Ultra-high-speed dynamics of acoustic droplet vaporization in soft biomaterials: Effects of viscoelasticity, frequency, and bulk boiling point.

Phase-shift droplets are a highly adaptable platform for biomedical applications of ultrasound. The spatiotemporal response of phase-shift droplets to focused ultrasound above a certain pressure threshold, termed acoustic droplet vaporization (ADV), is influenced by intrinsic features (e.g., bulk boiling point) and extrinsic factors (e.g., driving frequency and surrounding media). A deep understanding of ADV dynamics is critical to ensure the robustness and repeatability of an ADV-assisted application. Here, we integrated ultra-high-speed imaging, at 10 million frames per second, and confocal microscopy for a full-scale (i.e., from nanoseconds to seconds) characterization of ADV. Experiments were conducted in fibrin-based hydrogels to mimic soft tissue environments. Effects of fibrin concentration (0.2 to 8 % (w/v)), excitation frequency (1, 2.5, and 9.4 MHz), and perfluorocarbon core (perfluoropentane, perfluorohexane, and perfluorooctane) on ADV dynamics were studied. Several fundamental parameters related to ADV dynamics, such as expansion ratio, expansion velocity, collapse radius, collapse time, radius of secondary rebound, resting radius, and equilibrium radius of the generated bubbles were extracted from the radius vs time curves. Diffusion-driven ADV-bubble growth was fit to a modified Epstein-Plesset equation, adding a material stress term, to estimate the growth rate. Our results indicated that ADV dynamics were significantly impacted by fibrin concentration, frequency, and perfluorocarbon liquid core. This is the first study to combine ultra-high-speed and confocal microscopy techniques to provide insights into ADV bubble dynamics in tissue-mimicking hydrogels.

Real-time spatiotemporal characterization of mechanics and sonoporation of acoustic droplet vaporization in acoustically responsive scaffolds.

Phase-shift droplets provide a flexible and dynamic platform for therapeutic and diagnostic applications of ultrasound. The spatiotemporal response of phase-shift droplets to focused ultrasound, via the mechanism termed acoustic droplet vaporization (ADV), can generate a range of bioeffects. Although ADV has been used widely in theranostic applications, ADV-induced bioeffects are understudied. Here, we integrated ultra-high-speed microscopy, confocal microscopy, and focused ultrasound for real-time visualization of ADV-induced mechanics and sonoporation in fibrin-based, tissue-mimicking hydrogels. Three monodispersed phase-shift droplets-containing perfluoropentane (PFP), perfluorohexane (PFH), or perfluorooctane (PFO)-with an average radius of ∼6 µm were studied. Fibroblasts and tracer particles, co-encapsulated within the hydrogel, were used to quantify sonoporation and mechanics resulting from ADV, respectively. The maximum radial expansion, expansion velocity, induced strain, and displacement of tracer particles were significantly higher in fibrin gels containing PFP droplets compared to PFH or PFO. Additionally, cell membrane permeabilization significantly depended on the distance between the droplet and cell (d), decreasing rapidly with increasing d. Significant membrane permeabilization occurred when d was smaller than the maximum radius of expansion. Both ultra-high-speed and confocal images indicate a hyper-local region of influence by an ADV bubble, which correlated inversely with the bulk boiling point of the phase-shift droplets. The findings provide insight into developing optimal approaches for therapeutic applications of ADV.

Ogden material calibration via magnetic resonance cartography, parameter sensitivity and variational system identification.

Contemporary material characterization techniques that leverage deformation fields and the weak form of the equilibrium equations face challenges in the numerical solution procedure of the inverse characterization problem. As material models and descriptions differ, so too must the approaches for identifying parameters and their corresponding mechanisms. The widely used Ogden material model can be comprised of a chosen number of terms of the same mathematical form, which presents challenges of parsimonious representation, interpretability and stability. Robust techniques for system identification of any material model are important to assess and improve experimental design, in addition to their centrality to forward computations. Using fully three-dimensional displacement fields acquired in silicone elastomers with our recently developed magnetic resonance cartography (MR-u) technique on the order of greater than [Formula: see text], we leverage partial differential equation-constrained optimization as the basis of variational system identification of our material parameters. We incorporate the statistical F-test to maintain parsimony of representation. Using a new, local deformation decomposition locally into mixtures of biaxial and uniaxial tensile states, we evaluate experiments based on an analytical sensitivity metric and discuss the implications for experimental design. This article is part of the theme issue 'The Ogden model of rubber mechanics: Fifty years of impact on nonlinear elasticity'.

Multi-time scale characterization of acoustic droplet vaporization and payload release of phase-shift emulsions using high-speed microscopy.

