The image-based ultrasonic cell shaking test.

Mechanical signals play a vital role in cell biology and is a vast area of research. Thus, there is motivation to understand cell deformation and mechanobiological responses. However, the ability to controllably deform cells in the ultrasonic regime and test their response is a noted challenge throughout the literature. Quantifying and eliciting an appropriate stimulus has proven to be difficult, resulting in methods that are either too aggressive or oversimplified. Furthermore, the ability to gain a real-time insight into cell deformation and link this with the biological response is yet to be achieved. One application of this understanding is in ultrasonic surgical cutting, which is a promising alternative to traditional methods, but with little understanding of its effect on cells. Here we present the image based ultrasonic cell shaking test, a novel method that enables controllable loading of cells and quantification of their response to ultrasonic vibrations. Practically, this involves seeding cells on a substrate that resonates at ultrasonic frequencies and transfers the deformation to the cells. This is then incorporated into microscopic imaging techniques to obtain high-speed images of ultrasonic cell deformation that can be analysed using digital image correlation techniques. Cells can then be extracted after excitation to undergo analysis to understand the biological response to the deformation. This method could aid in understanding the effects of ultrasonic stimulation on cells and how activated mechanobiological pathways result in physical and biochemical responses.

An Acoustic Device for Ultra High-Speed Quantification of Cell Strain During Cell-Microbubble Interaction.

Microbubbles utilize high-frequency oscillations under ultrasound stimulation to induce a range of therapeutic effects in cells, often through mechanical stimulation and permeabilization of cells. One of the largest challenges remaining in the field is the characterization of interactions between cells and microbubbles at therapeutically relevant frequencies. Technical limitations, such as employing sufficient frame rates and obtaining sufficient image resolution, restrict the quantification of the cell's mechanical response to oscillating microbubbles. Here, a novel methodology was developed to address many of these limitations and improve the image resolution of cell-microbubble interactions at high frame rates. A compact acoustic device was designed to house cells and microbubbles as well as a therapeutically relevant acoustic field while being compatible with a Shimadzu HPV-X camera. Cell viability tests confirmed the successful culture and proliferation of cells, and the attachment of DSPC- and cationic DSEPC-microbubbles to osteosarcoma cells was quantified. Microbubble oscillation was observed within the device at a frame rate of 5 million FPS, confirming suitable acoustic field generation and ultra high-speed image capture. High spatial resolution in these images revealed observable deformation in cells following microbubble oscillation and supported the first use of digital image correlation for strain quantification in a single cell. The novel acoustic device provided a simple, effective method for improving the spatial resolution of cell-microbubble interaction images, presenting the opportunity to develop an understanding of the mechanisms driving the therapeutic effects of oscillating microbubbles upon ultrasound exposure.

Modelling of stress transfer in root-reinforced soils informed by four-dimensional X-ray computed tomography and digital volume correlation data.

Vegetation enhances soil shearing resistance through water uptake and root reinforcement. Analytical models for soils reinforced with roots rely on input parameters that are difficult to measure, leading to widely varying predictions of behaviour. The opaque heterogeneous nature of rooted soils results in complex soil-root interaction mechanisms that cannot easily be quantified. The authors measured, for the first time, the shear resistance and deformations of fallow, willow-rooted and gorse-rooted soils during direct shear using X-ray computed tomography and digital volume correlation. Both species caused an increase in shear zone thickness, both initially and as shear progressed. Shear zone thickness peaked at up to 35 mm, often close to the thickest roots and towards the centre of the column. Root extension during shear was 10-30% less than the tri-linear root profile assumed in a Waldron-type model, owing to root curvature. Root analogues used to explore the root-soil interface behaviour suggested that root lateral branches play an important role in anchoring the roots. The Waldron-type model was modified to incorporate non-uniform shear zone thickness and growth, and accurately predicted the observed, up to sevenfold, increase in shear resistance of root-reinforced soil.

Measurement of Internal Implantation Strains in Analogue Bone Using DVC.

