Spatiotemporal control of micromechanics and microstructure in acoustically-responsive scaffolds using acoustic droplet vaporization.

Acoustically-responsive scaffolds (ARSs), which are composite fibrin hydrogels, have been used to deliver regenerative molecules. ARSs respond to ultrasound in an on-demand, spatiotemporally-controlled manner via a mechanism termed acoustic droplet vaporization (ADV). Here, we study the ADV-induced, time-dependent micromechanical and microstructural changes to the fibrin matrix in ARSs using confocal fluorescence microscopy as well as atomic force microscopy. ARSs, containing phase-shift double emulsion (PSDE, mean diameter: 6.3 µm), were exposed to focused ultrasound to generate ADV - the phase transitioning of the PSDE into gas bubbles. As a result of ADV-induced mechanical strain, localized restructuring of fibrin occurred at the bubble-fibrin interface, leading to formation of locally denser regions. ADV-generated bubbles significantly reduced fibrin pore size and quantity within the ARS. Two types of ADV-generated bubble responses were observed in ARSs: super-shelled spherical bubbles, with a growth rate of 31 µm per day in diameter, as well as fluid-filled macropores, possibly as a result of acoustically-driven microjetting. Due to the strain stiffening behavior of fibrin, ADV induced a 4-fold increase in stiffness in regions of the ARS proximal to the ADV-generated bubble versus distal regions. These results highlight that the mechanical and structural microenvironment within an ARS can be spatiotemporally modulated using ultrasound, which could be used to control cellular processes and further the understanding of ADV-triggered drug delivery for regenerative applications.

Molecular, dynamic, and structural origin of inhomogeneous magnetization transfer in lipid membranes.

PURPOSE: To elucidate the dynamic, structural, and molecular properties that create inhomogeneous magnetization transfer (ihMT) contrast. METHODS: Amphiphilic lipids, lamellar phospholipids with cholesterol, and bovine spinal cord (BSC) specimens were examined along with nonlipid systems. Magnetization transfer (MT), enhanced MT (eMT, obtained with double-sided radiofrequency saturation), ihMT (MT - eMT), and dipolar relaxation, T1D , were measured at 2.0 and 11.7 T. RESULTS: The amplitude of ihMT ratio (ihMTR) is positively correlated with T1D values. Both ihMTR and T1D increase with increasing temperature in BSC white matter and in phospholipids and decrease with temperature in other lipids. Changes in ihMTR with temperature arise primarily from alterations in MT rather than eMT. Spectral width of MT, eMT, and ihMT increases with increasing carbon chain length. CONCLUSIONS: Concerted motions of phospholipids in white matter decrease proton spin diffusion leading to increased proton T1D times and increased ihMT amplitudes, consistent with decoupling of Zeeman and dipolar spin reservoirs. Molecular specificity and dynamic sensitivity of ihMT contrast make it a suitable candidate for probing myelin membrane disorders. Magn Reson Med 77:1318-1328, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Imaging and sensing based on dual-pulse nonlinear photoacoustic contrast: a preliminary study on fatty liver.

The feasibility of diagnostic imaging and tissue characterization based on a new contrast realized by dual-pulse photoacoustic measurement was studied. Unlike current photoacoustic methods which are mostly focused on the measurement of tissue optical absorption, this contrast revealed by a dual-pulse laser excitation process takes advantage of the temperature dependence of the Grüneisen parameter of tissue. The first laser pulse heats the sample and causes a temperature rise in the target tissue, which leads to a change of the Grüneisen parameter and the amplitude of the photoacoustic signal from the second laser pulse. This new contrast is then quantified by percentile change in the second pulse signal as a result of the first laser pulse. Since the temperature-dependent Grüneisen parameter is tissue specific and closely relevant to chemical and molecular properties of the sample, the dual-pulse photoacoustic measurement can differentiate various tissue types and conditions. The preliminary study on phantoms and a mouse model has suggested the capability of the proposed contrast in the characterization of fatty livers and the potential for future clinical diagnosis of liver conditions.

Acceleration of ultrasound thermal therapy by patterned acoustic droplet vaporization.

