopy (SAM) and synchrotron radiation microCT (SR-microCT) were used to assess microstructural parameters, acoustic impedance Z and tissue degree of mineralization of bone (DMB) in site-matched regions of interest in femoral bone of two inbred strains. Transverse femoral sections taken from 5 C57BL/6J@Ico (B6) and 5 C3H/HeJ@Ico (C3H) mice (5.5 months old) were explored. Mass density rho, elastic coefficient c(11) and Young's modulus E(1) were locally derived in the distal epiphysis, distal metaphysis for trabecular bone and mid-diaphysis for cortical bone using a rule-of-mixture model. Structural parameter estimations obtained from X-ray tomographic and acoustic images were almost identical. Both strains had the same bone diameter, but the C3H mice had greater cortical thickness and smaller cancellous diameter than did B6 mice. The average DMB and impedance values were in the range between 1.13 and 1.33 g cm(-3) and 5.8 and 7.8 Mrayl, respectively. All tissue parameters were lower in B6 mice than in C3H mice. However, interstrain differences of DMB were much less (up to 3.8%) than differences of Z (up to 13.2%). SAM and SR-microCT fulfill the requirement for a simultaneous evaluation of cortical bone microstructure and material properties at the tissue level. However, SAM provides a quantitative estimate of elastic properties at the tissue level that cannot be captured by SR-microCT. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.

Bone microstructure and elastic tissue properties are reflected in QUS axial transmission measurements.

Accurate clinical interpretation of the sound velocity derived from axial transmission devices requires a detailed understanding of the propagation phenomena involved and of the bone factors that have an impact on measurements. In the low megahertz range, ultrasonic propagation in cortical bone depends on anisotropic elastic tissue properties, porosity and the cortical geometry (e.g., thickness). We investigated 10 human radius samples from a previous biaxial transmission study using a 50-MHz scanning acoustic microscope (SAM) and synchrotron radiation microcomputed tomography. The relationships between low-frequency axial transmission sound speed at 1 and 2 MHz, structural properties (cortical width Ct.Wi, porosity, Haversian canal density and material properties (acoustic impedance, mineral density) on site-matched cross-sections were investigated. Significant linear multivariate regression models (1 MHz: R(2) = 0.84, p < 10(-4), root-mean-square error (RMSE) = 38 m/s, 2 MHz: R(2) = 0.65, p < 10(-4), RMSE = 48 m/s) were found for the combination of Ct.Wi with porosity and impedance. A new model was derived that accounts for the nonlinear dispersion relation with Ct.Wi and predicts axial transmission velocities measured at different ultrasonic frequencies (R(2) = 0.69, p < 10(-4), RMSE = 52 m/s).

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 model's 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.

High-resolution ultrasonography for analysis of age- and disease-related cartilage changes.

Because of their limited spatial resolution, current clinical noninvasive imaging modalities (radiography, computed tomography, conventional echography, and magnetic resonance imaging) are able to detect only the late stages of the cartilage degradation. To detect early lesions and follow their evolution in time with imaging, higher resolution is necessary. Recent work suggest that high-frequency ultrasound may serve as a useful means for the investigation of cartilage matrix structural changes occurring under various experimental and clinical circumstances, like the growing process and osteoarthritis. In this chapter, an experimental 50-100-MHz ultrasound scanner is described for high-resolution echographic imaging of articular cartilage. The procedures of data acquisition and signal processing are detailed for the quantitative evaluation of ultrasonic reflection and backscatter coefficients, which have been reported to be sensitive to subtle surface and internal disease-related alterations. Further technological developments and miniaturization of the echographic probes may lead to extension of this technique to the study of living small animals or to the clinical field in combination with conventional arthoscopy.

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.

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).

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.

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, ß-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.

Adaptive remodeling of trabecular bone core cultured in 3-D bioreactor providing cyclic loading: an acoustic microscopy study.

