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.

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.

Therapeutic oxygen delivery by perfluorocarbon-based colloids.

After the protocol-related indecisive clinical trial of Oxygent, a perfluorooctylbromide/phospholipid nanoemulsion, in cardiac surgery, that often unduly assigned the observed untoward effects to the product, the development of perfluorocarbon (PFC)-based O2 nanoemulsions ("blood substitutes") has come to a low. Yet, significant further demonstrations of PFC O2-delivery efficacy have continuously been reported, such as relief of hypoxia after myocardial infarction or stroke; protection of vital organs during surgery; potentiation of O2-dependent cancer therapies, including radio-, photodynamic-, chemo- and immunotherapies; regeneration of damaged nerve, bone or cartilage; preservation of organ grafts destined for transplantation; and control of gas supply in tissue engineering and biotechnological productions. PFC colloids capable of augmenting O2 delivery include primarily injectable PFC nanoemulsions, microbubbles and phase-shift nanoemulsions. Careful selection of PFC and other colloid components is critical. The basics of O2 delivery by PFC nanoemulsions will be briefly reminded. Improved knowledge of O2 delivery mechanisms has been acquired. Advanced, size-adjustable O2-delivering nanoemulsions have been designed that have extended room-temperature shelf-stability. Alternate O2 delivery options are being investigated that rely on injectable PFC-stabilized microbubbles or phase-shift PFC nanoemulsions. The latter combine prolonged circulation in the vasculature, capacity for penetrating tumor tissues, and acute responsiveness to ultrasound and other external stimuli. Progress in microbubble and phase-shift emulsion engineering, control of phase-shift activation (vaporization), understanding and control of bubble/ultrasound/tissue interactions is discussed. Control of the phase-shift event and of microbubble size require utmost attention. Further PFC-based colloidal systems, including polymeric micelles, PFC-loaded organic or inorganic nanoparticles and scaffolds, have been devised that also carry substantial amounts of O2. Local, on-demand O2 delivery can be triggered by external stimuli, including focused ultrasound irradiation or tumor microenvironment. PFC colloid functionalization and targeting can help adjust their properties for specific indications, augment their efficacy, improve safety profiles, and expand the range of their indications. Many new medical and biotechnological applications involving fluorinated colloids are being assessed, including in the clinic. Further uses of PFC-based colloidal nanotherapeutics will be briefly mentioned that concern contrast diagnostic imaging, including molecular imaging and immune cell tracking; controlled delivery of therapeutic energy, as for noninvasive surgical ablation and sonothrombolysis; and delivery of drugs and genes, including across the blood-brain barrier. Even when the fluorinated colloids investigated are designed for other purposes than O2 supply, they will inevitably also carry and deliver a certain amount of O2, and may thus be considered for O2 delivery or co-delivery applications. Conversely, O2-carrying PFC nanoemulsions possess by nature a unique aptitude for 19F MR imaging, and hence, cell tracking, while PFC-stabilized microbubbles are ideal resonators for ultrasound contrast imaging and can undergo precise manipulation and on-demand destruction by ultrasound waves, thereby opening multiple theranostic opportunities.

Spatially-directed cell migration in acoustically-responsive scaffolds through the controlled delivery of basic fibroblast growth factor.

Hydrogels are commonly used in regenerative medicine for the delivery of growth factors (GFs). The spatial and temporal presentations of GFs are critical for directing regenerative processes, yet conventional hydrogels do not enable such control. We have developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), where release of a GF is non-invasively and spatiotemporally-controlled using focused ultrasound. The ARS consists of a fibrin matrix doped with a GF-loaded, phase-shift emulsion. The GF is released when the ARS is exposed to suprathreshold ultrasound via a mechanism termed acoustic droplet vaporization. In this study, we investigate how different spatial patterns of suprathreshold ultrasound can impact the biological response upon in vivo implantation of an ARS containing basic fibroblast growth factor (bFGF). ARSs were fabricated with either perfluorohexane (bFGF-C6-ARS) or perflurooctane (bFGF-C8-ARS) within the phase-shift emulsion. Ultrasound generated stable bubbles in bFGF-C6-ARS, which inhibited matrix compaction, whereas transiently stable bubbles were generated in bFGF-C8-ARS, which decreased in height by 44% within one day of implantation. The rate of bFGF release and distance of host cell migration were up to 6.8-fold and 8.1-fold greater, respectively, in bFGF-C8-ARS versus bFGF-C6-ARS. Ultrasound increased the formation of macropores within the fibrin matrix of bFGF-C8-ARS by 2.7-fold. These results demonstrate that spatially patterning suprathreshold ultrasound within bFGF-C8-ARS can be used to elicit a spatially-directed response from the host. Overall, these findings can be used in developing strategies to spatially pattern regenerative processes. STATEMENT OF SIGNIFICANCE: Hydrogels are commonly used in regenerative medicine for the delivery of growth factors (GFs). The spatial and temporal presentations of GFs are critical for directing regenerative processes, yet conventional hydrogels do not enable such control. We have developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), where GF release is non-invasively and spatiotemporally-controlled using focused ultrasound. The ARS consists of a fibrin matrix doped with a phase-shift emulsion loaded with GF, which is released when the ARS is exposed to ultrasound. In this in vivo study, we demonstrate that spatially patterning ultrasound within an ARS containing basic fibroblast growth factor (bFGF) can elicit a spatially-directed response from the host. Overall, these findings can be used in developing strategies to spatially pattern regenerative processes.

