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The R. Bruce Lindsay Award (formerly the Biennial Award) is presented in the Spring to a member of the Society who is no more than 10 years post terminal degree on 1 July at the time of Award acceptance and who, during a period of two or more years immediately preceding the award, has been active in the affairs of the Society and has contributed substantially, through published papers, to the advancement of theoretical or applied acoustics, or both. The award was presented biennially until 1986. It is now an annual award.

Elastic, anisotropic tissue such as tendon has proven resistant to mechanical fractionation by histotripsy, a subset of focused ultrasound that uses the creation, oscillation and collapse of cavitation bubbles to fractionate tissue. Our objective was to fabricate an optically transparent hydrogel that mimics tendon for evaluation of histotripsy bubble dynamics. Ex vivo bovine deep digital flexor tendons were obtained (n = 4), and varying formulations of polyacrylamide (PA), collagen and fibrin hydrogels (n = 3 each) were fabricated. Axial sound speeds were measured at 1 MHz for calculation of anisotropy. All samples were treated with a 1.5-MHz focused ultrasound transducer with 10-ms pulses repeated at 1 Hz (p

Safety of biomedical ultrasound largely depends on controlling cavitation bubbles in vivo, yet bubble nuclei in biological tissues remain unexplored compared to water. This study evaluates the effects of elastic modulus (E) and impurities on bubble nuclei available for cavitation in tissue-mimicking polyacrylamide (PA) hydrogels. A 1.5 MHz focused ultrasound transducer with f# = 0.7 was used to induce cavitation in 17.5%, 20%, and 22.5% v/v PA hydrogels using 10-ms pulses with pressures up to peak negative pressure (p−) = 35 MPa. Cavitation was monitored at 0.075 ms through high-speed photography at 40 000 fps. At p− = 29 MPa for all hydrogels, cavitation occurred at random locations within the −6 dB focal area [9.4 × 1.2 mm (p−)]. Increasing p− to 35 MPa increased bubble location consistency and caused shock scattering in the E = 282 MPa hydrogels; as the E increased to 300 MPa, bubble location consistency decreased ( p = 0.045). Adding calcium phosphate or cholesterol at 0.25% w/v or bovine serum albumin at 5% or 10% w/v in separate 17.5% PA as impurities decreased the cavitation threshold from p− = 13.2 MPa for unaltered PA to p− = 11.6 MPa, p− = 7.3 MPa, p− = 9.7 MPa, and p− = 7.5 MPa, respectively. These results suggest that both E and impurities affect the bubble nuclei available for cavitation in tissue-mimicking hydrogels.

The development of tunable, ultrasound-responsive hydrogels that can deliver protein payload on-demand when exposed to focused ultrasound is described in this study. Reversible Diels-Alder linkers, which undergo a retro reaction when stimulated with ultrasound, were used to cross-link chitosan hydrogels with entrapped FITC-BSA as a model protein therapeutic payload. Two Diels-Alder linkage compositions with large differences in the reverse reaction energy barriers were compared to explore the influence of linker composition on ultrasound response. Selected physicochemical properties of the hydrogel construct, its basic degradation kinetics, and its cytocompatibility were measured with respect to Diels-Alder linkage composition. Focused ultrasound initiated the retro Diels-Alder reaction, controlling the release of the entrapped payload while also allowing for real-time visualization of the ongoing process. Additionally, increasing the focused ultrasound amplitude and time correlated with an increased rate of protein release, indicating stimuli responsive control.

Tendon injury is prevalent and costly in the United States, comprising 45% of the 66 million musculoskeletal injuries and costing $114 billion annually. Surgical and therapeutic methods, such as arthroscopic surgery, dry needling, and physical therapy, produce mixed success in reintroducing a healing response in tendinopathy due in part to inconsistent dosing and monitoring. Ultrasound is one therapeutic modality that has been shown to noninvasively induce bioeffects in tendon that may help promote healing. However, results from this modality have also been mixed. This review compares the current state of the field in therapeutic ultrasound and shockwave therapy, including low-intensity therapeutic ultrasound, extracorporeal shockwave therapy, and radial shockwave therapy, and evaluates the efficacy in treating tendinopathies with ultrasound. We found that the mixed successes may be attributed to the wide variety of achievable parameters within each broader treatment type and the lack of standardization in measurements and reporting. Despite mixed outcomes, all three therapies show potential as an alternative therapy with lower-risk adverse effects than more invasive methods like surgery. There is currently insufficient evidence to conclude which ultrasound modality or settings are most effective. More research is needed to understand the healing effects of these different therapeutic ultrasound and shockwave modalities.