One application of acoustic droplet vaporization (ADV), a method of converting biocompatible microdroplets into microbubbles, is to enhance locally high intensity focused ultrasound (HIFU) therapy. Two objectives are pursued here: (1) the controlled creation of a bubble trench prior to HIFU using ADV and (2) use of the trench for increasing ablation volumes, lowering acoustic powers, and decreasing therapy duration. Thermally responsive phantoms were made with perfluorocarbon emulsion. Compound lesions were formed in a laboratory setting and a clinical magnetic resonance imaging (MRI)-guided HIFU system. Linear and spiral patterned compound lesions were generated in trenches. A larger fraction of the HIFU beam is contained to increase the generation of heat. Using the laboratory system, a 90 mm linear length spiral trench was formed in 30 s with mechanical beam steering. Comparatively, the clinical HIFU system formed a 19.9 mm linear length spiral trench in approximately 1 s with electronic beam steering. Lesions were imaged optically and with MRI. A uniform thermal ablation volume of 3.25 mL was achieved in 55.4 s (4-times faster than standard clinical HIFU and 14-times larger volume versus sum of individual lesions). Single lesions showed a 400% volume increase.

Acoustically responsive scaffolds: Unraveling release kinetics and mechanisms for sustained, steady drug delivery.

Hydrogels can serve as local drug delivery depots that protect the biological activity of labile therapeutics. However, drug release from conventional hydrogels is typically rapid, which is not ideal for many therapeutic agents. We developed a composite hydrogel that enables sustained drug release in response to ultrasound. The composite, termed an acoustically responsive scaffold (ARS), consists of a fibrin hydrogel and a phase-shift emulsion. Upon exposure to ultrasound, the emulsion is vaporized into bubbles, which leads to release of drugs contained within the emulsion. Previously, ARSs have been used in regenerative applications to stimulate blood vessel growth. Here, we characterize the release kinetics and mechanisms of ARSs. Release exhibits a triphasic pattern compromising a slow phase prior to ultrasound exposure; a transient, fast phase immediately after ultrasound exposure that follows a sigmoidal profile; and a sustained, steady phase. In each phase, we demonstrate how derived kinetics parameters are impacted by the ARS composition (e.g., fibrin and emulsion concentrations) and ultrasound properties (e.g., acoustic pressure, pulse duration). Using confocal microscopy, protein assays, and B-mode ultrasound imaging, we demonstrate that drug release from an ARS is independent of fibrin degradation and dependent on bubble growth. These results are critical in optimizing ARSs for delivery of therapeutic agents.

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.

The radial dynamics and acoustic emissions of phase-shift droplets are impacted by mechanical properties of tissue-mimicking hydrogels.

Acoustic droplet vaporization (ADV) offers a dynamic approach for generating bubbles on demand, presenting new possibilities in biomedical applications. Although ADV has been investigated in various biomedical applications, its potential in tissue characterization remains unexplored. Here, we investigated the effects of surrounding media on the radial dynamics and acoustic emissions of ADV bubbles using theoretical and experimental methodologies. For theoretical studies, bubble dynamics were combined with the Kelvin-Voigt material constitutive model, accounting for viscoelasticity of the media. The radial dynamics and acoustic emissions of the ADV-bubbles were recorded via ultra-high-speed microscopy and passive cavitation detection, respectively. Perfluoropentane phase-shift droplets were embedded in tissue-mimicking hydrogels of varying fibrin concentrations, representing different elastic moduli. Radial dynamics and the acoustic emissions, both temporal and spectral, of the ADV-bubbles depended significantly on fibrin elastic modulus. For example, an increase in fibrin elastic modulus from ≈0.2 kPa to ≈6 kPa reduced the maximum expansion radius of the ADV-bubbles by 50%. A similar increase in the elastic modulus significantly impacted both linear (e.g., fundamental) and nonlinear (e.g., subharmonic) acoustic responses of the ADV-bubbles, by up to 10 dB. The sensitivity of ADV to the surrounding media was dependent on acoustic parameters such as driving pressure and the droplets concentration. Further analysis of the acoustic emissions revealed distinct ADV signal characteristics, which were significantly influenced by the surrounding media.

Simultaneous viewing of individual cells and ambient microvasculature using optical absorption and fluorescence contrasts.

