Ingrid Leguerney

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.

Contrast Ultrasonography: Necessity of Linear Data Processing for the Quantification of Tumor Vascularization

PURPOSE This study is intended to compare the value of uncompressed ultrasonic data, obtained after linear power detection of the ultrasonic radiofrequencies that we call linear data, with usual compressed video data for the quantification of tumor perfusion, particularly for monitoring antivascular therapy. MATERIALS AND METHODS To form a clinically useful ultrasonic image, the detected power of the received signals (linear data) is compressed in a quasi-logarithmic fashion in order to match the limited dynamic range of the video monitor. The resulting reduced range of signals from an injected contrast agent may limit the sensitivity to changes in the time-intensity curves. Following a theoretical evaluation of the effects of compression on time-intensity curves and as an in vivo example, we measured at different times the effects of an antivascular drug administered to mice bearing melanoma tumors. The mean time-intensity curves within the tumors after bolus injection of a contrast agent were determined using both linear and video data. Linearized data was recovered using the inverse of the true scanners compression law, which was experimentally determined. Three features were extracted from the time-intensity curves: peak intensity (PI), time to peak intensity (TPI) and area under the curve in the wash-in phase (AUC (wash-in)). When contrast reached its maximum value, the coefficient of variation reflecting the heterogeneity of the intensity of contrast uptake within the tumor, was computed using both data sets. RESULTS TPI was found to be similar with either data set (r = 0.98, p < 0.05, factor of 1.09). Linear PI and AUC (wash-in) had significantly earlier decreases after drug administration than video data (p = 0.015 and p = 0.03, respectively). The coefficient of variation was significantly lower when using video rather than linear data (p < 10 (-4)). CONCLUSION In conclusion, the use of linear data is the only mathematically valid methodology for determining a tumors time-intensity curve and, in practice, it allows earlier demonstration of responses to antivascular drugs.

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.

Assessment of Quantitative Perfusion Parameters by Dynamic Contrast-Enhanced Sonography Using a Deconvolution Method An In Vitro and In Vivo Study

The purpose of this study was to investigate the impact of the arterial input on perfusion parameters measured using dynamic contrast‐enhanced sonography combined with a deconvolution method after bolus injections of a contrast agent.

Dynamic contrast-enhanced ultrasound parametric maps to evaluate intratumoral vascularization.

ObjectivesThe purposes of this study were to assess the reliability of parametric maps from dynamic contrast-enhanced ultrasound (DCE-US) to reflect the heterogeneous distribution of intratumoral vascularization and to predict the tissue features linked to vasculature. This study was designed to compare DCE-US parametric maps with histologic vascularity measurements. Materials and MethodsDynamic contrast-enhanced ultrasound was performed on 17 melanoma-bearing nude mice after a 0.1-mL bolus injection of SonoVue (Bracco SPA, Milan, Italy). The parametric maps were developed from raw linear data to extract pixelwise 2 semiquantitative parameters related to perfusion and blood volume, namely, area under the curve (AUC) and peak intensity (PI). The mathematical method to fit the time-intensity curve for each pixel was a polynomial model used in clinical routine and patented by the team. Regions of interest (ROIs) were drawn on DCE-US parametric maps for whole tumors and for several local areas of 15 mm2 within each tumor (iROI), the latter reflecting the heterogeneity of intratumoral blood volume. As the criterion standard correlation, microvessel densities (MVDs) were determined for both ROI categories. In detail, for all iROI of 15 mm2, MVD and maturity were divided separately for vessels of 0 to 10 &mgr;m, 10 to 40 &mgr;m, and greater than 40 &mgr;m in diameter, and the results were correlated with the ultrasound findings. ResultsAmong the 17 studied mice, a total of 64 iROIs were analyzed. For the whole-tumor ROI set, AUC and PI values significantly correlated with MVD (rAUC = 0.52 [P = 0.0408] and rPI = 0.70 [P = 0.0026]). In the case of multiple iROI, a strong linear correlation was observed between the DCE-US parameters and the density of vessels ranging in their diameter from 0 to 10 &mgr;m (rAUC = 0.68 [P < 0.0001]; rPI = 0.63 [P < 0.0001]), 10 to 40 &mgr;m (rAUC = 0.98 [P = 0.0003]; rPI = 0.98 [P = 0.0004]), and greater than 40 &mgr;m (rAUC = 0.86 [P = 0.0120]; rPI = 0.92 [P = 0.0034]), respectively. However, the DCE-US parameter values of perfusion and blood volume were not significantly different according to the diameters (AUC: P = 0.1731; PI: P = 0.2918) and maturity of blood vessels. ConclusionsParametric maps of DCE-US can be reliably established from raw linear data and reflect the heterogeneous histological measures of vascularization within tumors. In contrast, the values of DCE-US parametric maps (AUC, PI) do not allow deduction of heterogeneous tissue features such as the diameters and maturity of vascular networks.