Around 30 million tendon injuries occur annually in the U.S. costing $ 114 billion. Conservative therapies, like dry needling, promote healing in chronically injured tendons by inducing microdamage but have mixed success rates. Focused ultrasound (fUS) therapy can noninvasively fractionate tissues through the creation, oscillation, and collapse of bubbles in a process termed histotripsy; however, highly collagenous tissues, like tendon, have shown resistance to mechanical fractionation. This study histologically evaluates whether fUS mechanical disruption is achievable in tendons. Ex vivo rat tendons (45 Achilles and 44 supraspinatus) were exposed to 1.5-MHz fUS operating with 0.1-10 ms pulses repeated at 1-100 Hz for 15-60 s with peak positive pressures89 MPa and peak negative pressures26 MPa; other tendons were exposed to dry needling or sham. Immediately after treatment, tendons were flash-frozen and stained with hematoxylin and eosin (HE) or alpha-nicotinamide adenine dinucleotide diaphorase ( α -NADH-d) and evaluated by two reviewers blinded to the exposure conditions. Results showed successful creation of bubbles for all fUS-treated samples; however, not all samples showed histological injury. When the injury was detected, parameter sets with shorter pulses (0.1-1 ms), lower acoustic pressures, or reduced treatment times showed mechanical disruption in the form of fiber separation and fraying with little to no thermal injury. Longer pulses or treatment times showed a combination of mechanical and thermal injury. These findings suggest that mechanical disruption is achievable in tendons within a small window of acoustic parameters, supporting the potential of fUS therapy in tendon treatment.

The color Doppler ultrasound twinkling artifact is a rapid color shift that appears on 43%-96% of kidney stones. Surface microbubbles on kidney stones are theorized to cause twinkling as exposure to elevated static pressures of 0.41-1.13 MPa (approximately 0.5-1 times diagnostic ultrasound pressure and 5-10 times ambient pressure) reduced twinkling. However, it is unclear what external and internal stone features support bubbles. Thirteen ex vivo kidney stones were scanned with color Doppler ultrasound at 2.5, 5 and 18.5 MHz. Select stones were imaged with environmental scanning electron microscopy or underwater micro-computed tomography to evaluate features that may cause twinkling. Results revealed that the lower frequencies produced larger volumes of twinkling. Condensation first occurred in the smallest (∼1 µm diameter) surface pores and may be indicative of where bubbles form. Gas pockets were seen inside two of three tested stones that may contribute to twinkling. Overall, these results provide evidence of cavity structures both externally and internally and their correlation to the twinkling artifact. This indicates that microbubbles may be present on and within kidney stones and may contribute to the twinkling artifact.

The inability to spatiotemporally guide proteins in tissues, and efficiently deliver them into cells, remain key barriers to realizing their full potential in precision medicine. Here, we report ul...

The elastic modulus, or slope of the stress-strain curve, is an important metric for evaluating tissue functionality, particularly for load-bearing tissues such as tendon. The applied force can be tracked directly from a mechanical testing system and converted to stress using the tissue cross-sectional area; however, strain can only be calculated in post-processing by tracking tissue displacement from video collected during mechanical testing. Manual tracking of Verhoeff stain lines pre-marked on the tissue is time-consuming and highly dependent upon the user. This paper details the development and testing of an automated processing method for strain calculations using Harris corner detection. The automated and manual methods were compared in a dataset consisting of 97 rat tendons (48 Achilles tendons, 49 supraspinatus tendons), divided into ten subgroups for evaluating the effects of different therapies on tendon mechanical properties. The comparison showed that average percent differences between the approaches were 0.89% and -2.10% for Achilles and supraspinatus tendons, respectively. The automated approach reduced processing time by 83% and produced similar results to the manual method when comparing the different subgroups. This automated approach to track tissue displacements and calculate elastic modulus improves post-processing time while simultaneously minimizing user dependency.

Electrical muscle stimulation is limited to surface muscle activation; however, focused ultrasound (fUS) has the potential to stimulate deep skeletal muscles. This study aims to evaluate the effects of fUS peripheral nerve stimulation on muscle mechanical properties in an in vivo murine model. The sciatic nerve of 19 Sprague-Dawley rats was insonified with 3.68-MHz fUS (p+ = 6.4 MPa, p− = 5 MPa) using 10-ms pulses (0.9-ms on/0.1-ms off, 10 times) repeated at 0.5-Hz for 30, 90, or 180 severy second day for two weeks. Stimulation was monitored using electromyography (EMG) with indwelling electrodes inserted in the gastrocnemius muscle belly. Bilateral muscles were harvested for tensile mechanical testing (n = 15) and histology (n = 4). Muscle contraction was confirmed via EMG in 16/19 rats. Elastic moduli of stimulated muscles (133.2 ± 42.1 kPa) and controls (101.4 ± 39.2 kPa) were not different (ANOVA, p = 0.053). A trend toward increased elastic modulus with number of muscle stimulations was observed until 450 stimulations (not significant: ANOVA, p = 0.437), after which elastic modulus began to decrease. No tissue damage was observed histologically. The results of this pilot study demonstrate that fUS can induce repeatable muscle contraction that may improve muscle strength and mechanical properties over time. [Work supported by PSU College of Engineering Seed Grant 2021.]

Collagenous, anisotropic tissues such as tendon are resilient to liquefaction by histotripsy, despite verification of cavitation activity using B-mode imaging. In previous published work, dehydrated fibrin gels were shown to mimic tendon bubble dynamics but are limited in thickness due to high opacity. Here, we aim to fabricate a thicker, more optically transparent hydrogel possessing similar cavitation dynamics to tendon. Highly transparent collagen hydrogels (TeloCol®, 10 mg/mL) were fabricated, and axial sound speeds were measured to determine anisotropy. Hydrogels were then exposed to 1.5-MHz focused ultrasound with 10-ms pulses at 1-Hz for 60 s with p + =127 MPa, p− = 35 MPa. Cavitation activity was monitored with high-speed photography and passive cavitation imaging using a Philips/ATL L7-4 transducer and Vantage® ultrasound system. Despite exhibiting low degrees of anisotropy like other non-dehydrated gels (20% lower than soft-tissue mimicking polyacrylamide. Additionally, both resilience to fractionation and transparency were notably improved compared to previously tested PureCol® collagen formulations. Different levels of collagen dehydration will be explored to further increase resilience to liquefaction and assess the role of water content in histotripsy fractionation. [Work supported by NIH R01EB032860.]