Viewing individual cells and ambient microvasculature simultaneously is crucial for understanding tumor angiogenesis and microenvironments. We developed a confocal fluorescence microscopy (CFM) and photoacoustic microscopy (PAM) dual-modality imaging system that can assess fluorescent contrast and optical absorption contrast in biologic samples simultaneously. After staining tissues with fluorescent dye at an appropriate concentration, each laser pulse can generate not only sufficient fluorescent signals from cells for CFM but also sufficient photoacoustic signals from microvessels for PAM. To explore the potential of this system for diagnosis of bladder cancer, experiments were conducted on a rat bladder model. The CFM image depicts the morphology of individual cells, showing not only large polygonal umbrella cells but also intracellular components. The PAM image acquired at the same time provides complementary information on the microvascular distribution in the bladder wall, ranging from large vessels to capillaries. This device provides an opportunity to realize both histologic assay and microvascular characterization simultaneously. The combination of the information of individual cells and local microvasculature in the bladder offers the capability of envisioning the viability and activeness of these cells and holds promise for more comprehensive study of bladder cancer in vivo.

Bubble nucleation and dynamics in acoustic droplet vaporization: a review of concepts, applications, and new directions.

The development of phase-shift droplets has broadened the scope of ultrasound-based biomedical applications. When subjected to sufficient acoustic pressures, the perfluorocarbon phase in phase-shift droplets undergoes a phase-transition to a gaseous state. This phenomenon, termed acoustic droplet vaporization (ADV), has been the subject of substantial research over the last two decades with great progress made in design of phase-shift droplets, fundamental physics of bubble nucleation and dynamics, and applications. Here, we review experimental approaches, carried out via high-speed microscopy, as well as theoretical models that have been proposed to study the fundamental physics of ADV including vapor nucleation and ADV-induced bubble dynamics. In addition, we highlight new developments of ADV in tissue regeneration, which is a relatively recently exploited application. We conclude this review with future opportunities of ADV for advanced applications such as in situ microrheology and pressure estimation.

Ultrasound Contrast Stability for Urinary Bladder Pressure Measurement.

The goal of this study was to evaluate ultrasound contrast microbubbles (MB) stability during a typical cystometrogram (CMG) for bladder pressure measurement application using the subharmonic-aided pressure estimation technique. A detailed study of MB stability was required given two unique characteristics of this application: first, bulk infusion of MBs into the bladder through the CMG infusion system, and second, duration of a typical CMG which may last up to 30 min. To do so, a series of size measurement and contrast-enhanced ultrasound imaging studies under different conditions were performed and the effects of variables that we hypothesized have an effect on MB stability, namely, i) IV bag air headspace, ii) MB dilution factor, and iii) CMG infusion system were investigated. The results verified that air volume in intravenous (IV) bag headspace was not enough to have a significant effect on MB stability during a CMG. We also showed that higher MB dosage results in a more stable condition. Finally, the results indicated that the CMG infusion system adversely affects MB stability. In summary, to ensure MB stability during the entire duration of a CMG, lower filling rates (limited by estimated bladder capacity in clinical applications) and/or higher MB dosage (limited by FDA regulations and shadowing artifact) and/or the consideration of alternative catheter design may be needed.

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.

Acoustic droplet vaporization for on-demand modulation of microporosity in smart hydrogels.

Microporosity in hydrogels is critical for directing tissue formation and function. We have developed a fibrin-based smart hydrogel, termed an acoustically responsive scaffold (ARS), which responds to focused ultrasound in a spatiotemporally controlled, user-defined manner. ARSs are highly flexible platforms due to the inclusion of phase-shift droplets and their tunable response to ultrasound through a mechanism termed acoustic droplet vaporization (ADV). Here, we demonstrated that ADV enabled consistent generation of micropores in ARSs, throughout the entire thickness (∼5.5 mm), utilizing perfluorooctane phase-shift droplets. Size characteristics of the generated micropores were quantified in response to critical parameters including acoustic properties, droplet size, and shear elastic modulus of fibrin using confocal microscopy. The findings showed that the length of the generated micropores correlated directly with excitation frequency, peak rarefactional pressure, pulse duration, droplet size, and indirectly with the shear elastic modulus of the fibrin matrix. The ADV-generated micropores in ARSs were further compared with cavitation-mediated micropores in fibrin gels without droplets. Additionally, the Keller-Miksis equation was used to predict an upper bound for micropore formation in ARSs at varying driving frequencies and droplet sizes. Finally, our in vivo studies showed that host cell migration following ADV-induced micropore formation was frequency-dependent, with up to 2.6 times higher cell migration at lower frequencies. Overall, these findings demonstrate a new potential application of ADV in hydrogels. STATEMENT OF SIGNIFICANCE: Interconnected micropores within a hydrogel can facilitate many cell-mediated processes. Most techniques for generating micropores are typically not biocompatible or do not enable controlled, in situ micropore formation. We used an ultrasound-based technique, termed acoustic droplet vaporization, to generate microporosity in smart hydrogels termed acoustically responsive scaffolds (ARSs). ARSs contain a fibrin matrix doped with a phase-shift droplet. We demonstrate that unique acoustic properties of phase-shift droplets can be tailored to yield spatiotemporally controlled, on-demand micropore formation. Additionally, the size characteristics of the ultrasound-generated micropores can be modulated by tuning ultrasound parameters, droplet properties, and bulk elastic properties of fibrin. Finally, we demonstrate significant, frequency-dependent host cell migration in subcutaneously implanted ARSs in mice following ultrasound-induced micropore formation in situ.

