Background. Deceased-donor kidneys experience cellular stress before undergoing transplantation. To alleviate this, preservation techniques were developed, including normothermic machine perfusion (NMP). Methods. Here, we performed kidney NMP on discarded human kidneys for up to 24 h. Volume management was regulated either by urine recirculation (UR) or urine replacement (NUR) with Ringer's lactate. Notably, UR led to longer perfusion times compared with NUR. To investigate kidney NMP metabolic traits with or without UR overtime, we performed longitudinal metabolomics analyses of perfusates of 8 NMP kidneys by 2-dimensional gas chromatography mass spectrometry (GCxGC-MS). Results. Over 600 metabolic features were profiled, from which 74 were identified and 54 consistently quantified across 26 perfusate samples. Most notably, elevated levels of disaccharides (different isomers), hydroxy-purines, urea, glutamate, and amino acids are associated with the perfusion factor UR. Moreover, donor estimated glomerular filtration rate correlated significantly with the accumulation of lactate and gluconate. Most strikingly, lactate levels seemed to be more balanced in UR-NMP perfusate, which otherwise accumulated rapidly within the first 6 h. Conclusions. Kidney preservation by NMP was previously limited to hours. UR-NMP affected kidney energy homeostasis, carbohydrate and purine metabolism, and the urea and citric acid cycles. These insights add value to explain how urine-driven adaptations contribute to prolonged kidney function under NMP.

The ability to quantify and compare different cavitational treatments is essential given their accelerating clinical adoption. Passive acoustic mapping (PAM) can reconstruct quantitative maps of cavitational radiated energy density (CRED) in real time, providing a powerful platform for cavitation dosimetry. However, significant imaging artefacts caused by a large and variable point spread function (PSF) limit the spatial resolvability of PAM-derived cavitation doses. The Lucy-Richardson deconvolution (LRD) can significantly reduce imaging artefacts, correct for gross overestimates of CRED values, and reduce setup-dependence of CRED estimates when applied to PAM images. However, the LRD condenses source energy densities into the central axis of the image, obscuring the underlying source distribution. Typically, a single central PSF is used to perform deconvolution, which additionally preserves biases in energy estimation due to PSF variation. This work investigates the underlying causes of these errors and presents four deconvolution approaches designed to preserve source localisation and improve spatial accuracy of PAM images. Each algorithm is tested using numerical simulations and experimental cavitational data monitoring cavitation in vitro as well as in normothermically perfused porcine liver, and show varied accuracy, robustness, and computational load. Modelling PSF variations over space to enable a shift-variant implementation of the LRD (svLRD-PAM) improves the spatial accuracy of cavitation doses and reduces imaging artefacts by 4-5 fold with a modest and parallelisable 36% increase in computation time relative to the standard delay-sum-integrate beamformer. svLRD-PAM shows great promise as the next step towards accurate, spatially resolvable cavitation dosimetry.

Passive Acoustic Mapping (PAM) is rapidly emerging as a ubiquitous tool for real-time localization and monitoring of therapeutic ultrasound treatments involving cavitation in the context of safety or efficacy. The ability of PAM to spatially quantify and resolve cavitation activity offers a unique opportunity to correlate the energy of cavitation phenomena with locally observed bioeffects. Objective: We aim to develop methods of measuring and reporting spatio-temporally varying cavitation energies that are energy-preserving, device-independent, and adequately normalized to the volume of tissue being affected by the reported cavitation activity. Methods: We introduce a new metric: cavitational radiated energy density (CRED), defined as the total energy of radiated acoustic emissions over a volume, divided by that volume. We show that image blurring caused by the instrumentation-dependent point spread function introduces significant image artefacts and CRED errors when using existing conventional and adaptive beamformers. We therefore introduce a novel PAM algorithm that utilizes the Lucy-Richardson deconvolution (LRD) technique to compensate for the point-spread function. Results: In single and multi-source simulations and experiments, LRD provides cavitation maps with reduced tail artefacts and improved energy estimates with < 5% increase in computation time relative to a conventional beamformer. Conclusion: We demonstrate the quantitative significance of CRED and the potential of the LRD-PAM algorithm to enable energy-preserving, quantitative, real-time monitoring of cavitation-based therapies independently of the instrumentation employed.

