Dynamics of crevice microbubbles that cause the twinkling artifact.

The Doppler ultrasound twinkling artifact, a rapid color shift, appears on pathological mineralizations and is theorized to arise from scattering off micron-sized crevice microbubbles. However, the influence of crevice number and size as well as the bubble dynamics on twinkling is not well-understood. Cylinders with diameters of 0.8-1.2 µm and depths of 1 µm were etched into a silicon wafer and crevice bubbles were driven at 0.75, 2.5, and 5.0 MHz while monitoring with high-speed photography. Experimental results were compared to a derived crevice bubble model. On three separate wafers, cylindrical crevices (10 or 100) with diameters of 1, 10, or 100 µm and depths of 10 µm were etched and imaged with a research ultrasound system in Doppler mode at 5, 7.8, and 18.5 MHz. Within the pressure ranges studied here (∼1MPa), no bubble oscillation was observed for the 0.8-1.2 µm crevice bubbles which matched computational results. Crevices with 1 and 10 µm diameters produced more twinkling than 100 µm crevices at 5 and 7.8 MHz. In contrast, 100 µm crevices produced more twinkling than 1 or 10 µm crevices at 18.5 MHz (p < 0.001 in all cases). These results provide better insight into how crevice bubbles cause twinkling on pathological mineralizations.

Focused ultrasound as an alternative to dry needling for the treatment of tendinopathies: A murine model.

Tendinopathies account for 30% of 102 million annual musculoskeletal injuries occurring annually in the United States. Current treatments, like dry needling, induce microdamage to promote healing but produce mixed success rates. Previously, we showed focused ultrasound can noninvasively create microdamage while preserving mechanical properties in ex vivo murine tendons. This present study compared growth factor, histological, and mechanical effects after focused ultrasound or dry needling treatments in an in vivo murine tendon injury model. Partial Achilles tenotomy was performed in 26 rats. One-week postsurgery, tendons were treated with focused ultrasound (1.5 MHz, 1-ms pulses at 10 Hz for 106 s, p+ = 49 MPa, p- = 19 MPa) or dry needling (30 G needle, 5 fenestrations over 20 s) and survived for 1 additional week. Blood was collected immediately before and after treatment and before euthanasia; plasma was assayed for growth factors. Treated tendons and contralateral controls were harvested for histology or mechanical testing. No differences were found between treatments in release of insulin growth factor 1 and transforming growth factor beta; vascular endothelial growth factor A concentrations were too low for detection. Histologically, focused ultrasound and dry needling tendons displayed localized fibroblast infiltration without collagen proliferation with no detectable differences between treatments. Mechanically, stiffness and percent relaxation of dry needling tendons were lower than controls (p = 0.0041, p = 0.0441, respectively), whereas stiffness and percent relaxation of focused ultrasound tendons were not different from controls. These results suggest focused ultrasound should be studied further to determine how this modality can be leveraged as a therapy for tendinopathies.

Effect of ambient gas and crystal features on Doppler ultrasound twinkling of pathological mineralizations.

Color Doppler twinkling on kidney stones and other pathological mineralizations is theorized to arise from stable microbubbles, which suggests twinkling will be sensitive to ambient gas. Here, lab-grown cholesterol, calcium phosphate, and uric acid crystals were imaged with ultrasound in water while varying oxygen, carbon dioxide, and nitrogen levels. Twinkling was found to increase on cholesterol in elevated oxygen, cholesterol and calcium phosphate in elevated carbon dioxide, and no crystals in elevated nitrogen. These results support the crevice microbubble theory of twinkling and suggest gases may be varied to enhance twinkling on some mineralizations.

Controlled Degradation of Polycaprolactone Polymers through Ultrasound Stimulation.

This study describes the development of an ultrasound-responsive polymer system that provides on-demand degradation when exposed to high-intensity focused ultrasound (HIFU). Diels-Alder cycloadducts were used to crosslink polycaprolactone (PCL) polymers and underwent a retro Diels-Alder reaction when stimulated with HIFU. Two Diels-Alder polymer compositions were explored to evaluate the link between reverse reaction energy barriers and polymer degradation rates. PCL crosslinked with isosorbide was also used as a non-Diels-Alder-based control polymer. An increase of HIFU exposure time and amplitude correlated with an increase of PCL degradation for Diels-Alder-based polymers. Ultrasound imaging during HIFU allowed for real-time visualization of the on-demand degradation through cavitation-based mechanisms. The temperature surrounding the sample was monitored with a thermocouple during HIFU stimulation; a minimal increase in temperature was observed. PCL polymers were characterized using Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), optical profilometry, and mechanical testing. PCL degradation byproducts were identified by mass spectrometry, and their cytocompatibility was evaluated in vitro. Overall, this study demonstrated that HIFU is an effective image-guided, external stimulus to control the degradation of Diels-Alder-based PCL polymers on-demand.