Acoustic droplet vaporization (ADV) is the phase-transitioning of perfluorocarbon emulsions, termed phase-shift emulsions, into bubbles using focused ultrasound. ADV has been utilized in many biomedical applications. For localized drug release, phase-shift emulsions with a bioactive payload can be incorporated within a hydrogel to yield an acoustically-responsive scaffold (ARS). The dynamics of ADV and associated drug release within hydrogels are not well understood. Additionally, emulsions used in ARSs often contain high molecular weight perfluorocarbons, which is unique relative to other ADV applications. In this study, we used ultra-high-speed brightfield and fluorescence microscopy, at frame rates up to 30 million and 0.5 million frames per second, respectively, to elucidate ADV dynamics and payload release kinetics in fibrin-based ARSs containing phase-shift emulsions with three different perfluorocarbons: perfluoropentane (PFP), perfluorohexane (PFH), and perfluorooctane (PFO). At an ultrasound excitation frequency of 2.5 MHz, the maximum expansion ratio, defined as the maximum bubble diameter during ADV normalized by the initial emulsion diameter, was 4.3 ± 0.8, 4.1 ± 0.6, and 3.6 ± 0.4, for PFP, PFH, PFO emulsions, respectively. ADV yielded stable bubble formation in PFP and PFH emulsions, though the bubble growth rate post-ADV was three orders of magnitudes slower in the latter emulsion. Comparatively, ADV generated bubbles in PFO emulsions underwent repeated vaporization/recondensation or fragmentation. Different ADV-generated bubble dynamics resulted in distinct release kinetics in phase-shift emulsions carrying fluorescently-labeled payloads. The results provide physical insight enabling the modulation of bubble dynamics with ADV and hence release kinetics, which can be used for both diagnostic and therapeutic applications of ultrasound.

Fiber splay precludes the direct identification of ligament material properties: Implications for ACL graft selection.

Anterior cruciate ligament (ACL) injuries typically require surgical reconstruction to restore adequate knee stability. The middle third of an injured patient's patellar tendon (PT) is a commonly used graft for ACL reconstruction. However, many clinicians and researchers question whether it is the best option, as several studies have suggested that it is a stiffer material than the ACL. Still, there is little to no consensus on even the most basic material property of ligaments/tendons: the tangent modulus in the fiber direction, or slope of the linear portion of the uniaxial stress-strain curve. In this study, we investigate the effect of fiber splay (the tendency of collagen fibers to spread out near the enthesis) on the apparent tangent modulus. Using a simplified theoretical model, we establish a quantity we call the splay ratio, which describes the relationship between splay geometry and the apparent tangent modulus. We then more rigorously investigate the effect of the splay ratio on the apparent tangent modulus of the ovine PT and anteromedial and posterolateral regions of the ACL using experimental and computational methods. Both approaches confirmed that splay geometry significantly affects the apparent material behavior. Because true material properties are independent of geometry, we conclude that the macroscopic response of ligaments and tendons is not sufficient for the characterization of their material properties, but rather is reflective of both material and structural properties. We further conclude that the PT is probably not a stiffer material than ACL, but that the PT graft is likely a stiffer structure than either ACL region.

Flagellar kinematics reveals the role of environment in shaping sperm motility.

Swimming spermatozoa from diverse organisms often have very similar morphologies, yet different motilities as a result of differences in the flagellar waveforms used for propulsion. The origin of these differences has remained largely unknown. Using high-speed video microscopy and mathematical analysis of flagellar shape dynamics, we quantitatively compare sperm flagellar waveforms from marine invertebrates to humans by means of a novel phylokinematic tree. This new approach revealed that genetically dissimilar sperm can exhibit strikingly similar flagellar waveforms and identifies two dominant flagellar waveforms among the deuterostomes studied here, corresponding to internal and external fertilizers. The phylokinematic tree shows marked discordance from the phylogenetic tree, indicating that physical properties of the fluid environment, more than genetic relatedness, act as an important selective pressure in shaping the evolution of sperm motility. More broadly, this work provides a physical axis to complement morphological and genetic studies to understand evolutionary relationships.

Application of mild hypothermia successfully mitigates neural injury in a 3D in-vitro model of traumatic brain injury.

Therapeutic hypothermia (TH) is an attractive target for mild traumatic brain injury (mTBI) treatment, yet significant gaps in our mechanistic understanding of TH, especially at the cellular level, remain and need to be addressed for significant forward progress to be made. Using a recently-established 3D in-vitro neural hydrogel model for mTBI we investigated the efficacy of TH after compressive impact injury and established critical treatment parameters including target cooling temperature, and time windows for application and maintenance of TH. Across four temperatures evaluated (31.5, 33, 35, and 37°C), 33°C was found to be most neuroprotective after 24 and 48 hours post-injury. Assessment of TH administration onset time and duration showed that TH should be administered within 4 hours post-injury and be maintained for at least 6 hours for achieving maximum viability. Cellular imaging showed TH reduced the percentage of cells positive for caspases 3/7 and increased the expression of calpastatin, an endogenous neuroprotectant. These findings provide significant new insight into the biological parameter space that renders TH effective in mitigating the deleterious effects of cellular mTBI and provides a quantitative foundation for the future development of animal and preclinical treatment protocols.