Passive acoustic mapping (PAM) is a powerful and widely used method of imaging cavitation activity. However, the presence of a container around a cavitating sample in experiments performed in vitro can introduce significant aberrations into recorded cavitation noise and resulting PAM images. These artifacts may lead to energy being incorrectly estimated or mapped to the wrong place, preventing accurate correlation between cavitation and bioeffects. In this work, we quantify these acoustic effects for six common types of sample containers using an acoustic reciprocity experiment, then use the results to inform the design of a new container with improved acoustic transparency. Existing vessels were found to introduce up to 13-dB broadband insertion loss and change the location and spread of energy in PAM images by up to 1 mm and 25%, respectively. The new container caused up to 1.4-dB insertion loss (the lowest of any container tested) and introduced no significant phase aberration, source location error, or change in energy spread to the PAM images. Testing the new container with real cavitation noise produced very similar insertion loss figures of up to 1.6 dB. These results highlight deficiencies in existing sample containers for the purposes of quantifying cavitation activity with PAM, which is increasingly desired as cavitation matures as a therapy. The guidelines for acoustic transparency developed here may assist researchers in avoiding container aberrations and enable accurate measurement of cavitation energy in future studies.

The acoustic emissions generated by oscillating gas bubbles (cavitation) provide an extremely useful means of treatment monitoring in ultrasound-mediated therapy. Metrics such as the relative proportions of harmonics and broadband noise in the acoustic emissions frequency spectra are widely used to categorize cavitation, e.g., as “inertial” or “stable.” It is unclear, however, whether these simple categories can adequately describe the wide range of bubble dynamics that may occur or the effects that these may produce in tissue. We have previously shown through simultaneous capture of high-speed video footage and acoustic radiation that even for single bubbles there is no simple correlation between different types of bubble behavior and the frequency content of the acoustic emissions. This suggests that measures such as the onset of broadband noise, or the appearance of sub- or ultra-harmonics are not appropriate for use as universal thresholds in therapeutic ultrasound. Binary categories such as stable and inertial cavitation, or the use of spectral characteristics to infer these, should similarly be avoided. In this talk, the implications of these findings for defining a cavitation dose and applying it to different types of therapy will be discussed.

Lower back pain is closely associated with intervertebral disc (IVD) degeneration and is a leading cause of global disability. Existing treatment options are unable to provide suitable long‐term outcomes, and emerging strategies employing injectable biomaterials are hindered by factors including limited native tissue integration and depth‐ or time‐constrained gelation mechanisms. To overcome these issues, the present research evaluates a new concept employing ultrasound to remotely trigger in situ implant formation. The concept centers around an implant precursor biomaterial consisting of an anionic polysaccharide solution containing thermally sensitive liposomes loaded with ionic crosslinkers. Ultrasound‐mediated heating to 4–5 °C above normal body temperature triggers liposomal release of the crosslinking species, thereby initiating hydrogel formation. Optimization studies define the implant precursor material (1.5% wt/v sodium alginate seeded with calcium‐loaded liposomes (10–15 m m calcium chloride) and 6% wt/v glass microspheres) and the ultrasound parameters (0.95 MHz, 1.6 MPa amplitude, 87% duty cycle). Proof‐of‐concept experiments in degenerated ex vivo bovine IVDs indicate partial restoration of biomechanical function, with the implanted biomaterial well‐integrated into the disc tissue and without material herniation. These results offer promise for treating intervertebral disc degeneration, with continued refinement of biomaterials and protocols being essential for achieving robust in‐disc efficacy.