The histotripsy spectrum: differences and similarities in techniques and instrumentation.

Since its inception about two decades ago, histotripsy - a non-thermal mechanical tissue ablation technique - has evolved into a spectrum of methods, each with distinct potentiating physical mechanisms: intrinsic threshold histotripsy, shock-scattering histotripsy, hybrid histotripsy, and boiling histotripsy. All methods utilize short, high-amplitude pulses of focused ultrasound delivered at a low duty cycle, and all involve excitation of violent bubble activity and acoustic streaming at the focus to fractionate tissue down to the subcellular level. The main differences are in pulse duration, which spans microseconds to milliseconds, and ultrasound waveform shape and corresponding peak acoustic pressures required to achieve the desired type of bubble activity. In addition, most types of histotripsy rely on the presence of high-amplitude shocks that develop in the pressure profile at the focus due to nonlinear propagation effects. Those requirements, in turn, dictate aspects of the instrument design, both in terms of driving electronics, transducer dimensions and intensity limitations at surface, shape (primarily, the F-number) and frequency. The combination of the optimized instrumentation and the bio-effects from bubble activity and streaming on different tissues, lead to target clinical applications for each histotripsy method. Here, the differences and similarities in the physical mechanisms and resulting bioeffects of each method are reviewed and tied to optimal instrumentation and clinical applications.

Real-Time, In Situ Imaging of Macrophages via Phase-Change Peptide Nanoemulsions.

Macrophages are specialized phagocytes that play central roles in immunity and tissue repair. Their diverse functionalities have led to an evolution of new allogenic and autologous macrophage products. However, realizing the full therapeutic potential of these cell-based therapies requires development of imaging technologies that can track immune cell migration within tissues in real-time. Such innovations will not only inform treatment regimens and empower interpretation of therapeutic outcomes but also enable prediction and early intervention during adverse events. Here, phase-changing nanoemulsion contrast agents are reported that permit real-time, continuous, and high-fidelity ultrasound imaging of macrophages in situ. Using a de novo designed peptide emulsifier, liquid perfluorocarbon nanoemulsions are prepared and show that rational control over interfacial peptide assembly affords formulations with tunable acoustic sensitivity, macrophage internalization, and in cellulo stability. Imaging experiments demonstrate that emulsion-loaded macrophages can be readily visualized using standard diagnostic B-mode and Doppler ultrasound modalities. This allows on-demand and long-term tracking of macrophages within porcine coronary arteries, as an exemplary model. The results demonstrate that this platform is poised to open new opportunities for non-invasive, contrast-enhanced imaging of cell-based immunotherapies in tissues, while leveraging the low-cost, portable, and safe nature of diagnostic ultrasound.

Dual-frequency boiling histotripsy in an ex vivo bovine tendinopathy model.

Histotripsy fractionates most soft tissues; however, healthy tendons have shown resistance to histotripsy fractionation. Prior work has shown that pre-heating tendons increases susceptibility to histotripsy fractionation; combining multiple driving frequencies may also allow successful fractionation of tendons. Here, we evaluate single- and dual-frequency histotripsy in four healthy and eight tendinopathic ex vivo bovine tendons. First, we evaluated single-frequency (1.07, 1.5, and 3.68 MHz) and dual-frequency (1.07 and 1.5 MHz or 1.5 and 3.68 MHz) bubble dynamics with high-speed photography in a tissue-mimicking phantom. Then, tendons were treated with histotripsy. Cavitation activity was monitored with a passive cavitation detector (PCD) and targeted areas were evaluated grossly and histologically. Results in tendinopathic tendons showed 1.5 MHz or 3.68 MHz single-frequency exposure caused focal disruption, whereas 1.5 and 3.68 MHz dual-frequency exposures caused fractionated holes; all treatments caused some thermal denaturation. Exposure to 1.07 MHz alone or combined with 1.5 MHz did not show fractionation in tendinopathic tendons. In healthy tendons, only thermal necrosis was observed for all tested exposures. PCD showed some differences in cavitation activity in tendinopathic tendons but did not predict successful fractionation. These results suggest that full histotripsy fractionation is possible using dual-frequency exposures in tendinopathic tendons.