The Doppler ultrasound twinkling artifact is a rapid color shift observed in ∼66% of kidney stones. Breathing oxygen at elevated pressures was shown to enhance twinkling on kidney stones in human subjects. Here, in humans and in ex vivo stones, we investigate the effects of elevated oxygen at ambient pressure. Recruited subjects with confirmed stones were scanned with a research ultrasound system and Philips/ATL C5-2 transducer while breathing ambient air for 2 min. Then, subjects were masked with 100% oxygen for 15 min and scanned again for the last 2 min. Similarly, ex vivo stones were imaged before and after bubbling oxygen into water. Average Doppler power was calculated for in vivo and ex vivo stones. In human subjects, Doppler power increased by 30% when subjects breathed pure oxygen compared to ambient air. Similarly, on ex vivo stones, an average increase in Doppler power of 98% was observed in elevated oxygen conditions; twinkling also became visible on stones that otherwise did not twinkle at elevated oxygen (50% more oxygen than control). These results suggest that elevated oxygen at ambient pressure may improve the consistency of twinkling to detect kidney stones. [Work supported by the PSU Center for Biodevices Seed Grant.]

The increasing prevalence of blended resident and distance education courses makes it difficult to implement in-class demonstrations or hands-on activities to enhance learning. Our objective here is to develop and evaluate the use of at-home activity kits in fundamental acoustics and vibration courses for graduate students. First-generation PAWkits includedfive1-ft PVC pipes, four PVC couplers, a PVC end cap, a PVC T-branch, two dental dams, a tuning fork, a bluetooth speaker with passive radiator, a lapel microphone, a thick string, a thin string, ten magnets, four magnetic hooks, four springs (two spring constants), and an alligator clip. The total cost of the kit per student was estimated at $66 plus shipping. The first-generation PAWkit was designed for five main activities includingmeasuring directivity of the speaker and tuning fork, waves in pipes including T-branch and low-pass filters, waves in membranes, waves in strings, and mass-spring vibration systems. Activities were tested across two required Penn State first-year required courses for graduate students. PAWkits are currently being evaluated through student surveys and, in the future, by tracking the performance of Ph.D. students on the qualifying exam. [Work supported by Penn State Leonhard Center Seed Grant.]

The twinkling artifact, or twinkling, appears when imaging kidney stones and other mineralizations with Doppler ultrasound and is caused by scattering off crevice microbubbles. Previous work found that twinkling on in vivo kidney stones increased or decreased with elevated oxygen or carbon dioxide, respectively. However, it is unclear whether these results are from biological adaptions to the changing respiratory gas and thus if in vitro mineralizations are similarly affected. Here, cholesterol, calcium phosphate, and uric acid crystals were grown in vitro and imaged in deionized water using a Vantage-128 research ultrasound system with a Philips/ATL L7-4 transducer. Five of each crystal composition were imaged for one minute at different dissolved gas concentrations. By bubbling gas through water, dissolved oxygen ranged from 2 to 20 mg/L while dissolved carbon dioxide ranged from 30 to 290 mg/L. Twinkling on cholesterol crystals increased by 70% and 140% when oxygen and carbon dioxide levels were maximized, respectively. In contrast, twinkling on calcium phosphate and uric acid was not significantly affected by either gas. These results suggest that changes in twinkling from increased gas concentrations are dependent on crystal composition and are likely affected by biological adaptations in vivo. [Work supported by NSF-CAREER-1943937 and NSF-GRFP-DGE1255832.]

Histotripsy has successfully fractionated most soft tissues; however, highly collagenous tissues like tendon have been resistant to histotripsy fractionation. Previously, we showed that some histotripsy parameters could create mild mechanical microdamage in healthy ex vivo rat tendons. Our objective here is to evaluate whether complete histotripsy fractionation is possible in tendons. Eight bovine tendons were injected with collagenase to induce tendinopathy; an additional four tendons were unaltered. Tendons were exposed to single- or dual-frequency histotripsy at 1.07-, 1.5-, and/or 3.68-MHz with 10-ms pulses delivered at 1 Hz for 60 s. Treatments were monitored with passive cavitation detection (PCD) and samples were evaluated for damage. Results show that exposure of tendinopathic tendons to 1.5- and 3.68-MHz dual-frequency histotripsy produces a distinct, fractionated hole; 1.5- and 3.68-MHz single-frequency histotripsy produces partial fractionation without a distinct hole. No fractionation was observed in healthy tendons, or in tendinopathic tendons exposed to 1.07 MHz single- or dual-frequency histotripsy. Histologically, all tendons showed evidence of thermal necrosis independent of whether a hole was observed. PCD showsincreases in amplitude and sustainment of cavitation in exposures that successfully fractionated tendon. These results suggest histotripsy fractionation is possible in tendinopathic tendons. [Work supported by NIH R21EB027886 and R01EB032860.]