Genetically encodable gas-filled particles, known as gas vesicles (GVs), have shown promise as a biomolecular contrast agent for ultrasound imaging and have the potential to be used as cavitation nuclei for ultrasound therapy. In this study, we used passive acoustic mapping techniques to characterize GV-seeded cavitation, utilizing 0.5 and 1.6 MHz ultrasound insonation over peak rarefactional pressures ranging from 100 to 2200 kPa. We found that GVs produce cavitation for the duration of the first applied pulse, up to at least 5000 cycles, but that bubble activity diminishes rapidly over subsequent pulses. At 0.5 MHz, the frequency content of cavitation emissions was predominantly broadband in nature, while at 1.6 MHz, narrowband content at harmonics of the main excitation frequency dominated. Simulations and high-speed camera imaging suggest that the received cavitation emissions come not from individual GVs but instead from the coalescence of GV-released gas into larger bubbles during the applied ultrasound pulse. These results will aid the future development of GVs as cavitation nuclei in ultrasound therapy.

Passive Acoustic Mapping (PAM) is a leading approach for the quantification of cavitation energy and can inform the safe and effective use of cavitation in medicine. To achieve quantitative measurements, the sensitivity and directivity of each element in a PAM array must be calibrated, and any issues in the calibration can propagate into poor resolution, incorrect magnitudes, and location errors in all subsequent PAM images. Accurate array calibrations are therefore crucial in the pursuit of a “cavitation dose” to correlate with bioeffects. The most commonly used calibration method, reported by Gray and Coussios in 2018, is a substitution approach using a fine wire scatterer to approximate a point source. Despite its apparent simplicity, this experiment features a number of complicating factors that can prohibit successful measurements, particularly for newer acousticians. Here, we describe the calibration experiment in detail, along with the impacts, causes, and solutions of a range of issues identified through the authors’ hard-won experience. Special consideration will be given to the selection of sources and hydrophones, alignment, management of signal-to-noise ratio, and data processing. We will also discuss common assumptions and their validity, measurement uncertainty, comparing results to theory, and future directions for this area of ultrasound metrology.

Passive Acoustic Mapping (PAM) makes it possible to qualify and quantify the spatio-temporal distribution of cavitation activity during therapeutic ultrasound treatments, in addition to providing a valuable tool for real-time treatment monitoring and cavitation imaging. To enable meaningful correlation of cavitation activity with the safety and efficacy of associated bioeffects, a cavitation dose metric is required that is energy-preserving, device-independent and adequately normalized to the tissue volume being affected by the reported cavitation activity. We introduce cavitational radiated energy density (CRED), defined as the total energy of either narrowband or broadband radiated acoustic emissions over a volume, divided by that volume. We show that image blurring caused by the instrumentation-dependentpoint spread function introduces significant image artifacts and CRED errors when using existing conventional and adaptive beamformers, and propose a novel PAM algorithm that utilizes the Lucy–Richardson deconvolution (LRD) technique to compensate for the point-spread function and thus provide cavitation maps with reduced tail artifacts and improved energy estimates in a computationally efficient manner. Using a combination of modeling and experimental approaches, we demonstrate the quantitative significance of CRED and the potential of the LRD-PAM algorithm to enable energy-preserving quantitative real-time monitoring of cavitation-based therapies, independently of the instrumentation employed.

Testing cell viability in three-dimensional (3D) in vitro cell cultures is not always straightforward. Reduction-based metabolic assays have been suggested as a rapid, reportedly non-toxic mechanism for monitoring cell viability in continuous culture experiments. However, in 3D cultures these assays have been predominantly used semiquantitatively to indicate relative trends in viability. Obtaining quantitative values of cell number is often challenging, particularly when performing continuous measurements of viability when compared with end-point assessments. This work presents experiments performed to robustly identify and address the sources of inconsistency in existing Presto Blue™ methods for 3D cultures. Using human dermal fibroblasts (HDFs) as a model cell line seeded onto type I collagen scaffolds, we examine the impact of Presto Blue™ assays on reported cell numbers in 2D versus 3D cultures. We separate the impact of potential dye-induced toxicity from manipulation-induced cell detachment by considering the effects of passage number, incubation period, modifications of substrate chemistry and duration of total dye exposure as independent variables. The work presented includes a finalised protocol that demonstrates that while resazurin-based reduction assays remain a valuable tool for rapid assessment of cell metabolism and/or viability, additional considerations of the substrate and sampling frequencies are needed in 3D cultures to ensure accurate and reproducible results.