Pressure Measurement in a Bladder Phantom Using Contrast-Enhanced Ultrasonography-A Path to a Catheter-Free Voiding Cystometrogram.

OBJECTIVES: The long-term goal of this study is to investigate the efficacy of a novel, ultrasound-based technique called subharmonic-aided pressure estimation (SHAPE) to measure bladder pressure as a part of a cystometrogram (CMG) in a urodynamic test (ie, pressure-flow study). SHAPE is based on the principle that subharmonic emissions from ultrasound contrast microbubbles (MBs) decrease linearly with an increase in ambient pressure. We hypothesize that, using the SHAPE technique, we can measure voiding bladder pressure catheter-free. This is of importance because the CMG catheter, due to its space-occupying property and non-physiological effects, can undermine the reliability of the test during voiding and cause misdiagnosis. In this study, we tested this hypothesis and optimized the protocol in a controlled benchtop environment. MATERIALS AND METHODS: A bladder phantom was designed and built, capable of simulating clinically relevant bladder pressures. Laboratory-made lipid-shelled MBs (similar in composition to the commercial agent, DEFINITY) was diluted in 0.9% normal saline and infused into the bladder phantom using the CMG infusion system. A typical simulated CMG consists of 1 filling and 4 post-filling events. During CMG events, the bladder phantom is pressurized multiple times at different clinically relevant levels (small, medium, and large) to simulate bladder pressures. Simultaneous with pressurization, MB subharmonic signal was acquired. For each event, the change in MB subharmonic amplitude was correlated linearly with the change in bladder phantom pressure, and the SHAPE conversion factor (slope of the linear fit) was determined. In doing so, a specific signal processing technique (based on a small temporal window) was used to account for time-decay of MB subharmonic signal during a simulated CMG. RESULTS: A strong inverse linear relationship was found to exist between SHAPE and bladder phantom pressures for each of the CMG filling and post-filling events ( r2> 0.9, root mean square error < 0.3 dB, standard error <0.01 dB, and P < 0.001). SHAPE showed a transient behavior in measuring bladder phantom pressure. The SHAPE conversion factor (in dB/cm H 2 O) varied between filling and post-filling events, as well as by post-filling time. The magnitude of the SHAPE conversion factor tended to increase immediately after filling and then decreases with time. CONCLUSIONS: Microbubble subharmonic emission is an excellent indicator of bladder phantom pressure variation. The strong correlation between SHAPE signal and bladder phantom pressure is indicative of the applicability of this method in measuring bladder pressure during a CMG. Our results suggest that different SHAPE conversion factors may be needed for different events during a CMG (ie, at different time points of a CMG). These findings will help us better protocolize this method for introduction into human subjects and allow us to take the next step toward developing a catheter-free voiding CMG using SHAPE.

In vivo microscopy of targeted vessel occlusion employing acoustic droplet vaporization.