Scanning acoustic microscopy (SAM) provides high-resolution mapping of acoustic impedance related to tissue stiffness. This study investigates changes in tissue acoustic impedance resulting from mechanical loading in trabecular bone cores cultured in 3-D bioreactor. Trabecular bone cores were extracted from bovine sternum (n = 15) and ulna metaphysis (n = 15). From each bone, the samples were divided in three groups. The basal control (BC) group was fixed post-extraction, the control (C) and loaded (L) groups were maintained as viable in a controlled culture-loading cell over three weeks. Samples of L group underwent a dynamic compressive strain, whereas C samples were left free from loading. After three weeks, L and C samples were embedded in polymethylmethacrylate and all samples were explored with a 200-MHz SAM. For each specimen, the acoustic impedance distribution was obtained over flat and polished section of bone blocks prepared parallel to the loading axis. Our results showed that in basal controls, the acoustic impedance varied with bone anatomical location and was 15% higher in weight-bearing ulna compared with nonweight-bearing sternum. The comparison between loaded and nonloaded groups showed that sternum-only exhibited significant change in acoustic impedance (L vs. C sternum: +9%). This result suggests that when the applied load is comparable with the stress naturally experienced by a weight-bearing bone (ulna), the tissue material properties (manifested by acoustic impedance) remained unchanged. In conclusion, SAM is a potentially relevant tool for the assessment of subtle changes in intrinsic microelastic properties of bone induced by adaptive remodeling process in response to mechanical loading.

Assessment of bone structure and acoustic impedance in C3H and BL6 mice using high resolution scanning acoustic microscopy.

Two hundred-MHz time-resolved scanning acoustic microscopy was applied for the investigation of acoustic and structural bone properties of mice from two inbred strains. Transverse sections of femur taken from 5 C57BL/6J@Ico and 5 C3H/HeJ@Ico mice were explored. Both strains had the same bone diameter, but the C3H/HeJ@Ico mice had greater cortical thickness, smaller cancellous diameter, and greater acoustic impedance values than C57BL/6J@Ico mice. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.

Effects of antiinflammatory drugs on arthritic cartilage: a high-frequency quantitative ultrasound study in rats.

OBJECTIVE: To evaluate the ability of 55-MHz quantitative ultrasound (US) to detect the in vivo effects of experimental arthritis, as well as those of two antiinflammatory drugs, naproxen (NPX) and dexamethasone (DEX), on cartilage and subchondral bone. METHODS: Arthritis was induced in both knees of 108 rats by intraarticular injection of zymosan (ZYM). Two groups of arthritic rats (n = 36 per group) were treated daily with either NPX (10 mg/kg/day) or DEX (0.1 mg/kg/day). Using a 3-dimensional US microscope, patellae were explored in vitro on days 5, 14, and 21 after injections. US assessment included the analysis of quantitative indices of local modifications involving cartilage and bone: integrated reflection coefficient (IRC) from the cartilage surface and apparent integrated backscatter from the cartilage internal structure (cartilage matrix) (AIB(cartilage)) and the cartilage-bone interface (AIB(bone)). RESULTS: ZYM induced articular surface fibrillation that resulted in a decrease in IRC at all times (P < 0.02) and in an increase in AIB(bone) on days 5 and 14 (P < 0.005). Fibrillation was not changed by NPX administration, while it disappeared following DEX treatment. Cartilage-bone interface alterations were prevented by DEX and partially compensated for by NPX. Cartilage matrix echogenicity decreased with time in all groups due to maturation (P < 0.05), except in DEX-treated rats. CONCLUSION: Quantitative 55 MHz US allowed detection of early cartilage and bone lesions due to experimental arthritis, and also allowed detection of the effects of antiinflammatory drugs. NPX seemed to have an effect on subchondral bone lesions, but not on cartilage. DEX appeared to repair articular surface and bone, but prevented animal growth and cartilage maturation.