The ability to quantify and compare different cavitational treatments is essential given their accelerating clinical adoption. Passive acoustic mapping (PAM) can reconstruct quantitative maps of cavitational radiated energy density (CRED) in real time, providing a powerful platform for cavitation dosimetry. However, when compared to optical imaging, the relatively longer acoustic wavelengths and longitudinal nature of 2-D PAM result in greater variability and a more significant spatial impact of the point spread function (PSF). The associated imaging artifacts limit the spatial localization of cavitation dose and introduce errors in source energy estimation that vary across source parameters and between setups. Deconvolving with a constant PSF can reduce many of these errors and minimize the setup dependence of CRED estimates, but can introduce iteration-dependent errors in the estimated source location. This work explores the challenges of deconvolving images with large and variable PSFs using algorithms designed for optical applications and proposes strategies for mitigating errors. These approaches will be evaluated with respect to their quantitative performance, computation time, and robustness to noise both in silico and in vitro, with the aim of unlocking the full capabilities of PAM as a spatially resolved, energy preserving, setup independent platform for cavitational dosimetry.

The science of acoustics has extraordinary potential to enable the development of new therapies ranging from non-invasive surgery to oncological drug delivery, neuromodulation, transdermal immunization, and immune-modulation. However, these relatively complex medical devices or combination products often challenge established clinical pathways and face considerable regulatory, usability, adoption, and reimbursement challenges. Using case studies drawn from the clinical translation of novel ultrasound technologies for drug delivery and tissue fractionation, as well as other medical technologies, some of the generic choices and considerations that should be identified and mitigated early on in the technological and commercial development process will be highlighted. For example, products involving ultrasound-responsive injectables require an in-depth understanding of their stability and stimulus-responsiveness, accompanied by the ability to confirm these characteristics post scale-up and sterilization. For all ultrasound technologies, pre-clinical testing also needs to be expanded beyond animal models to address scale and other challenges unique to humans in order to ensure preparedness for a successful clinical trial. Last but not least, the design of early feasibility trials needs to enable confirmation of the mechanism of action of the proposed new therapy and quantification of its potential added benefit relative to the most appropriate current or emerging standard-of-care.

As cavitation-based therapies continue to enter the clinic, there is growing demand for methods to quantify the energy released by cavitating bubbles. Passive Acoustic Mapping (PAM) can reconstruct the energy and distribution of cavitation from multi-sensor recordings of bubble emissions, but its accuracy is impaired in vitro by the aberrating presence of a sample container between the cavitating media (nuclei/cells/tissues) and detectors. To our knowledge, the effects of these vessels on PAM have never been studied. Additionally, the typical need for sterility and a large number of samples makes low cost essential for sample containers in cavitation experiments. Here, we characterize the effects of common laboratory vessels in the range 3–14 MHz via an acoustic reciprocity experiment, then describe the design and testing of a novel container with improved acoustic transparency. The new device reduced worst-case magnitude and phase errors by 13.7 dB and 6.6 radians respectively, compared to ordinary 2 ml centrifuge tubes. We will also present quantitative measures of container effects on PAM energy measurement and localization. The new containers are manufactured from 100 micron polymer film via vacuum forming, are quick and easy to make in any shape in a normal laboratory, and cost US$0.02 each.