OBJECTIVE: Embolotherapy is a potential means to treat a variety of cancers. Our approach-gas embolotherapy-introduces the droplets upstream from the tumor and then acoustically activates them to form bubbles for occlusion-a process known as ADV. We wanted to provide the first optical documentation of ADV, lodged bubbles, or vessel occlusion in vivo. METHODS: We used the rat cremaster muscle for in vivo microscopy. Perfluorocarbon droplets were administered into the aortic arch. Ultrasound exposures in the cremaster induced vaporization. The cremaster was examined pre- and post-exposure for ADV-related effects. Two sets of experiments compared the effect of exposure in the capillaries versus the first order arteriole. RESULTS: Bubbles that lodge following capillary exposure are significantly larger (76 µm mean length, 36 µm mean diameter) than those following feeder vessel exposure (25 µm mean length, 11 µm mean diameter). Despite the differing sizes in bubbles, the ratio of bubble length to the hydraulic diameter of all lodged bubbles was 2.11 (±0.65; n = 112), which agrees with theoretical predictions and experimental observations. CONCLUSIONS: Our results provide the first optical evidence of targeted vessel occlusion through ADV. These findings could lay the groundwork for the advancement of gas embolotherapy.

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.

Slow-Flow Ultrasound Localization Microscopy Using Recondensation of Perfluoropentane Nanodroplets.

Ultrasound localization microscopy (ULM) is an emerging, super-resolution imaging technique for detailed mapping of the microvascular structure and flow velocity via subwavelength localization and tracking of microbubbles. Because microbubbles rely on blood flow for movement throughout the vascular space, acquisition times can be long in the smallest, low-flow microvessels. In addition, detection of microbubbles in low-flow regions can be difficult because of minimal separation of microbubble signal from tissue. Nanoscale, phase-change contrast agents (PCCAs) have emerged as a switchable, intermittent or persisting contrast agent for ULM via acoustic droplet vaporization (ADV). Here, the focus is on characterizing the spatiotemporal contrast properties of less volatile perfluoropentane (PFP) PCCAs. The results indicate that at physiological temperature, nanoscale PFP PCCAs with diameters less than 100 nm disappear within microseconds after ADV with high-frequency ultrasound (16 MHz, 5- to 6-MPa peak negative pressure) and that nanoscale PFP PCCAs have an inherent deactivation mechanism via immediate recondensation after ADV. This "blinking" on-and-off contrast signal allowed separation of flow in an in vitro flow phantom, regardless of flow conditions, although with a need for some replenishment at very low flow conditions to maintain count rate. This blinking behavior allows for rapid spatial mapping in areas of low or no flow with ULM, but limits velocity tracking because there is no stable bubble formation with nanoscale PFP PCCAs.

Micropatterning of acoustic droplet vaporization in acoustically-responsive scaffolds using extrusion-based bioprinting.

Acoustically-responsive scaffolds (ARSs) are composite hydrogels that respond to ultrasound in an on-demand, spatiotemporally-controlled manner due to the presence of a phase-shift emulsion. When exposed to ultrasound, a gas bubble is formed within each emulsion droplet via a mechanism termed acoustic droplet vaporization (ADV). In previous in vitro and in vivo studies, we demonstrated that ADV can control regenerative processes by releasing growth factors and/or modulating micromechanics in ARSs. Precise, spatial patterning of emulsion within an ARS could be beneficial for ADV-induced modulation of biochemical and biophysical cues. However, precise patterning is limited using conventional bulk polymerization techniques. Here, we developed an extrusion-based method for bioprinting ARSs with micropatterned structures. Emulsions were loaded within bioink formulations containing fibrin, hyaluronic acid and/or alginate. Experimental as well as theoretical studies elucidated the interrelations between printing parameters, needle geometry, rheological properties of the bioink, and the process-induced mechanical stresses during bioprinting. The shear thinning properties of the bioinks enabled use of lower extrusion pressures resulting in decreased shear stresses and shorter residence times, thereby facilitating high viability for cell-loaded bioinks. Bioprinting yielded greater alignment of fibrin fibers in ARSs compared to conventionally polymerized ARSs. Bioprinted ARSs also enabled generation of ADV at high spatial resolutions, which were otherwise not achievable in conventional ARSs, and acoustically-driven collapse of ADV-induced bubbles. Overall, bioprinting could aid in optimizing ARSs for therapeutic applications.

Spatiotemporal control of myofibroblast activation in acoustically-responsive scaffolds via ultrasound-induced matrix stiffening.

