In Vitro Evaluation of Real-Time Viscoelastic and Coagulation Properties of Various Classes of Topical Hemostatic Agents Using a Novel Contactless Nondestructive Technology.

Hemorrhaging is the main cause of death among combat and civilian injuries and has significant clinical and economic consequences. Despite their vital roles in bleeding management, an optimal topical hemostatic agent (HA) has yet to be developed for a particular scenario. This is partly due to a lack of an overarching quantitative testing technology to characterize the various classes of HAs in vitro. Herein, the feasibility of a novel, contactless, and nondestructive technique to quantitatively measure the shear storage modulus (G') and clotting properties of whole blood in contact with different dosages of eight topical HAs, including particulates and gauze-like and sponge-like systems, was assessed. The real-time G'-time profiles of these blood/HA systems revealed their distinct biomechanical behavior to induce and impact coagulation. These were analyzed to characterize the clot initiation time, clotting rate, clotting time, and apparent stiffness of the formed clots (both immediately and temporally), which were correlated with their reported hemostatic mechanisms of action. Moreover, the HAs that worked independently from the natural blood clotting cascade were identified and quantified through this technology. In sum, this study indicated that the nondestructive nature of the technology may offer a promising tool for accurate, quantitative in vitro measurements of the clotting properties of various classes of HAs, which may be used to better predict their in vivo outcomes.

Validation and application of a nondestructive and contactless method for rheological evaluation of biomaterials.

Hydrogels are extensively used for tissue engineering, cell therapy or controlled release of bioactive factors. Nondestructive techniques that can follow their viscoelastic properties during polymerization, remodeling, and degradation are needed, since these properties are determinant for their in vivo efficiency. In this work, we proposed the viscoelastic testing of bilayered materials (VeTBiM) as a new method for nondestructive and contact-less mechanical characterization of soft materials. The VeTBiM method measures the dynamic displacement response of a material, to a low amplitude vibration in order to characterize its viscoelastic properties. We validated VeTBiM by comparing data obtained on various agar and chitosan hydrogels with data from rotational rheometry, and compression tests. We then investigated its potential to follow the mechanical properties of chitosan hydrogels during gelation and in the presence of papain and lysozyme that induce fast or slow enzymatic degradation. Due to this nondestructive and contactless approach, samples can be removed from the instrument and stored in different conditions between measurements. VeTBiM is well adapted to follow biomaterials alone or with cells, over long periods of time. This new method will help in the fine tuning of the mechanical properties of biomaterials used for cell therapy and tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2565-2573, 2017.

Rheological assessment of a polymeric spherical structure using a three-dimensional shear wave scattering model in dynamic spectroscopy elastography.

With the purpose of assessing localized rheological behavior of pathological tissues using ultrasound dynamic elastography, an analytical shear wave scattering model was used in an inverse problem framework. The proposed method was adopted to estimate the complex shear modulus of viscoelastic spheres from 200 to 450 Hz. The inverse problem was formulated and solved in the frequency domain, allowing assessment of the complex viscoelastic shear modulus at discrete frequencies. A representative rheological model of the spherical obstacle was determined by comparing storage and loss modulus behaviors with Kelvin-Voigt, Maxwell, Zener, and Jeffrey models. The proposed inversion method was validated by using an external vibrating source and acoustic radiation force. The estimation of viscoelastic properties of three-dimensional spheres made softer or harder than surrounding tissues did not require a priori rheological assumptions. The proposed method is intended to be applied in the context of breast cancer imaging.

Design of a phased array for the generation of adaptive radiation force along a path surrounding a breast lesion for dynamic ultrasound elastography imaging.

This work demonstrates, with numerical simulations, the potential of an octagonal probe for the generation of radiation forces in a set of points following a path surrounding a breast lesion in the context of dynamic ultrasound elastography imaging. Because of the in-going wave adaptive focusing strategy, the proposed method is adapted to induce shear wave fronts to interact optimally with complex lesions. Transducer elements were based on 1-3 piezocomposite material. Three-dimensional simulations combining the finite element method and boundary element method with periodic boundary conditions in the elevation direction were used to predict acoustic wave radiation in a targeted region of interest. The coupling factor of the piezocomposite material and the radiated power of the transducer were optimized. The transducer's electrical impedance was targeted to 50 Ω. The probe was simulated by assembling the designed transducer elements to build an octagonal phased-array with 256 elements on each edge (for a total of 2048 elements). The central frequency is 4.54 MHz; simulated transducer elements are able to deliver enough power and can generate the radiation force with a relatively low level of voltage excitation. Using dynamic transmitter beamforming techniques, the radiation force along a path and resulting acoustic pattern in the breast were simulated assuming a linear isotropic medium. Magnitude and orientation of the acoustic intensity (radiation force) at any point of a generation path could be controlled for the case of an example representing a heterogeneous medium with an embedded soft mechanical inclusion.

Viscoelastic characterization of elliptical mechanical heterogeneities using a semi-analytical shear-wave scattering model for elastometry measures.

