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Jingle Cell Rock: Steering Cellular Activity With Low-Intensity Pulsed Ultrasound (LIPUS) to Engineer Functional Tissues in Regenerative Medicine.
Acoustic manipulation or perturbation of biological soft matter has emerged as a promising clinical treatment for a number of applications within regenerative medicine, ranging from bone fracture repair to neuromodulation. The potential of ultrasound (US) endures in imparting mechanical stimuli that are able to trigger a cascade of molecular signals within unscathed cells. Particularly, low-intensity pulsed ultrasound (LIPUS) has been associated with bio-effects such as activation of specific cellular pathways and alteration of cell morphology and gene expression, the extent of which can be modulated by fine tuning of LIPUS parameters including intensity, frequency and exposure time. Although the molecular mechanisms underlying LIPUS are not yet fully elucidated, a number of studies clearly define the modulation of specific ultrasonic parameters as a means to guide the differentiation of a specific set of stem cells towards adult and fully differentiated cell types. Herein, we outline the applications of LIPUS in regenerative medicine and the in vivo and in vitro studies that have confirmed the unbounded clinical potential of this platform. We highlight the latest developments aimed at investigating the physical and biological mechanisms of action of LIPUS, outlining the most recent efforts in using this technology to aid tissue engineering strategies for repairing tissue or modelling specific diseases. Ultimately, we detail tissue-specific applications harnessing LIPUS stimuli, offering insights over the engineering of new constructs and therapeutic modalities. Overall, we aim to lay the foundation for a deeper understanding of the mechanisms governing LIPUS-based therapy, to inform the development of safer and more effective tissue regeneration strategies in the field of regenerative medicine.
Rapid Production of Nanoscale Liposomes Using a 3D-Printed Reactor-In-A-Centrifuge: Formulation, Characterisation, and Super-Resolution Imaging.
Nanoscale liposomes have been extensively researched and employed clinically for the delivery of biologically active compounds, including chemotherapy drugs and vaccines, offering improved pharmacokinetic behaviour and therapeutic outcomes. Traditional laboratory-scale production methods often suffer from limited control over liposome properties (e.g., size and lamellarity) and rely on laborious multistep procedures, which may limit pre-clinical research developments and innovation in this area. The widespread adoption of alternative, more controllable microfluidic-based methods is often hindered by complexities and costs associated with device manufacturing and operation, as well as the short device lifetime and the relatively low liposome production rates in some cases. In this study, we demonstrated the production of liposomes comprising therapeutically relevant lipid formulations, using a cost-effective 3D-printed reactor-in-a-centrifuge (RIAC) device. By adjusting formulation- and production-related parameters, including the concentration of polyethylene glycol (PEG), temperature, centrifugation time and speed, and lipid concentration, the mean size of the produced liposomes could be tuned in the range of 140 to 200 nm. By combining selected experimental parameters, the method was capable of producing liposomes with a therapeutically relevant mean size of ~174 nm with narrow size distribution (polydispersity index, PDI ~0.1) at a production rate of >8 mg/min. The flow-through method proposed in this study has potential to become an effective and versatile laboratory-scale approach to simplify the synthesis of therapeutic liposomal formulations.
Ultrasound-activated microbubbles as a novel intracellular drug delivery system for urinary tract infection.