Objective. Pathological mineralizations form throughout the body and can be difficult to detect using conventional imaging methods. Color Doppler ultrasound twinkling highlights ∼60% of kidney stones with a rapid color shift and is theorized to arise from crevice microbubbles as twinkling disappears on kidney stones at elevated pressures and scratched acrylic balls in ethanol. Twinkling also sometimes appears on other pathological mineralizations; however, it is unclear whether the etiology of twinkling is the same as for kidney stones. Approach. In this study, five cholesterol, calcium phosphate, and uric acid crystals were grown in vitro and imaged in Doppler mode with a research ultrasound system and L7-4 transducer in water. To evaluate the influence of pressure on twinkling, the same crystals were imaged in a high-pressure chamber. Then, the effect of surface tension on twinkling was evaluated by imaging crystals in different concentrations of surfactant (1%, 2%, 3%, 4%) and ethanol (10%, 30%, 50%, 70%), artificial urine, bovine blood, and a tissue-mimicking phantom. Main results. Results showed that all crystals twinkled in water, with cholesterol twinkling significantly more than calcium phosphate and uric acid. When the ambient pressure was increased, twinkling disappeared for all tested crystals when pressures reached 7 MPa (absolute) and reappeared when returned to ambient pressure (0.1 MPa). Similarly, twinkling across all crystals decreased with surface tension when imaged in the surfactant and ethanol (statistically significant when surface tension −1) and decreased in blood (surface tension = 52.7 mN m−1) but was unaffected by artificial urine (similar surface tension to water). In the tissue-mimicking phantom, twinkling increased for cholesterol and calcium phosphate crystals with no change observed in uric acid crystals. Significance. Overall, these results support the theory that bubbles are present on crystals and cause twinkling, which could be leveraged to improve twinkling for the detection of other pathological mineralizations.

Tendon injuries are extremely common, resulting in mechanically weaker tendons that could lead to tendon rupture. Dry needling (DN) is widely used to manage pain and function after injury. However, DN is invasive and high inter-practitioner variability has led to mixed success rates. Focused ultrasound (fUS) is a non-invasive medical technology that directs ultrasound energy into a well-defined focal volume. fUS can induce thermal ablation or mechanical fractionation, with bioeffect type controlled through ultrasound parameters. Tendons must withstand high physiological loads, thus treatments maintaining tendon mechanical properties while promoting healing are needed. Our objective was to evaluate mechanical effects of DN and 3 fUS parameter sets, chosen to prioritize mechanical fractionation, on Achilles and supraspinatus tendons. Ex vivo rat Achilles and supraspinatus tendons (50 each) were divided into sham, DN, fUS-1, fUS-2, and fUS-3 (n = 10/group). Following treatment, tendons were mechanically tested. Elastic modulus of supraspinatus tendons treated with DN (126.64 ± 28.1 MPa) was lower than sham (153.02 ± 29.3 MPa; p = 0.0280). Stiffness and percent relaxation of tendons treated with DN (Achilles: 114.40 ± 31.6 N/mm; 49.10 ± 6.1%; supraspinatus: 109.53 ± 30.8 N/mm; 50.17 ± 7.6%) were lower (all p 0.0334) than sham (Achilles: 141.34 ± 20.9 N/mm; 60.30 ± 7.7%; supraspinatus: 135.14 ± 30.2 N/mm; 60.85 ± 15.4%). Modulus of Achilles and supraspinatus tendons treated with fUS-1 (159.88 ± 25.7 MPa; 150.12 ± 22.0 MPa, respectively) were similar to sham (156.35 ± 23.0 MPa; 153.02 ± 29.3 MPa, respectively). These results suggest that fUS preserves mechanical properties better than DN, with fUS-1 performing better than fUS-2 and fUS-3. fUS should be studied further to fully understand its mechanical and healing effects to help evaluate fUS as an alternative, non-invasive treatment for tendon injuries.

One of my favorite homework problems for waves in fluids or a general acoustics class is Blackstock 9-3, which involves a submarine positioned 50 m below the ocean surface and 500 m above the seafloor. The problem asks students to calculate the impedance of the seafloor given a frequency and known sound pressure levels received by the submarine after bouncing off the ocean surface or the seafloor. In part a, the calculation is made assuming a lossless medium and then in part b, using attenuation. The reasons this is one of my favorite problems are that it is real-world derived, combines many concepts, and requires understanding of wave propagation physics to solve; there is no simple equation (that I have found at least) that will immediately provide the correct answer. Students often struggle with what type of wave is emitted (plane versus spherical), then, if spherical waves, how to account for the boundary—especially on the seafloor where one must calculate the reflection coefficient. Every year this problem allows for good discussion in class and homework review sections.

The twinkling artifact, or rapid color shifts, are observed when imaging kidney stones and other mineralizations. The current theory is that scattering from bubbles in micron-sized crevices causes twinkling. Our objective is to investigate the effect of ultrasound frequency and crevice size and number on twinkling of silicon wafers. Ten or 100 randomly positioned cylindrical crevices with diameters of 1, 10, or 100 μm and depths of 10 μm were dry-etched on wafers. A research ultrasound system was used to image wafers at 5, 7.8, and 18.5 MHz; IQ data were post-processed to calculate twinkling or Doppler power. Twinkling for all crevice sizes and numbers followed the trend 5 MHz>7.8 MHz>18.5 MHz. For crevice size, at 5 and 7.8 MHz, twinkling was higher for 1- or 10-μm crevices compared to 100 μm; at 18.5 MHz, twinkling was only observed on 100-μm crevices. Increasing the number of crevices increased twinkling for 1 μm crevices at 5 and 7.8 MHz and 10 μm crevices at 7.8 MHz; no differences were noted at 18.5 MHz. These results support previous work showing twinkling is frequency-dependent and suggest that even 10, 1-μm crevices are sufficient to cause twinkling. [Work supported by SURIEA, NSF CAREER-1943937.]