Motivation: Lower back pain (LBP) is one of the leading causes of global disability. LBP and related radicular leg pain are closely linked to intervertebral disc (IVD) degeneration, which accounts for approximately 40% of the estimated 619 million LBP cases worldwide. Currently, there are two diametrically opposed treatment options for this condition: conservative physiotherapy to provide temporary relief, or major surgical intervention. Neither has proven to provide suitable long-term outcomes. Emerging strategies focus on injectable biomaterials to provide structural support and facilitate tissue repair, although they are still largely experimental and face several limitations, including limited integration with native tissue. Moreover, the implant formation mechanism may be depth-limited (light curing) or time-constrained (self-curing). Aim: The objective of this research is to demonstrate a new option to restore spinal function through the use of extracorporeal ultrasound to remotely trigger in situ implant formation on demand, such that the clinician can control the process with the timing and location of their choosing. The system concept centers around an implant precursor material consisting of an anionic polysaccharide matrix seeded with thermally sensitive liposomes. Ultrasound-mediated heating on the order of 3-5 degrees above normal body temperature triggers the release of crosslinking species from the liposomes, thereby initiating hydrogel formation. Methods: Candidate polysaccharide and liposome formulations were evaluated for their injectability, loading efficiency, and post-gelation mechanical properties. Ultrasound parameters (frequency, pressure, duty cycle) were optimized for targeted heating efficiency. Techniques for treatment process monitoring and control were independently investigated using thermometry and acoustic cavitation emissions. The material constructs and ultrasound protocols were used together in a series of proof-of-concept experiments using ex vivo bovine IVD specimens, with biomechanical analysis across three states: intact, degenerated, and after ultrasound-triggered implant formation.Results: Extensive testing revealed an optimized implant precursor material consisting of sodium alginate (1.5 wt/v%) seeded with calcium-loaded liposomes (157±9 nm) to enable hyperthermia-triggered release and glass microspheres (6 wt/v%) to ensure preferential ultrasound absorption for safe heating. No significant difference was found between hydrogels heated with an incubator or ultrasound, suggesting comparable calcium release between both methods. Optimal ultrasound parameters for precursor gel heating were found to be 0.95 MHz, 1.6 MPa (peak negative), and 87% duty cycle. Automated treatment control using temperature or cavitation emission measurements both were successfully implemented, with cavitation being preferable for non-invasive implementation. Proof of concept experiments indicated partial restoration of biomechanical function in ex vivo bovine IVDs, with implant material well-integrated into the disc tissue, and without material herniation.Conclusion: We have demonstrated the feasibility of ultrasound-guided hydrogel gelation in situ. These results offer promise for treating spinal disc degeneration, with continued refinement of materials and protocols essential for achieving robust in-disc efficacy.

Dimethylsulfoxide (DMSO) has conventionally been used for cell cryopreservation both in research and in clinical applications, but has long-term cytotoxic effects. Trehalose, a natural disaccharide, has been proposed as a non-toxic cryoprotectant. However, the lack of specific cell membrane transporter receptors inhibits transmembrane transport and severely limits its cryoprotective capability. This research presents a method to successfully deliver trehalose into mesenchymal stem cells (MSCs) using ultrasound in the presence of microbubbles. The optimised trehalose concentration was shown to be able to not only preserve membrane integrity and cell viability but also the multipotency of MSCs, which are essential for stem cell therapy. Confocal imaging revealed that rhodamine-labelled trehalose was transported into cells rather than simply attached to the membrane. Additionally, the membranes were successfully preserved in lyophilised cells. This study demonstrates that ultrasonication with microbubbles facilitated trehalose delivery, offering promising cryoprotective capability without the cytotoxicity associated with DMSO-based methods.

Homogeneous nutrient distribution throughout three-dimensional (3-D) scaffolds remains a key challenge in tissue engineering. The buildup of cells on scaffold edges and rapid nutrient uptake along the periphery often cause large regions of the centre to be left unoccupied by cells. Microstreaming associated with acoustic cavitation has been exploited to enhance mass transport in oncological drug delivery and transdermal vaccination, making it an attractive mechanism for promoting nutrient and oxygen distribution in tissue engineering scaffolds. In this work, we seek to use protein cavitation nuclei to synthesize ultrasound-responsive collagen scaffolds. Cavitation nuclei were embedded into the scaffold during fabrication and then exposed to 0.5 MHz focused ultrasound at peak negative pressures ranging from 0.5 to 2.7 MPa to induce inertial cavitation. Acoustic data was collected using passive cavitation detection (PCD) and post processed to isolate harmonics and broadband emissions. We compare the benefits and disadvantages of including cavitation nuclei in the scaffold fabrication process versus adding them to surrounding media during ultrasound exposure, discussing potential use of embedded protein nuclei to induce cell migration and differentiation. Additionally, we examine the effects of cavitation, exposure time, and peak negative pressure on the microstructure of collagen scaffolds, namely, pore size, interconnectivity, and percolation diameter.