Robust high resolution strain imaging by alternating pulsed field gradient stimulated echo imaging (APGSTEi) at 7 Tesla.

A novel displacement-encoding spin-echo-stimulated-echo MRI sequence (APGSTEi) was used to obtain full-volume 3D strain fields in samples of two soft materials, a silicone elastomer and an ovine ligament. The samples were stretched cyclically and imaged synchronously. The multi-slice imaging sequence employed a combination of hard and soft spin-echos with bipolar gradient pulses for spatial encoding and decoding, combined with rapid multi-slice spin echo readouts. The sequence minimized undesirable signal loss due to T2∗ and T2 decays, which occur in polymeric materials or in the presence of appreciable air-solid susceptibility contrast, a particular concern for irregularly shaped samples in high magnetic fields. The images' magnitudes were T1-weighted; their phase encoded displacements which occurred during a Δ = 400 ms storage interval separating encoding and decoding pulses. Unwanted residual signals were filtered using a Gaussian filter tailored to attain the desired noise floor. The experiments measured 3D deformation with a nominal resolution of 290 µm × 250 µm × 250 µm in a sample volume of 5.6 cm × 1.6 cm × 1.6 cm, in less than an hour.

Modular approach for resolving and mapping complex neural and other cellular structures and their associated deformation fields in three dimensions.

Understanding the biological implications of cellular mechanotransduction, especially in the context of pathogenesis, requires the accurate resolution of material deformation and strain fields surrounding the cells. This is particularly challenging for cells displaying branched, 3D architectures. Here, we provide a modular approach for 3D image segmentation and strain mapping of topologically complex structures. We describe how to use our approach, using neural cells and networks as an example. In addition to describing how to implement the computational analysis, we provide details of a cell culture protocol that can be used to generate neural networks for analysis and experimentation. This protocol allows for transformation of matrix-induced strains, and their full resolution across single cells or networks in three dimensions. The protocol also provides analyses to compute both the locally varying cytoskeletal strains and the average strain experienced by cells. An additional module allows spatial correlation of these strain maps with cytoskeletal features, including neurite disruptions such as neuronal blebs. Image processing and strain mapping take ≥3 h, with the exact time required being dependent on use case, software familiarity, and file size.

Strain and rate-dependent neuronal injury in a 3D in vitro compression model of traumatic brain injury.

In the United States over 1.7 million cases of traumatic brain injury are reported yearly, but predictive correlation of cellular injury to impact tissue strain is still lacking, particularly for neuronal injury resulting from compression. Given the prevalence of compressive deformations in most blunt head trauma, this information is critically important for the development of future mitigation and diagnosis strategies. Using a 3D in vitro neuronal compression model, we investigated the role of impact strain and strain rate on neuronal lifetime, viability, and pathomorphology. We find that strain magnitude and rate have profound, yet distinctively different effects on the injury pathology. While strain magnitude affects the time of neuronal death, strain rate influences the pathomorphology and extent of population injury. Cellular injury is not initiated through localized deformation of the cytoskeleton but rather driven by excess strain on the entire cell. Furthermore we find that, mechanoporation, one of the key pathological trigger mechanisms in stretch and shear neuronal injuries, was not observed under compression.

Mean deformation metrics for quantifying 3D cell-matrix interactions without requiring information about matrix material properties.

Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamide- and polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress-strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix's displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels.

Differences in Morphology and Traction Generation of Cell Lines Representing Different Stages of Osteogenesis.

Osteogenesis is the process by which mesenchymal stem cells differentiate to osteoblasts and form bone. The morphology and root mean squared (RMS) traction of four cell types representing different stages of osteogenesis were quantified. Undifferentiated D1, differentiated D1, MC3T3-E1, and MLO-A5 cell types were evaluated using both automated image analysis of cells stained for F-actin and by traction force microscopy (TFM). Undifferentiated mesenchymal stem cell lines were small, spindly, and exerted low traction, while differentiated osteoblasts were large, had multiple processes, and exerted higher traction. Size, shape, and traction all correlated with the differentiation stage. Thus, cell morphology evolved and RMS traction increased with differentiation. The results provide a foundation for further work with these cell lines to study the mechanobiology of bone formation.