Liquid atomization and fountain formation by focused ultrasound was first published by Wood and Loomis [1]. Since then, the cavitation-wave hypothesis emerged to explain atomization in a fountain, which states atomization arises from a combination of surface capillary waves and the collapse of cavitation bubbles. More recently, high intensity focused ultrasound (HIFU) has been shown to fractionate tissue through either pulsed-cavitation or millisecond boiling histotripsy therapies; however it is unclear how millimeter-size boiling bubbles or cavitation bubble clouds fractionate tissue into submicron-size fragments. The objective of this work is to test the hypothesis experimentally that atomization and fountain formation occurs similarly in liquids and tissues and results in tissue erosion. A 2-MHz HIFU transducer operating at peak in situ pressures of 50 MPa and −11 MPa (linear intensity = 14,000 W/cm(2)) was focused at the interface between a liquid or tissue and air. A high-speed camera was used to monitor atomization and fountain formation in water, ethanol, glycerol, bovine liver, and porcine blood clots. The in situ linear intensity threshold for consistent atomization in one 10-ms pulse increased in the order: ethanol (180 W/cm(2)) < blood clot (250 W/cm(2)) < water (350 W/cm(2)) < liver (6200 W/cm(2)); glycerol did not atomize. Average jet velocities for the initial spray at the maximum acoustic intensity were similar for all materials and on the order of 20 m/s. The tissue erosion rate of liver approached saturation at around 300 10-ms pulses repeated at 1 Hz, which had an average erosion volume of 25.7±10.9 mm(3). While tissue atomization and fountain formation does not completely mimic what is observed in liquids, atomization provides a plausible explanation of how tissue is fractionated in millisecond boiling and possibly even cavitation cloud histotrispy therapies.

Boiling histotripsy (BH) is a High Intensity Focused Ultrasound (HIFU) method for precise mechanical disintegration of target tissue using millisecond-long pulses containing shocks. BH treatments with real-time ultrasound (US) guidance allowed by BH-generated bubbles were previously demonstrated ex vivo and in vivo in exposed porcine liver and small animals. Here, the feasibility of US-guided transabdominal and partially transcostal BH ablation of kidney and liver in an acute in vivo swine model was evaluated for 6 animals. BH parameters were: 1.5 MHz frequency, 5–30 pulses of 1–10 ms duration per focus, 1% duty cycle, peak acoustic powers 0.9–3.8 kW, sonication foci spaced 1–1.5 mm apart in a rectangular grid with 5–15 mm linear dimensions. In kidneys, well-demarcated volumetric BH lesions were generated without respiratory gating and renal medulla and collecting system were more resistant to BH than cortex. The treatment was accelerated 10-fold by using shorter BH pulses of larger peak power without affecting the quality of tissue fractionation. In liver, respiratory motion and aberrations from subcutaneous fat affected the treatment but increasing the peak power provided successful lesion generation. These data indicate BH is a promising technology for transabdominal and transcostal mechanical ablation of tumors in kidney and liver.

Tissue mimicking hydrogels can help us understand how viscoelastic properties (stiffness, elastic modulus, etc.) of biological tissues impact bubble nucleation. Adding impurities to hydrogels introduces inhomogeneities and increases their similarity to biological tissues. In this study, we evaluated the effect of stiffness and impurities on bubble nucleation in polyacrylamide (PA) hydrogels. Bubble nuclei were evaluated in 17.5%, 20%, and 22.5% v/v PA hydrogels, after which 0.25% w/v cholesterol crystals (maximum dimension = 0.6 mm) were embedded in the gels (n = 3 each). A 1.5 MHz focused ultrasound transducer was used to induce cavitation using 10-ms pulses with pressures ranging up to p+ = 89 MPa, and p− = 26 MPa and −6 dB focal dimensions of 9.4 × 1.2 mm (p−). Image analysis from high-speed photography showed bubble nucleation increases with increasing peak negative pressure and decreasing hydrogel stiffness. Adding cholesterol crystals largely decreases the acoustic cavitation threshold from p− = 19 MPa for 17.5% v/v hydrogels with no added impurities to p− = 12 MPa for the same concentration hydrogel with added cholesterol crystals. This suggests that hydrophobic cholesterol crystals weaken the gel or trap bubble nuclei, thus lowering the cavitation threshold. Future work includes investigating bubble nuclei in rat hepatocytes. [Work supported by NSF CAREER 1943937 and PSU Riess Fellowship.]