The development of new modalities for high-efficiency intracellular drug delivery is a priority for a number of disease areas. One such area is urinary tract infection (UTI), which is one of the most common infectious diseases globally and which imposes an immense economic and healthcare burden. Common uropathogenic bacteria have been shown to invade the urothelial wall during acute UTI, forming latent intracellular reservoirs that can evade antimicrobials and the immune response. This behaviour likely facilitates the high recurrence rates after oral antibiotic treatments, which are not able to penetrate the bladder wall and accumulate to an effective concentration. Meanwhile, oral antibiotics may also exacerbate antimicrobial resistance and cause systemic side effects. Using a human urothelial organoid model, we tested the ability of novel ultrasound-activated lipid microbubbles to deliver drugs into the cytoplasm of apical cells. The gas-filled lipid microbubbles were decorated with liposomes containing the non-cell-permeant antibiotic gentamicin and a fluorescent marker. The microbubble suspension was added to buffer at the apical surface of the bladder model before being exposed to ultrasound (1.1 MHz, 2.5 Mpa, 5500 cycles at 20 ms pulse duration) for 20 s. Our results show that ultrasound-activated intracellular delivery using microbubbles was over 16 times greater than the control group and twice that achieved by liposomes that were not associated with microbubbles. Moreover, no cell damage was detected. Together, our data show that ultrasound-activated microbubbles can safely deliver high concentrations of drugs into urothelial cells, and have the potential to be a more efficacious alternative to traditional oral antibiotic regimes for UTI. This modality of intracellular drug delivery may prove useful in other clinical indications, such as cancer and gene therapy, where such penetration would aid in treatment.
Ultrasound-compatible 3D-printed Franz diffusion system for sonophoresis with microbubbles.
Sonophoresis is a topical drug delivery approach that utilises ultrasound as a physical stimulus to enhance permeation of active pharmaceutical ingredients through the skin. Only limited research has however been conducted to evaluate the potential of ultrasound-responsive drug carriers, such as gas microbubbles, in sonophoresis. Franz diffusion cells have been extensively used for measuring drug permeation in vitro; however, traditional systems lack compatibility with ultrasound and only limited characterisation of their acoustical behaviour has been carried out in previous research. To overcome this limitation, we designed and manufactured a novel Franz cell donor compartment coupled with a conventional glass receptor, and performed a functional characterisation of the assembly for application in sonophoresis with ultrasound-responsive agents (specifically imiquimod-loaded gas microbubbles). The donor was fabricated using a photoreactive resin via 3D printing and was designed to enable integration with a therapeutically relevant ultrasound source. The assembly was capable of effectively retaining liquids during prolonged incubation and the absorption of imiquimod onto the 3D-printed material was comparable to the one of glass. Moreover, a predictable ultrasound field could be generated at a target surface without any significant spatial distortion. Finally, we demonstrated applicability of the developed assembly in sonophoresis experiments with StratM®, wherein ultrasound stimulation in the presence of microbubbles resulted in significantly enhanced drug permeation through and partitioning within the membrane (2.96 ± 0.25 μg and 3.84 ± 0.39 μg) compared to passive diffusion alone (1.74 ± 0.29 μg and 2.29 ± 0.32 μg), over 24 h.
Research data for: Understanding the dynamics of superparamagnetic particles under the influence of high field gradient arrays
The archive file, "SW Experiments 170316.zip", contains all images obtained on 17 Mar 2016. Each file should involve images captured using a microscope of magnetic microbeads being conveyed in a glass capillary channel, focused into a single trajectory by a standing wave ultrasound field, and then magnetically deflected towards a magnet array, following the method described in the related publication. The .zip archive can be accessed along with all other parts ("SW Experiments 170316.z01" and "SW Experiments 170316.z02"), and the image files are organized inside the archive based on the nominal distance between the glass channel and magnet array, and labelled based on the experimental parameters set while the image was captured, including volumetric flow rate, run number and signal generator voltage. The .opj files can be opened using Origin (OriginLab, MA, USA). The file "Numerical simulations with linear Halbach Array.opj" contains data resulting from numerical simulations that are described and reported in section 3.1 of the related publication. The file "Numerical simulations with other magnetic systems.opj" contains data resulting from numerical simulations that are described and reported in section 3.3 of the related publication. The file "Magnetometry.opj" contains data resulting from magnetometry measurements of an ensemble of magnetic microbeads; the results are described and reported in appendix A of the related publication. The file "Analytical capture efficiencies with different initial distributions.opj" contains data resulting from semi-analytical simulations that are described and reported in appendix C of the related publication. The file, "Glass capillary device.mph" can be opened using COMSOL Multiphysics (COMSOL, Inc, Burlington, MA, USA) and contains a finite element model of the device used to generate an ultrasound standing wave inside a glass capillary channel. Results of simulations using this model are described and reported in appendix B of the related publication.