Hydrogels are often used to study the impact of biomechanical and topographical cues on cell behavior. Conventional hydrogels are designed a priori, with characteristics that cannot be dynamically changed in an externally controlled, user-defined manner. We developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), that enables non-invasive, spatiotemporally controlled modulation of mechanical and morphological properties using focused ultrasound. An ARS consists of a phase-shift emulsion distributed in a fibrin matrix. Ultrasound non-thermally vaporizes the emulsion into bubbles, which induces localized, radial compaction and stiffening of the fibrin matrix. In this in vitro study, we investigate how this mechanism can control the differentiation of fibroblasts into myofibroblasts, a transition correlated with substrate stiffness on 2D substrates. Matrix compaction and stiffening was shown to be highly localized using confocal and atomic force microscopies, respectively. Myofibroblast phenotype, evaluated by α-smooth muscle actin (α-SMA) immunocytochemistry, significantly increased in matrix regions proximal to bubbles compared to distal regions, irrespective of the addition of exogenous transforming growth factor-ß1 (TGF-ß1). Introduction of the TGF-ß1 receptor inhibitor SB431542 abrogated the proximal enhancement. This approach providing spatiotemporal control over biophysical signals and resulting cell behavior could aid in better understanding fibrotic disease progression and the development of therapeutic interventions for chronic wounds. STATEMENT OF SIGNIFICANCE: Hydrogels are used in cell culture to recapitulate both biochemical and biophysical aspects of the native extracellular matrix. Biophysical cues like stiffness can impact cell behavior. However, with conventional hydrogels, there is a limited ability to actively modulate stiffness after polymerization. We have developed an ultrasound-based method of spatiotemporally-controlling mechanical and morphological properties within a composite hydrogel, termed an acoustically-responsive scaffold (ARS). Upon exposure to ultrasound, bubbles are non-thermally generated within the fibrin matrix of an ARS, thereby locally compacting and stiffening the matrix. We demonstrate how ARSs control the differentiation of fibroblasts into myofibroblasts in 2D. This approach could assist with the study of fibrosis and the development of therapies for chronic wounds.

Ultrasound-Induced Mechanical Compaction in Acoustically Responsive Scaffolds Promotes Spatiotemporally Modulated Signaling in Triple Negative Breast Cancer.

Cancer cells continually sense and respond to mechanical cues from the extracellular matrix (ECM). Interaction with the ECM can alter intracellular signaling cascades, leading to changes in processes that promote cancer cell growth, migration, and survival. The present study used a recently developed composite hydrogel composed of a fibrin matrix and phase-shift emulsion, termed an acoustically responsive scaffold (ARS), to investigate effects of local mechanical properties on breast cancer cell signaling. Treatment of ARSs with focused ultrasound drives acoustic droplet vaporization (ADV) in a spatiotemporally controlled manner, inducing local compaction and stiffening of the fibrin matrix adjacent to the matrix-bubble interface. Combining ARSs and live single cell imaging of triple-negative breast cancer cells, it is discovered that both basal and growth-factor stimulated activities of protein kinase B (also known as Akt) and extracellular signal-regulated kinase (ERK), two major kinases driving cancer progression, negatively correlate with increasing distance from the ADV-induced bubble both in vitro and in a mouse model. Together, these data demonstrate that local changes in ECM compaction regulate Akt and ERK signaling in breast cancer and support further applications of the novel ARS technology to analyze spatial and temporal effects of ECM mechanics on cell signaling and cancer biology.

Drug delivery monitoring by photoacoustic tomography with an ICG encapsulated double emulsion.

The absorption spectrum of indocyanine green (ICG), a nontoxic dye used for medical diagnostics, depends upon its concentration as well as the nature of its environment, i.e., the solvent medium into which it is dissolved. In blood, ICG binds with plasma proteins, thus causing changes in its photoacoustic spectrum. We successfully encapsulated ICG in an ultrasound-triggerable perfluorocarbon double emulsion that prevents ICG from binding with plasma proteins. Photoacoustic spectral measurements on point target as well as 2-D photoacoustic images of blood vessels revealed that the photoacoustic spectrum changes significantly in blood when the ICG-loaded emulsion undergoes acoustic droplet vaporization (ADV), which is the conversion of liquid droplets into gas bubbles using ultrasound. We propose that these changes in the photoacoustic spectrum of the ICG emulsion in blood, coupled with photoacoustic tomography, could be used to spatially and quantitatively monitor ultrasound initiated drug delivery. In addition, we suggest that the photoacoustic spectral change induced by ultrasound exposure could also be used as contrast in photoacoustic imaging to obtain a background free image.