Imaging crystalline structures with Doppler ultrasound can produce a rapid color shift, termed the twinkling artifact, that can assist in diagnosing pathological mineralizations such as kidney stones, heterotopic ossification, gout, and breast microcalcifications. Twinkling is theorized to arise from scattering off surface crevice microbubbles, which are affected by the surface tension between the bubble and surrounding medium. In this study, we evaluated the effect of surface tension on twinkling in pure crystals. Cholesterol, calcium phosphate, and uric acid crystals were grown in vitro (n = 5 each) and imaged with a Philips/ATL L7-4 transducer and Vantage-128 research ultrasound system. Crystals were imaged in water while varying surfactant concentration (0%–4%) leading to surface tensions that ranged from 45–72 mN/m. Surface tension of the solution was determined by measuring the contact angle of 0.1-mL droplet on an acrylic sheet exposed to air. As the concentration of surfactant increased, twinkling was found to decrease by ∼20% for cholesterol and calcium phosphate and ∼10% for uric acid. These results continue to support the crevice bubble theory of twinkling and suggest the importance of surface tension when evaluating minerals with the twinkling artifact. [Work supported by NSF-CAREER-1943937 and NSF-GRFP-DGE1255832.]

Tendinopathy remains a common and debilitating condition that shows mixed success rates in response to treatment. Focused ultrasound (fUS) histotripsy is a non-invasive therapy that can mechanically fractionate soft tissues through bubble creation, oscillation, and collapse. Although collagenous tissues show resistance to full mechanical fractionation, previous studies in ex vivo murine tendons found acoustic parameters that create microdamage while preserving mechanical properties. Our objective is to compare dry needling (DN) or fUS treatments in a murine tendinopathy model. Achilles tenotomy was performed in 26 rats. One week later, tendons were treated with DN (30G needle, five fenestrations over 20 s) or fUS (1.5MHz, 1-ms pulses at 10 Hz for 60 s, p+ = 89 MPa, p− = 26 MPa). Blood was collected from the tail vein immediately before and after treatment and 1-week post-intervention. Tendons were harvested for histology or mechanical testing. Results show no differences between fUS and DN in the release of IGF-I and TGF-β healing factors. No differences were found between interventions and controls in H&E-stained histology. Stiffness and percent relaxation of DN-treated tendons were lower than controls (p = 0.0041, p = 0.0441, respectively), whereas fUS-treated properties were similar. Fromthese results, fUS treatment may be a viable alternative to DN in the treatment of tendinopathy. [Funding: NSF-GRFP-DGE1255832 and NIH-NIBIB-EB027886]

INTRODUCTION AND OBJECTIVE:The color Doppler ultrasound twinkling artifact, which highlights kidney stones with rapidly changing color, has been shown to increase the sensitivity and specificity of...

The inability to spatiotemporally guide proteins in tissues and efficiently deliver them into cells remains a key barrier to realizing their full potential in precision medicine. Here, we report ultrasound-sensitive fluoro-protein nanoemulsions which can be acoustically tracked, guided, and activated for on-demand cytosolic delivery of proteins, including antibodies, using clinically relevant diagnostic ultrasound. This advance is accessed through the discovery of a family of fluorous tags, or FTags, that transiently mask proteins to mediate their efficient dispersion into ultrasound-sensitive liquid perfluorocarbons, a phenomenon akin to dissolving an egg in liquid Teflon. We identify the biochemical basis for protein fluorous masking and confirm FTag coatings are shed during delivery, without disrupting the protein structure or function. Harnessing the ultrasound sensitivity of fluorous emulsions, real-time imaging is used to simultaneously monitor and activate FTag-protein complexes to enable controlled cytosolic antibody delivery

Heterotopic ossification (HO), or the growth of bone in soft tissue where bone does not usually exist, can occur after any musculoskeletal trauma. While the exact cause of HO is unknown, it can take 2i–3 months before HO can be detected on x-ray or computed tomography (CT). The color ultrasound Doppler twinkling artifact is a rapid color shift that was discovered when imaging kidney stones. Here, our objective is to determine whether twinkling can be used to detect the earliest signs of cellular mineralization in HO. A Vantage-128 research ultrasound system with Philips/ATL L7-4, L12-5, and Vermon L22-14 transducers was used to image human bone marrow-derived stem cells (hBMSCs) cultured in osteogenic media and supersaturated solutions of calcium phosphate. Preliminary results show a 50% increase in Doppler magnitude and a 250% increase in Doppler variance in the presence of cellular mineralization or calcium phosphate crystals compared to images of cells without minerals or in non-crystalline solutions. The L7-4 was found to be more sensitive to twinkling from mineralization than the higher frequency transducers. The increase in Doppler magnitude and variance with cellular mineralization suggests twinkling may be a sensitive early detector of HO. [Work supported by DoD CDMRP PR201164.]

The color Doppler ultrasound twinkling artifact has been found to improve detection of kidney stones with ultrasound; however, it appears on only ∼60% of stones. Evidence from ex vivo kidney stones suggests twinkling arises from microbubbles stabilized in crevices on the stone surface. Yet it is unknown whether these bubbles are present on stones in humans. Here, we used a research ultrasound system to quantify twinkling in humans with kidney stones in a hyperbaric chamber. Eight human patients with non-obstructive kidney stones previously observed to twinkle were exposed to a maximum pressure of 4 atmospheres absolute (ATA) while breathing air, except during the 10-min pause at 1.6 ATA and while the pressure decreased to 1 ATA, during which patients breathed oxygen to minimize the risk of decompression sickness. A paired one-way t-test was used to compare the mean twinkle power at each pressure pause with baseline twinkling, with p0.05 considered to indicate significance. Results revealed that exposure to 3 and 4 ATA of pressure significantly reduced twinkle power by averages of 35% and 39%, respectively, in 7 patients (p = 0.04); data from the eighth patient were excluded because of corruption. This study supports the theory that microbubbles are present on kidney stones in humans.