Magnetic targeting to enhance microbubble delivery in an occluded micro arterial bifurcation
The data were generated in the course of the numerical and practical experimental wok described in the accompanying paper. They consist of JPG image files and excel spreadsheets detailing the results of this work and were generated between 2015 and 2017. They can be read by standard image and/or spreadsheet software
Spectral Imaging Toolbox v1.0
Background: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internal-ized fluorescent probes. Results: Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internal-ized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is deter-mined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for model and cell membranes and microbubbles with carboxyl-modified Laurdan (C-Laurdan) and Di-4-AN(F)EPPTEA (FE), common environmentally-sensitive probes for quantification of the local lateral density of lipids or lipid packing. Conclusions: The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membranes segmentation and no ability in programming required.
Spectral Imaging Toolbox
NB: This dataset has since been superseded by Spectral Imaging Toolbox v1.0 (DOI: 10.5287/bodleian:gp8XN80Pw). The Spectral Imaging Toolbox is designed to facilitate batch processing and analysis of image stacks and hyperstacks produced by spectral imaging. Key highlights include reliable membrane segmentation, batch processing, 3D reconstruction, spectra generation, generalized polarization (GP) maps and histograms, GP calibration factor, and .xls summary output. Demo images and instructions as well as a .pdf user manual are included in the download. Note: the Spectral Imaging Toolbox requires MATLAB and the MATLAB Image Processing Toolbox.
Research data for: Optimized shapes of magnetic arrays for drug targeting applications
The .opj files can be opened using Origin (OriginLab, MA, USA). The file, "Volume dep study.opj" contains designs, data and analysis of optimized magnet arrays reported or referred to in the related publication, specifically in section 3.1. The file "POI dep study.opj" contains designs, data and analysis of optimized magnet arrays reported or referred to in the related publication, specifically in sections 3.2 and 3.5. The file "DOF dep study.opj" contains designs, data and analysis of optimized magnet arrays reported or referred to in the related publication, specifically in sections 3.3 and 3.6. The file "COMSOL particle trajectory simulations data and models.zip" is a compressed archive containing finite element model files, in .mph format, which can be opened using COMSOL Multiphysics (COMSOL, Inc, Burlington, MA, USA) and supporting look-up tables in spreadsheet format. The results generated from simulations using these model files are contained in the file "COMSOL particle tracing v2.opj", together with supporting data and analysis, following the methods outlined in sections 2.3 and 3.4 of the related publication.
Research data for: Halbach arrays consisting of cubic elements optimised for high field gradients in magnetic drug targeting applications
The file, Magnetic_microbubble_retention_experiments_data.zip contains all videos obtained on 4 Dec 2014. Each video should involve magnetic targeting of microbubbles against flow. The Excel file in this compressed folder lists the type of magnet used during each video in terms of the time of day that the video was obtained. All other experimental parameters are detailed in the related publication. The .fig files, accessible using Matlab (MathWorks, Natick, MA, USA), display the regions of interest used to analyze each video and are named in terms of the time of day when the associated video was obtained. The .opj files can be opened using Origin (OriginLab, MA, USA), one of which stores analysis of results from the magnetic microbubble retention experiments and the other two give raw data and analysis of microbubble and ferrofluid magnetometry data obtained following the method described in the related publication. The Origin Graph file, Analysis_of_optimized_designs.opj contains designs, data and analysis of optimized magnet arrays reported or referred to in the related publication, as well as experimental Gaussmeter measurements of the assembled array, obtained following the methods described in the related publication. The COMSOL Model file, Assembled_array.mph can be opened using COMSOL Multiphysics (COMSOL, Inc, Burlington, MA, USA) and contains a finite element model of the assembled array reported in the related publication to model its magnetic properties.