This paper presents a semi-analytical model of shear wave scattering by a viscoelastic elliptical structure embedded in a viscoelastic medium, and its application in the context of dynamic elastography imaging. The commonly used assumption of mechanical homogeneity in the inversion process is removed introducing a priori geometrical information to model physical interactions of plane shear waves with the confined mechanical heterogeneity. Theoretical results are first validated using the finite element method for various mechanical configurations and incidence angles. Secondly, an inverse problem is formulated to assess viscoelastic parameters of both the elliptic inclusion and its surrounding medium, and applied in vitro to characterize mechanical properties of agar-gelatin phantoms. The robustness of the proposed inversion method is then assessed under various noise conditions, biased geometrical parameters and compared to direct inversion, phase gradient and time-of-flight methods. The proposed elastometry method appears reliable in the context of estimating confined lesion viscoelastic parameters.

Hyper-frequency viscoelastic spectroscopy of biomaterials.

With the emergence of new biomaterials and elastography imaging techniques, there is a need for innovative instruments dedicated to viscoelasticity measurements. In this work, we introduce a novel hyper-frequency viscoelastic spectroscopy (HFVS) technique dedicated to characterize soft media subjected to mid-to-very-high frequency stress ranges (or, equivalently, to probe short-to-very-short relaxation times). HFVS, which has been implemented in an analytical instrument performing non-contact measurements in less than 1 s between 10 and 1000 Hz, is a suitable tool to study viscoelasticity for bio-applications. In this context, HFVS has been compared to classical oscillatory rheometry on several classes of soft materials currently encountered in tissue repair, bioengineering and elastography imaging on a frequency range between 10 and 100 Hz. After having demonstrated the good correspondence between HFVS and rheometry, this study has been completed by exploring the sensitivity of HFVS to physicochemically induced variations of viscoelasticity. HFVS opens promising perspectives in the challenging field of biomaterial science and for viscoelasticity-based quality control of materials.

Characterization of blood clot viscoelasticity by dynamic ultrasound elastography and modeling of the rheological behavior.

Dynamic elastography (DE) is a new tool to study mechanical behavior of soft tissues via their motion response to propagating shear waves. This technique characterized viscoelasticity of 9 porcine whole blood samples (3 animals) during coagulation for a shearing frequency of 70Hz, and after complete clot formation between 50 and 160Hz. Clot storage (G') and loss (G″) moduli were calculated from shear wave velocity and attenuation. Temporal evolutions of G' and G″ during coagulation were typified with 4 parameters: maximum change in elasticity (G' slope(max)), elasticity after 120min of coagulation (G'(max)), time occurrence of G″ maximum (t(e)) and G″ at the plateau (G″(plateau)). G' and G″ frequency dependence of completely formed blood clots was fitted with 5 standard rheological models: Maxwell, Kelvin-Voigt, Jeffrey, Zener and third-order generalized Maxwell. DE had sufficient sensitivity to follow the coagulation kinetics described by a progressive increase in G', while G″ transitory increased followed by a rapid stabilization. Inter- and intra-animal dispersions (InterAD and IntraAD) of G'(max) (InterAD=15.9%, IntraAD=9.1%) showed better reproducibility than G' slope(max) (InterAD=40.4%, IntraAD=21.9%), t(e) (InterAD=27.4%, IntraAD=18.7%) and G″(plateau) (InterAD=58.6%, IntraAD=40.2%). G' evolution within the considered range of frequency exhibited an increase, followed by stabilization to a plateau, whereas G″ presented little variations with convergence at a quasi-constant value at highest frequencies. Residues χ(⁎), describing the goodness of fit between models and experimental data, showed statistically (p<0.05) that the Kelvin-Voigt model was less in agreement with experimental data than other models. The Zener model is recommended to predict G' and G″ dispersion of coagulated blood over the explored frequency range.

Ultrasound dynamic micro-elastography applied to the viscoelastic characterization of soft tissues and arterial walls.

Quantitative noninvasive methods that provide in vivo assessment of mechanical characterization of living tissues, organs and artery walls are of interest because information on their viscoelastic properties in the presence of disease can affect diagnosis and treatment options. This article proposes the dynamic micro-elastography (DME) method to characterize viscoelasticity of small homogeneous soft tissues, as well as the adaptation of the method for vascular applications [vascular dynamic micro-elastography (VDME)]. The technique is based on the generation of relatively high-frequency (240-1100 Hz) monochromatic or transient plane shear waves within the medium and the tracking of these waves from radio-frequency (RF) echoes acquired at 25 MHz with an ultrasound biomicroscope (Vevo 770, Visualsonics). By employing a dedicated shear wave gated strategy during signal acquisition, postprocessed RF sequences could achieve a very high frame rate (16,000 images per s). The proposed technique successfully reconstructed shear wave displacement maps at very high axial (60 mum) and lateral (250 mum) spatial resolutions for motions as low as a few mum. An inverse problem formulated as a least-square minimization, involving analytical simulations (for homogenous and vascular geometries) and experimental measurements were performed to retrieve storage (G') and loss (G'') moduli as a function of the shearing frequency. Viscoelasticity measurements of agar-gelatin materials and of a small rat liver were proven feasible. Results on a very thin wall (3 mm thickness) mimicking artery enabled to validate the feasibility and the reliability of the vascular inverse problem formulation. Subsequently, the G' and G'' of a porcine aorta showed that both parameters are strongly dependent on frequency, suggesting that the vascular wall is mechanically governed by complex viscoelastic laws.