Tendon injuries comprise 45% of the 66 million musculoskeletal injuries each year with annual costs of $114 billion. Conservative therapies to treat injured tendon produce mixed success rates. Histotripsy is a high intensity focused ultrasound modality that creates and oscillates bubbles to fractionate soft tissues in a well-defined focal volume; however, highly collagenous tissues like tendon have proven resistant to histotripsy. The goal here is to explore acoustic parameters for mechanical fractionation of tendons. Large animal Achilles tendons were exposed to HIFU at 1.07-3.68 MHz with 0.005-20 ms pulses repeated at 1-1000 Hz for 15-60 s with acoustic pressures up to p+ = 97 MPa, p- = 27 MPa. A Verasonics research ultrasound imaging system and ATL L7-4 transducer was used to monitor bubble activity during exposure. Tendon samples were fixated and stained with Hematoxylin and Eosin (HE) for cellular morphology analysis and alpha-nicotinamide dinucleotide diaphorase (αNADH-d) for enzymatic activity analysis. Results showed successful bubble creation in the focal region; however, only 1/90 samples showed focal coagulative necrosis histologically. Future work involves an evaluation of whether bubbles are of insufficient size or collapse strength to mechanically fractionate tendon. [Work supported by Penn State Seed Grant, NIH EB027886, and NSF DGE1255832].

Deep vein thrombosis (DVT) is a life-threatening blood clotting condition that, if undetected, can cause deadly pulmonary embolisms. Critical to its clinical management is the ability to rapidly detect, monitor, and treat thrombosis. However, current diagnostic imaging modalities lack the resolution required to precisely localize vessel occlusions and enable clot monitoring in real time. Here, we rationally design fibrinogen-mimicking fluoropeptide nanoemulsions, or nanopeptisomes (NPeps), that allow contrast-enhanced ultrasound imaging of thrombi and synchronous inhibition of clot growth. The theranostic duality of NPeps is imparted via their intrinsic binding to integrins overexpressed on platelets activated during coagulation. The platelet-bound nanoemulsions can be vaporized and oscillate in an applied acoustic field to enable contrast-enhanced Doppler ultrasound detection of thrombi. Concurrently, nanoemulsions bound to platelets competitively inhibit secondary platelet-fibrinogen binding to disrupt further clot growth. Continued development of this synchronous theranostic platform may open new opportunities for image-guided, non-invasive, interventions for DVT and other vascular diseases.

In the U.S., approximately 14 million tendon and ligament injuries are reported each year. Dry needling (DN) is a conservative treatment introduced to alleviate pain and restore function; however, it is invasive and has mixed success. Focused ultrasound (fUS) is a non-invasive technology that directs ultrasound energy into a well-defined focal volume. fUS induces thermal and/or mechanical bioeffects which can be controlled by the choice of ultrasound parameters. fUS could be an alternative to DN for treatment of tendon injuries, but the bioeffects must be established. Thus, the purpose of this pilot study was to compare the effect of DN and fUS on the mechanical properties and cell morphology of 30 ex vivo rat Achilles tendons. Tendons were randomly assigned to sham, DN, or fUS, with 10 tendons per group. Within each group, 5 tendons were evaluated mechanically, and 5 tendons were analyzed histologically. Elastic modulus in the DN (74.05 ± 15.0 MPa) group was significantly lower than sham (149.84 ± 59.1 MPa; p = 0.0094) and fUS (128.84 ± 28.3 MPa; p = 0.0453) groups. Stiffness in DN (329.05 ± 236.8 N/mm; p = 0.0034) and fUS (315.26 ± 68.9 N/mm; p = 0.0027) groups were significantly lower than sham (786.10 ± 238.7 N/mm) group. Histologically, localized necrosis was observed in 3 out of 5 tendons exposed to fUS, with surrounding tissue unharmed; no evidence of cellular injury was observed in DN or sham groups. These results suggest that fUS preserves the mechanical properties of tendon better than DN. Further studies are needed to evaluate fUS as an alternative, noninvasive treatment modality for tendon injuries.

Collagenous, anisotropic tissue such as tendon has proven resistant to fractionation by histotripsy, prompting the need to better understand and quantify bubble dynamics in anisotropic tissue. The elastic moduli in each axial direction were measured for collagen, fibrin, and polyacrylamide hydrogels prior to applying histotripsy; high-speed photography and cavitation emission data were used to evaluate bubble size and collapse strength. [Work supported by NIH R21EB027886.]

Before clinical translation, all therapeutic ultrasound therapies require testing in pre-clinical models that can be broadly categorized as in vitro (cells), tissue-mimicking phantoms, ex vivo (tissues), and in vivo (animals) to establish safety and effectiveness. Choice of pre-clinical models is governed by the target application, the desired bioeffect, and the types of data that can be collected; other considerations include cost, access, and time. In this tutorial session, an overview of the four main pre-clinical categories will be presented, including common formulations used for therapeutic ultrasound. Within each category, experimental design considerations as well as the relative advantages and disadvantages to each will be discussed.

