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.

Effects of droplet size and perfluorocarbon boiling point on the frequency dependence of acoustic vaporization threshold.

Phase shift liquid perfluorocarbon (PFC) droplets vaporizable by ultrasound into echogenic microbubble above a threshold pressure, termed acoustic droplet vaporization (ADV), are used for therapeutic and diagnostic applications. This study systematically investigated the effect of excitation frequency (2.25, 10, and 15 MHz) on the ADV and inertial cavitation (IC) thresholds of lipid-coated PFC droplets of three different liquid cores-perfluoropentane (PFP), perfluorohexane (PFH), and perfluorooctyl bromide (PFOB)-and of two different sizes-average diameters smaller than 3 µm and larger than 10 µm-in a tubeless setup. This study found that the ADV threshold increases with frequency for the lowest boiling point liquid, PFP, for both large and small size droplets. For higher boiling point liquids, PFH and PFOB, this study did not detect vaporization for small size droplets at the excitation levels (maximum 4 MPa peak negative) studied here. The large PFOB droplets experienced ADV only at the highest excitation frequency 15 MHz. For large PFH droplets, ADV threshold decreases with frequency that could possibly be due to the superharmonic focusing being a significant effect at larger sizes and the higher excitation pressures. ADV thresholds at all the frequencies studied here occurred at lower rarefactional pressures than IC thresholds indicating that phase transition precedes inertial cavitation.

Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying pore geometries (square and hexagonal channels) were investigated. The scaffold with square pores resulted in higher hMSC growth and chondrogenic differentiation than a solid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz were found to be 100 mW/cm2 and 20% duty cycle. LIPUS stimulation increased proliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilage biomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen and total collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% for scaffolds with square and hexagonal patterns, respectively, after 2 weeks. Additionally, type II collagen production increased by 60% and 40% for the same patterns, respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUS stimulation, which has already been approved by FDA for treatment of bone fracture, could be a highly efficient tool for tissue engineering in combination with 3D printing and hMSCs to regenerate damaged cartilage tissues.

Acoustic vaporization threshold of lipid-coated perfluoropentane droplets.

Phase shift droplets vaporizable by acoustic stimulation offer the advantages of producing microbubbles as contrast agents in situ as well as higher stability and the possibility of achieving smaller sizes. Here, the acoustic droplet vaporization (ADV) threshold of a suspension of droplets with a perfluoropentane (PFP) core (diameter 400-3000 nm) is acoustically measured as a function of the excitation frequency in a tubeless setup at room temperature. The changes in scattered responses-fundamental, sub-, and second harmonic-are investigated, a quantitative criterion is used to determine the ADV phenomenon, and findings are discussed. The average threshold obtained using three different scattered components increases with frequency-1.05 ± 0.28 MPa at 2.25 MHz, 1.89 ± 0.57 MPa at 5 MHz, and 2.34 ± 0.014 MPa at 10 MHz. The scattered response from vaporized droplets was also found to qualitatively match with that from an independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the determined ADV threshold value.

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.

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.

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.

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.

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.

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.

Material Properties, Dissolution and Time Evolution of PEGylated Lipid-Shelled Microbubbles: Effects of the Polyethylene Glycol Hydrophilic Chain Configurations.

Polyethylene glycol (PEG) is often added to the lipid coating of a contrast microbubble to prevent coalescence and improve circulation. At high surface density, PEG chains are known to undergo a transition from a mushroom configuration to an extended brush configuration. We investigated the effects of PEG chain configuration on attenuation and dissolution of microbubbles by varying the molar ratio of the PEGylated lipid in the shell with three (0%, 2% and 5%) in the mushroom configuration and two (10% and 20%) in the brush configuration. We measured attenuation through the bubble suspensions and used it to obtain the characteristic rheological properties of their shells according to two interfacial rheological models. The interfacial elasticity was found to be significantly lower in the brush regime (∼0.6 N/m) than in the mushroom regime (∼1.3 N/m), but similar in value within each regime. The dissolution behavior of microbubbles under acoustic excitation inside an air-saturated medium was studied by measuring the time-dependent attenuation. Total attenuation recorded a transient increase because of growth resulting from air influx and an eventual decrease caused by dissolution. Microbubble shell composition with varying PEG concentrations had significant effects on dissolution dynamics.

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.

Spatially-directed angiogenesis using ultrasound-controlled release of basic fibroblast growth factor from acoustically-responsive scaffolds.

Vascularization is a critical step following implantation of an engineered tissue construct in order to maintain its viability. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for anastomosis and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially- controlling regenerative processes using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). A therapeutic payload, which is initially contained within the PSDE, is released by an ultrasound-mediated process called acoustic droplet vaporization (ADV). During ADV, the perfluorocarbon (PFC) phase within the PSDE is vaporized into a gas bubble. In this study, we generated ex situ four different spatial patterns of ADV within ARSs containing basic fibroblast growth factor (bFGF), which were subcutaneously implanted in mice. The PFC species within the PSDE significantly affected the morphology of the ARS, based on the stability of the gas bubble generated by ADV, which impacted host cell migration. Irrespective of PFC, significantly greater cell proliferation (i.e., up to 2.9-fold) and angiogenesis (i.e., up to 3.7-fold) were observed adjacent to +ADV regions of the ARSs compared to -ADV regions. The morphology of the PSDE, macrophage infiltration, and perfusion in the implant region were also quantified. These results demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis. Overall, these finding can be applied to improve strategies for spatially-controlling vascularization. STATEMENT OF SIGNIFICANCE: Vascularization is a critical step following implantation of an engineered tissue. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for inosculation and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially-controlling angiogenesis using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). An ultrasound-mediated process called acoustic droplet vaporization (ADV) was used to release basic fibroblast growth factor (bFGF), which was initially contained within the PSDE. We demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis in vivo. Overall, these finding can improve strategies for spatially-controlling vascularization.