The survivorship of cementless orthopaedic implants may be related to their initial stability; insufficient press-fit can lead to excessive micromotion between the implant and bone, joint pain, and surgical revision. However, too much interference between implant and bone can produce excessive strains and damage the bone, which also compromises stability. An understanding of the nature and mechanisms of strain generation during implantation would therefore be valuable. Previous measurements of implantation strain have been limited to local discrete or surface measurements. In this work, we devise a Digital Volume Correlation (DVC) methodology to measure the implantation strain throughout the volume. A simplified implant model was implanted into analogue bone media using a customised loading rig, and a micro-CT protocol optimised to minimise artefacts due to the presence of the implant. The measured strains were interpreted by FE modelling of the displacement-controlled implantation, using a bilinear elastoplastic constitutive model for the analogue bone. The coefficient of friction between the implant and bone was determined using the experimental measurements of the reaction force. Large strains at the interface between the analogue bone and implant produced localised deterioration of the correlation coefficient, compromising the ability to measure strains in this region. Following correlation coefficient thresholding (removing strains with a coefficient less than 0.9), the observed strain patterns were similar between the DVC and FE. However, the magnitude of FE strains was approximately double those measured experimentally. This difference suggests the need for improvements in the interface failure model, for example, to account for localised buckling of the cellular analogue bone structure. A further recommendation from this work is that future DVC experiments involving similar geometries and structures should employ a subvolume size of 0.97 mm as a starting point.

Characterising the compressive anisotropic properties of analogue bone using optical strain measurement.

The validity of conclusions drawn from pre-clinical tests on orthopaedic devices depends upon accurate characterisation of the support materials: frequently, polymer foam analogues. These materials often display anisotropic mechanical behaviour, which may considerably influence computational modelling predictions and interpretation of experiments. Therefore, this study sought to characterise the anisotropic mechanical properties of a range of commonly used analogue bone materials, using non-contact multi-point optical extensometry method to account for the effects of machine compliance and uneven loading. Testing was conducted on commercially available 'cellular', 'solid' and 'open-cell' Sawbone blocks with a range of densities. Solid foams behaved largely isotropically. However, across the available density range of cellular foams, the average Young's modulus was 23%-31% lower (p < 0.005) perpendicular to the foaming direction than parallel to it, indicating elongation of cells with foaming. The average Young's modulus of open-celled foams was 25%-59% higher (p < 0.05) perpendicular to the foaming direction than parallel to it. This is thought to result from solid planes of material that were observed perpendicular to the foaming direction, stiffening the bulk material. The presented data represent a reference to help researchers design, model and interpret tests using these materials.

Sensitivity-based virtual fields for the non-linear virtual fields method.

The virtual fields method is an approach to inversely identify material parameters using full-field deformation data. In this manuscript, a new set of automatically-defined virtual fields for non-linear constitutive models has been proposed. These new sensitivity-based virtual fields reduce the influence of noise on the parameter identification. The sensitivity-based virtual fields were applied to a numerical example involving small strain plasticity; however, the general formulation derived for these virtual fields is applicable to any non-linear constitutive model. To quantify the improvement offered by these new virtual fields, they were compared with stiffness-based and manually defined virtual fields. The proposed sensitivity-based virtual fields were consistently able to identify plastic model parameters and outperform the stiffness-based and manually defined virtual fields when the data was corrupted by noise.

Elastic stiffness characterization using three-dimensional full-field deformation obtained with optical coherence tomography and digital volume correlation.

This paper presents a methodology for stiffness identification from depth-resolved three-dimensional (3-D) full-field deformation fields. These were obtained by performing digital volume correlation on optical coherence tomography volume reconstructions of silicone rubber phantoms. The effect of noise and reconstruction uncertainties on the performance of the correlation algorithm was first evaluated through stationary and rigid body translation tests to give an indication of the minimum strain that can be reliably measured. The phantoms were then tested under tension, and the 3-D deformation fields were used to identify the elastic constitutive parameters using a 3-D manually defined virtual fields method. The identification results for the cases of uniform and heterogeneous strain fields were compared with those calculated analytically through the constant uniaxial stress assumption, showing good agreement.