In the fight against a broad spectrum of human diseases, cavitation techniques show great promise for overcoming physical barriers that lead to suboptimal uptake of passively administered therapeutics. However, small animal testing of candidate therapies remains a poor predictor of clinical success. Here we demonstrate ultrasound-mediated drug delivery in normal and tumour-bearing human livers infused with protein-based cavitation nuclei (PCaN). Whole and partial human livers were obtained immediately from hepatectomy surgeries and were normothermically sustained using a clinically approved perfusion system (OrganOx Metra). Ultrasound was applied using a 0.5 MHz focused source (Sonic Concepts H107) and was monitored with a calibrated linear array (ATS L7-4) for real time structural and cavitational imaging implemented on an array controller (Verasonics Vantage 256). Specifically, the therapy process was monitored using passive acoustic mapping (PAM) of broadband cavitation emissions, employing a non-adaptive beamformer that deconvolves the array point spread function. Levels of fluorescently labelled drugs incorporated in and co-administered with the PCaN were quantified in blood and tissue samples collected during and following treatment, respectively. This presentation highlights PAM observations of broadband cavitation persistence and drug delivery in untargeted and targeted tissues, including the first ever experiments in tumour-bearing human livers.

BACKGROUND:Liver disease is the third leading cause of premature death in the UK. Transplantation is the only successful treatment for end-stage liver disease but is limited by a shortage of suitable donor organs. As a result, up to 20% of patients on liver transplant waiting lists die before receiving a transplant. A third of donated livers are not suitable for transplant, often due to steatosis. Hepatic steatosis, which affects 33% of the UK population, is strongly associated with obesity, an increasing problem in the potential donor pool. We have recently tested defatting interventions during normothermic machine perfusion (NMP) in discarded steatotic human livers that were not transplanted. A combination of therapies including forskolin (NKH477) and L-carnitine to defat liver cells and lipoprotein apheresis filtration were investigated. These interventions resulted in functional improvement during perfusion and reduced the intrahepatocellular triglyceride (IHTG) content. We hypothesise that defatting during NMP will allow more steatotic livers to be transplanted with improved outcomes. METHODS:In the proposed multi-centre clinical trial, we will randomly assign 60 livers from donors with a high-risk of hepatic steatosis to either NMP alone or NMP with defatting interventions. We aim to test the safety and feasibility of the defatting intervention and will explore efficacy by comparing ex-situ and post-reperfusion liver function between the groups. The primary endpoint will be the proportion of livers that achieve predefined functional criteria during perfusion which indicate potential suitability for transplantation. These criteria reflect hepatic metabolism and injury and include lactate clearance, perfusate pH, glucose metabolism, bile composition, vascular flows and transaminase levels. Clinical secondary endpoints will include proportion of livers transplanted in the two arms, graft function; cell-free DNA (cfDNA) at follow-up visits; patient and graft survival; hospital and ITU stay; evidence of ischemia-reperfusion injury (IRI); non-anastomotic biliary strictures and recurrence of steatosis (determined on MRI at 6 months). DISCUSSION:This study explores ex-situ pharmacological optimisation of steatotic donor livers during NMP. If the intervention proves effective, it will allow the safe transplantation of livers that are currently very likely to be discarded, thereby reducing waiting list deaths. TRIAL REGISTRATION:ISRCTN ISRCTN14957538. Registered in October 2022.