Release of basic fibroblast growth factor from acoustically-responsive scaffolds promotes therapeutic angiogenesis in the hind limb ischemia model.

Pro-angiogenic growth factors have been studied as potential therapeutics for cardiovascular diseases like critical limb ischemia (CLI). However, the translation of these factors has remained a challenge, in part, due to problems associated with safe and effective delivery. Here, we describe a hydrogel-based delivery system for growth factors where release is modulated by focused ultrasound (FUS), specifically a mechanism termed acoustic droplet vaporization. With these fibrin-based, acoustically-responsive scaffolds (ARSs), release of a growth factor is non-invasively and spatiotemporally-controlled in an on-demand manner using non-thermal FUS. In vitro studies demonstrated sustained release of basic fibroblast growth factor (bFGF) from the ARSs using repeated applications of FUS. In in vivo studies, ARSs containing bFGF were implanted in mice following induction of hind limb ischemia, a preclinical model of CLI. During the 4-week study, mice in the ARS + FUS group longitudinally exhibited significantly more perfusion and less visible necrosis compared to other experimental groups. Additionally, significantly greater angiogenesis and less fibrosis were observed for the ARS + FUS group. Overall, these results highlight a promising, FUS-based method of delivering a pro-angiogenic growth factor for stimulating angiogenesis and reperfusion in a cardiovascular disease model. More broadly, these results could be used to personalize the delivery of therapeutics in different regenerative applications by actively controlling the release of a growth factor.

Stable and transient bubble formation in acoustically-responsive scaffolds by acoustic droplet vaporization: theory and application in sequential release.

Acoustically-responsive scaffolds (ARSs), which are fibrin hydrogels containing monodispersed perfluorocarbon (PFC) emulsions, respond to ultrasound in an on-demand, spatiotemporally-controlled manner via a mechanism termed acoustic droplet vaporization (ADV). Previously, ADV has been used to control the release of bioactive payloads from ARSs to stimulate regenerative processes. In this study, we used classical nucleation theory (CNT) to predict the nucleation pressure in emulsions of different PFC cores as well as the corresponding condensation pressure of the ADV-generated bubbles. According to CNT, the threshold bubble radii above which ADV-generated bubbles remain stable against condensation were 0.4 µm and 5.2 µm for perfluoropentane (PFP) and perfluorohexane (PFH) bubbles, respectively, while ADV-generated bubbles of any size in perfluorooctane (PFO) condense back to liquid at ambient condition. Additionally, consistent with the CNT findings, stable bubble formation from PFH emulsion was experimentally observed using confocal imaging while PFO emulsion likely underwent repeated vaporization and recondensation during ultrasound pulses. In further experimental studies, we utilized this unique feature of ADV in generating stable or transient bubbles, through tailoring the PFC core and ultrasound parameters (excitation frequency and pulse duration), for sequential delivery of two payloads from PFC emulsions in ARSs. ADV-generated stable bubbles from PFH correlated with complete release of the payload while transient ADV resulted in partial release, where the amount of payload release increased with the number of ultrasound exposure. Overall, these results can be used in developing drug delivery strategies using ARSs.

Standing wave-assisted acoustic droplet vaporization for single and dual payload release in acoustically-responsive scaffolds.

An ultrasound standing wave field (SWF) has been utilized in many biomedical applications. Here, we demonstrate how a SWF can enhance drug release using acoustic droplet vaporization (ADV) in an acoustically-responsive scaffold (ARS). ARSs are composite fibrin hydrogels containing payload-carrying, monodispersed perfluorocarbon (PFC) emulsions and have been used to stimulate regenerative processes such as angiogenesis. Elevated amplitudes in the SWF significantly enhanced payload release from ARSs containing dextran-loaded emulsions (nominal diameter: 6 µm) compared to the -SWF condition, both at sub- and suprathreshold excitation pressures. At 2.5 MHz and 4 MPa peak rarefactional pressure, the cumulative percentage of payload released from ARSs reached 84.1 ± 5.4% and 66.1 ± 4.4% under + SWF and -SWF conditions, respectively, on day 10. A strategy for generating a SWF for an in situ ARS is also presented. For dual-payload release studies, bi-layer ARSs containing a different payload within each layer were exposed to temporally staggered ADV at 3.25 MHz (day 0) and 8.6 MHz (day 4). Sequential payload release was demonstrated using dextran payloads as well as two growth factors relevant to angiogenesis: basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB (PDGF-BB). In addition, bubble growth and fibrin degradation were characterized in the ARSs under +SWF and -SWF conditions. These results highlight the utility of a SWF for modulating single and dual payload release from an ARS and can be used in future therapeutic studies.