The effect of crystal composition and environment on the color Doppler ultrasound twinkling artifact.

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 grownin vitroand 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 <22 mN m-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.

High intensity focused ultrasound atomization and erosion in healthy and tendinopathic tendons.

Objective.High-intensity focused ultrasound (HIFU) can induce thermal and mechanical mechanisms in a well-defined focal volume of tissues. Histotripsy is a form of mechanical HIFU that can initiate and interact with bubble(s) to cause shock scattering and perhaps atomization within the bubble(s) to fractionate most soft tissues. Ultrasonic atomization, or the ejection of fine droplets from an acoustically-excited liquid exposed to air, has been shown to erode planar soft tissue surfaces, which has led to theories that atomization is a mechanism in histotripsy. However, healthy tendons show resistance to conventional histotripsy; pre-treatment of tendons with heat increases susceptibility to histotripsy fractionation. This study investigates ultrasonic atomization and erosion from planar healthy and tendinopathic tendon surfaces as we evaluate HIFU parameters for histotripsy in tendons.Approach.Forty-sixex vivobovine tendon-air interfaces were pre-conditioned to surface wetting, heat baths of 20 °C (unaltered), 37 °C (body temperature), and 58 °C (collagen degradation), collagenase soaks for 1, 3, 5, and 24 h (mimicking tendinopathic tendons), and phosphate buffered saline soaks for 24 h. Ejected fragments, histology, and gross analysis determined erosion success. Tissue displacement from the HIFU radiation force was monitored with high-speed photography, and tissue relaxation was pixel-tracked and fit to a Kelvin-Voigt model to evaluate changes in viscoelastic properties.Main results.Results showed that atomization produced holes in 24 h collagenase tendons and surface pitting in 58 °C, 3 h, and 5 h collagenase tendons. Increased mound heights and viscoelastic constants in pre-heated (to 58 °C) and collagenase-soaking (3+ hours) tendinopathic models caused a decrease in elasticity and/or increase in viscosity, increasing susceptibility to erosion by HIFU atomization.Significance.Therefore, tendons with chronic tendinopathies may be more susceptible than healthy tendons to histotripsy fractionation.

Histotripsy Bubble Dynamics in Elastic, Anisotropic Tissue-Mimicking Phantoms.

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+ = 127 MPa, p- = 35 MPa); treatments were monitored with passive cavitation imaging and high-speed photography. Dehydrated fibrin gels were found to be the most similar to tendon in cavitation emission energy (fibrin = 0.69 ± 0.24, tendon = 0.64 ± 0.19 [× 1010 V2]) and anisotropy (fibrin = 3.16 ± 1.12, tendon = 19.4). Bubble cloud area in dehydrated fibrin (0.79 ± 0.14 mm2) was significantly smaller than most other tested hydrogels. Finally, anisotropy was found to have moderately strong linear relationships with cavitation energy and bubble cloud size (r = -0.65 and -0.80, respectively). Dehydrated fibrin shows potential as a repeatable, transparent, tissue-mimicking hydrogel for evaluation of histotripsy bubble dynamics in elastic, anisotropic tissues.

The effects of elastic modulus and impurities on bubble nuclei available for acoustic cavitation in polyacrylamide hydrogels.

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.

Therapeutic Ultrasound and Shockwave Therapy for Tendinopathy: A Narrative Review.

ABSTRACT: 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.

Ultrasound-Responsive Hydrogels for On-Demand Protein Release.

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.

Automated Tissue Strain Calculations Using Harris Corner Detection.

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.

Effects of focused ultrasound and dry needling on tendon mechanical properties.

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.

Ultrasound-Responsive Nanopeptisomes Enable Synchronous Spatial Imaging and Inhibition of Clot Growth in Deep Vein Thrombosis.

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.

Focused Ultrasound Mechanical Disruption of Ex Vivo Rat Tendon.

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 pressures <89 MPa and peak negative pressures <26 MPa; other tendons were exposed to dry needling or sham. Immediately after treatment, tendons were flash-frozen and stained with hematoxylin and eosin (H&E) 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.

Comparison between dry needling and focused ultrasound on the mechanical properties of the rat Achilles tendon: A pilot study.

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.

Evaluation of Stone Features That Cause the Color Doppler Ultrasound Twinkling Artifact.

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.

Evidence of Microbubbles on Kidney Stones in Humans.

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 p < 0.05 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.