MRI-guided transurethral insonation of silica-shell phase-shift emulsions in the prostate with an advanced navigation platform.

PURPOSE: In this study, the efficacy of transurethral prostate ablation in the presence of silica-shell ultrasound-triggered phase-shift emulsions (sUPEs) doped with MR contrast was evaluated. The influence of sUPEs on MR imaging assessment of the ablation zone was also investigated. METHODS: sUPEs were doped with a magnetic resonance (MR) contrast agent, Gd2 O3 , to assess ultrasound transition. Injections of saline (sham), saline and sUPEs alone, and saline and sUPEs with Optison microbubbles were performed under guidance of a prototype interventional MRI navigation platform in a healthy canine prostate. Treatment arms were evaluated for differences in lesion size, T1  contrast, and temperature. In addition, non-perfused areas (NPAs) on dynamic contrast-enhanced (DCE) MRI, 55°C isotherms, and areas of 240 cumulative equivalent minutes at 43°C (CEM43 ) dose or greater computed from MR thermometry were measured and correlated with ablated areas indicated by histology. RESULTS: For treatment arms including sUPEs, the computed correlation coefficients between the histological ablation zone and the NPA, 55°C isotherm, and 240 CEM43 area ranged from 0.96-0.99, 0.98-0.99, and 0.91-0.99, respectively. In the absence of sUPEs, the computed correlation coefficients between the histological ablation zone and the NPA, 55°C isotherm, and 240 CEM43 area were 0.69, 0.54, and 0.50, respectively. Across all treatment arms, the areas of thermal tissue damage and NPAs were not significantly different (P = 0.47). Areas denoted by 55°C isotherms and 240 CEM43 dose boundaries were significantly larger than the areas of thermal damage, again for all treatment arms (P = 0.009 and 0.003, respectively). No significant differences in lesion size, T1 contrast, or temperature were observed between any of the treatment arms (P > 0.0167). Lesions exhibiting thermal fixation on histological analysis were present in six of nine insonations involving sUPE injections and one of five insonations involving saline sham injections. Significantly larger areas (P = 0.002), higher temperatures (P = 0.004), and more frequent ring patterns of restricted diffusion on ex vivo diffusion-weighted imaging (P = 0.005) were apparent in lesions with thermal fixation. CONCLUSIONS: T1 contrast suggesting sUPE transition was not evident in sUPE treatment arms. The use of MR imaging metrics to predict prostate ablation was not diminished by the presence of sUPEs. Lesions generated in the presence of sUPEs exhibited more frequent thermal fixation, though there were no significant changes in the ablation areas when comparing arms with and without sUPEs. Thermal fixation corresponded to some qualitative imaging features.

Dissolved Oxygen Scavenging by Acoustic Droplet Vaporization using Intravascular Ultrasound.

Modification of dissolved gas content by acoustic droplet vaporization (ADV) has been proposed for several therapeutic applications. Reducing dissolved oxygen (DO) during reperfusion of ischemic tissue during coronary interventions could inhibit reactive oxygen species production and rescue myocardium. The objective of this study was to determine whether intravascular ultrasound (IVUS) can trigger ADV and reduce DO. Perfluoropentane emulsions were created using high-speed shaking and microfluidic manufacturing. High-speed shaking resulted in a polydisperse droplet distribution ranging from less than 1 micron to greater than 16 microns in diameter. Microfluidic manufacturing produced a narrower size range of droplets with diameters between 8.0 microns and 9.6 microns. The DO content of the fluids was measured before and after ADV triggered by IVUS exposure. Duplex B-mode and passive cavitation imaging was performed to assess nucleation of ADV. An increase in echogenicity indicative of ADV was observed after exposure with a clinical IVUS system. In a flow phantom, a 20% decrease in DO was measured distal to the IVUS transducer when droplets, formed via high-speed shaking, were infused. In a static fluid system, the DO content was reduced by 11% when droplets manufactured with a microfluidic chip were exposed to IVUS. These results demonstrate that a reduction of DO by ADV is feasible using a clinical IVUS system. Future studies will assess the potential therapeutic efficacy of IVUS-nucleated ADV and methods to increase the magnitude of DO scavenging.