Ultrasonic Backscatter and Attenuation (11–27 MHz) Variation with Collagen Fiber Distribution in Ex Vivo Human Dermis

This ex vivo study explores the relationship of ultrasonic attenuation and backscatter to dermal microarchitecture by comparing ultrasonic measurements of these parameters (11–27 MHz) to a microscopic analysis of three parameters describing the collagen distribution (mean thickness and spacing of collagen bundles along the insonification direction and the percent area occupied by collagen). Skin samples (N = 31) were obtained from patients undergoing breast or abdominal reduction surgery. Radio-frequency (rf) signals were acquired in a B-scan format using an ultrasound system developed for skin imaging (Ultrasons Technologies, Tours, France). Ultrasonic data were analyzed to calculate average integrated backscatter (IBS in dB) and frequency dependence of backscatter (n, dimensionless) of each specimen at depths centered approximately 370, 620 and 880 μm beneath the skin surface. Average integrated attenuation coefficient (IA in dB.cm−1) and frequency dependence of attenuation coefficient (β in dB.cm−1.MHz−1) were estimated across the depth between 240 and 1,000 μm. The three collagen distribution parameters were estimated using digitized microcopic fields from matched regions of histological sections stained with hematoxylin-eosin-saffron. No significant correlation was identified between collagen distribution parameters and IA or β. For the most superficial depth studied in abdominal skin, n was inversely correlated to collagen bundle thickness (r = −0.67, p = 0.002) and percent area (r = −0.65, p = 0.003). At the same depth, IBS was inversely correlated to percent area of collagen (r = −0.51, p = 0.03). The rather high collagen packing (48 to 82% area) measured in histological sections and the inverse relationship observed between IBS and percent area of collagen suggest that a packing factor should be included in models relating skin collagen distribution to ultrasound spectral parameters. A better understanding of the relationship between ultrasound parameters and the microarchitecture of the dermis should help to interpret changes in ultrasonic parameters observed during in vivo ultrasonic skin examinations.

Molecular Ultrasound Imaging Using Contrast Agents Targeting Endoglin, Vascular Endothelial Growth Factor Receptor 2 and Integrin

Expression levels of endoglin, αv integrin and vascular endothelial growth factor receptor 2 (VEGFR2) were investigated using targeted, contrast-enhanced ultrasonography in murine melanoma tumor models. Microvasculature and expression levels of biomarkers were investigated using specific contrast agents conjugated with biotinylated monoclonal antibodies. Ultrasound signal intensity from bound contrast agents was evaluated in two groups of mice: control mice and mice treated with sorafenib. Expression levels were analyzed by immunohistochemistry. Endoglin biomarkers were more highly expressed than αv integrin and VEGFR2. Endoglin decreased in the sorafenib group, whereas it tended to increase with time in the control group. Targeted ultrasound contrast agents may be used for non-invasive longitudinal evaluation of tumor angiogenesis during tumor growth or therapeutic treatment in preclinical studies. Endoglin protein, which plays an important role in angiogenesis, seems to be a target of interest for detection of cancer and for prediction of therapeutic efficacy.

Impact of the arterial input function on microvascularization parameter measurements using dynamic contrast-enhanced ultrasonography

AIM To evaluate the sources of variation influencing the microvascularization parameters measured by dynamic contrast-enhanced ultrasonography (DCE-US). METHODS Firstly, we evaluated, in vitro, the impact of the manual repositioning of the ultrasound probe and the variations in flow rates. Experiments were conducted using a custom-made phantom setup simulating a tumor and its associated arterial input. Secondly, we evaluated, in vivo, the impact of multiple contrast agent injections and of examination day, as well as the influence of the size of region of interest (ROI) associated with the arterial input function (AIF). Experiments were conducted on xenografted B16F10 female nude mice. For all of the experiments, an ultrasound scanner along with a linear transducer was used to perform pulse inversion imaging based on linear raw data throughout the experiments. Semi-quantitative and quantitative analyses were performed using two signal-processing methods. RESULTS In vitro, no microvascularization parameters, whether semi-quantitative or quantitative, were significantly correlated (P values from 0.059 to 0.860) with the repositioning of the probe. In addition, all semi-quantitative microvascularization parameters were correlated with the flow variation while only one quantitative parameter, the tumor blood flow, exhibited P value lower than 0.05 (P = 0.004). In vivo, multiple contrast agent injections had no significant impact (P values from 0.060 to 0.885) on microvascularization parameters. In addition, it was demonstrated that semi-quantitative microvascularization parameters were correlated with the tumor growth while among the quantitative parameters, only the tissue blood flow exhibited P value lower than 0.05 (P = 0.015). Based on these results, it was demonstrated that the ROI size of the AIF had significant influence on microvascularization parameters: in the context of larger arterial ROI (from 1.17 ± 0.6 mm(3) to 3.65 ± 0.3 mm(3)), tumor blood flow and tumor blood volume were correlated with the tumor growth, exhibiting P values lower than 0.001. CONCLUSION AIF selection is an essential aspect of the deconvolution process to validate the quantitative DCE-US method.

Numerical modelling of the flow of the ultrasound contrast agents in tumour microvasculature

Functional imaging of tumour microvasculature is a major challenge in oncology to assess early therapeutic efficacy of new drugs, with detection of microcirculation modifications before change in tumour volume. The ultrasound imaging method is based on the study of microvascular enhancement by injection of microbulles as contrast agents (CA) to characterise some parameters according to blood volume and blood flow. This new modality called dynamic contrast-enhanced ultrasonography (DCE-US) has been validated recently (Lassau et al. 2012) and is included in International guidelines 2012 (Claudon et al. 2012). Nevertheless, the different methods of quantification of the microbubbles are being developed and improved. In this context, we developed the first numerical modelling (NM) of the CA flow to study the ability of quantification methods to evaluate the tumour microvasculature, and to apprehend their variations according to the tumour growth, the hemodynamic configurations and different types of injections of CA. The purposes of this study are as follows:

Assessment of Bone Properties Using High Resolution Scanning Acoustic Microscopy

Bone mechanical properties are determined by an ensemble of complex characteristics, such as microstructure (organization of collagen fibers and hydroxyapatite crystals, integrity of collagen-mineral interface), degree of mineralization, micro-architecture, and tissue elasticity. These characteristics strongly contribute by varying degrees to bone strength and resistance to fracture.