Collagenous tissues, such as tendon, have proven resistant to mechanical fractionation by histotripsy. Evidence on B-mode ultrasound images suggests the successful creation of boiling bubbles and/or cavitation bubble clouds in these collagenous tissues; however, the oscillation and collapse of the bubbles does not result in tissue fractionation. Here, tissue-mimicking collagen gels were placed at the focus of a 1.5-MHz HIFU transducer and insonified at various pulse lengths and repetition frequencies. Cavitation activity was monitored passively with a Philips/ATL L7-4 imaging transducer (Bothell, WA USA) and Vantage® research ultrasound system (Verasonics, Kirkland, WA, USA) and compared to high-speed photographs (Photron Nova S-9, Tokyo, Japan). Preliminary results in the tissue-mimicking collagen phantoms show violent cavitation activity and rapid gel fractionation with conventional histotripsy parameters, which is dissimilar to what is observed in highly collagenous tissues. To address this limitation, we will explore phantoms with superior fiber alignment, such as fibrin gels, which will allow for evaluation of histotripsy parameters that promote mechanical fractionation in highly collagenous tissues. [Work supported by NIH R21EB027886 and NSF GRF #DGE1255832.]

This paper summarizes the special session, “Shock Waves and Ultrasound for Calculus Fragmentation,” that took place during the 176th Meeting of the Acoustical Society of America and 2018 Acoustics Week in Canada. The sessions were cosponsored by the Biomedical Acoustics and Physical Acoustics Technical Committees and consisted of ten talks by industry and academic researchers from institutions in the United States, France, and the Russian Federation. The sessions described basic and applied acoustic research to manage urinary stones.This paper summarizes the special session, “Shock Waves and Ultrasound for Calculus Fragmentation,” that took place during the 176th Meeting of the Acoustical Society of America and 2018 Acoustics Week in Canada. The sessions were cosponsored by the Biomedical Acoustics and Physical Acoustics Technical Committees and consisted of ten talks by industry and academic researchers from institutions in the United States, France, and the Russian Federation. The sessions described basic and applied acoustic research to manage urinary stones.

Ultrasound is currently the only noninvasive technology able to completely diagnose and manage kidney stones.

The color Doppler ultrasound twinkling artifact, which highlights kidney stones with rapidly changing color, has the potential to improve stone detection; however, its inconsistent appearance has limited its clinical utility. Recently, it was proposed stable crevice bubbles on the kidney stone surface cause twinkling; however, the hypothesis is not fully accepted because the bubbles have not been directly observed. In this paper, the micron or submicron-sized bubbles predicted by the crevice bubble hypothesis are enlarged in kidney stones of five primary compositions by exposure to acoustic rarefaction pulses or hypobaric static pressures in order to simultaneously capture their appearance by high-speed photography and ultrasound imaging. On filming stones that twinkle, consecutive rarefaction pulses from a lithotripter caused some bubbles to reproducibly grow from specific locations on the stone surface, suggesting the presence of pre-existing crevice bubbles. Hyperbaric and hypobaric static pressures were found to modify the twinkling artifact; however, the simple expectation that hyperbaric exposures reduce and hypobaric pressures increase twinkling by shrinking and enlarging bubbles, respectively, largely held for rough-surfaced stones but was inadequate for smoother stones. Twinkling was found to increase or decrease in response to elevated static pressure on smooth stones, perhaps because of the compression of internal voids. These results support the crevice bubble hypothesis of twinkling and suggest the kidney stone crevices that give rise to the twinkling phenomenon may be internal as well as external.

The color Doppler ultrasound twinkling artifact, or localized areas of rapid color shifts, has been used to aid in the diagnosis of kidney stones. Gallstones and some atherosclerotic plaques have also been shown to twinkle, suggesting that crystals, regardless of chemical composition, may harbor the stable microbubbles that are hypothesized to cause twinkling. Here, uric acid, cholesterol, and calcium phosphate crystals, which are often present in sites of pathological biomineralization, were grown in the lab using previously developed techniques and imaged with the Verasonics® research ultrasound system using Philips/ATL P4-2 and Verasonics L22-14 transducers. The magnitude of the Doppler signals was quantified and compared amongst the three crystal chemical compositions. Preliminary results show that all three crystal types displayed twinkling with the strongest magnitude appearing in the cholesterol crystals. These results suggest that twinkling is very sensitive in the presence of crystals regardless of the chemical composition. Future work will explore the use of histotripsy to mechanically fractionate crystals as a novel treatment for pathological biomineralization.The color Doppler ultrasound twinkling artifact, or localized areas of rapid color shifts, has been used to aid in the diagnosis of kidney stones. Gallstones and some atherosclerotic plaques have also been shown to twinkle, suggesting that crystals, regardless of chemical composition, may harbor the stable microbubbles that are hypothesized to cause twinkling. Here, uric acid, cholesterol, and calcium phosphate crystals, which are often present in sites of pathological biomineralization, were grown in the lab using previously developed techniques and imaged with the Verasonics® research ultrasound system using Philips/ATL P4-2 and Verasonics L22-14 transducers. The magnitude of the Doppler signals was quantified and compared amongst the three crystal chemical compositions. Preliminary results show that all three crystal types displayed twinkling with the strongest magnitude appearing in the cholesterol crystals. These results suggest that twinkling is very sensitive in the presence of crystals regardless ...