Nanoparticle-loaded protein-polymer nanodroplets for improved stability and conversion efficiency in ultrasound imaging and drug delivery
These data were created on 15th July of 2015 for the paper to be published in Advanced Materials. We have developed a new formulation of volatile nanodroplets, stabilised by a protein and polymer coating. The nanodroplets are prepared from hydrophobic perfluoropentane (PFP) and coated with human serum albumin and polyethylene glycol modified N-hydrosuccinimide conjugated in dichloromethane using an oil-in-water emulsification method. The resulting nanodroplets have an average diameter of 344 nm and are stable at 37oC for several days. These properties offer advantages both for storage of the droplets and their ability to extravasate and permeate target tissue as compared with microbubble agents. Upon exposure to ultrasound the nanodroplets undergo a phase change, generating microbubbles. The efficiency of this process was increased by a factor of 2.8 when iron oxide nanocrystals were added to the PFP. In addition, hydrophobic drugs can be incorporated into the droplet core and the release characteristics of an anti-cancer drug, paclitaxel, were studied. The rate of drug release was found to increase by 70.7 % compared to a control formulation without PFP upon exposure to ultrasound for 180 seconds. Finally the effect of the nanodroplets on breast cancer cells was compared with that of the control formulation, and it was found that the droplets showed 38%p enhanced cytotoxicity than that of free drug.
Repurposing antimicrobials with ultrasound-triggered nanoscale systems for targeted biofilm drug delivery.
Chronic infections represent a major clinical challenge due to the enhanced antimicrobial tolerance of biofilm-dwelling bacteria. To address this challenge, an ultrasound-responsive nanoscale drug delivery platform (nanodroplets) is presented in this work, loaded with four different antimicrobial agents, capable of simultaneous biofilm disruption and targeted antimicrobial delivery. When loaded, a robust protective effect against clinically-derived MRSA and ESBL Gram-positive and Gram-negative planktonic isolates was shown in vitro. Upon application of therapeutic ultrasound, an average 7.6-fold, 44.4-fold, and 25.5-fold reduction was observed in the antibiotic concentrations compared to free drug required to reach the MBC, MBEC and complete persister eradication levels, respectively. Nanodroplets substantially altered subcellular distribution of encapsulated antimicrobials, enhancing accumulation of antimicrobials by 11.1-fold within the biofilm-residing bacteria's cytoplasm compared to treatment with unencapsulated drugs. These findings illustrate the potential of this multifunctional platform to overcome the critical penetration and localization limitations of antimicrobials within biofilms, opening potential new avenues in the treatment of chronic clinical infections.
Optimising the manufacture of perfluorocarbon nanodroplets through varying sonication parameters.
Perfluorocarbon nanodroplets (PFC-NDs) are promising ultrasound-responsive theranostic agents with applications in both diagnostic imaging and drug delivery. The acoustic vaporisation threshold, extravasation potential, and stability of PFC-NDs are all affected by their size. However, methods to ensure reproducible size and concentration during production by sonication are lacking. To address this need, we examined the effect of temperature, sonication time, sonication intensity, PFC concentration and sonicator tip height on ND characteristics. PFC-NDs with a perfluoro-n-pentane (PFP) core and a phospholipid shell were manufactured by probe-sonication. Pulsed sonication was used to maintain the sample temperature below the boiling point of PFP. Median particle diameter was measured using nanoparticle tracking analysis. PFC-ND diameter increased with increasing PFP concentration, with a stronger relationship as sonicator tip height increased. Above 5% v/v PFP, there was a qualitative increase in the number of particles visible by light microscopy. Increasing the sonication duration did not yield a significant change in ND size. A minimum amplitude of 60% was required for mixing to occur, with amplitudes of 80% and 100% resulting in foam production. Sonicator power output was linear with respect to time but differed depending on sample volume, composition, and vessel geometry. This study indicates that controlling the processing parameters can facilitate reproducible manufacturing of PFC-NDs.
Artificial urinary bladder model.
Technological advancements in the medical field are often slow and expensive, sometimes due to complexities associated with pre-clinical testing of medical devices and implants. There is therefore a growing need for new test beds that can mimic more closely the in vivo environment of physiological systems. In the present study, a novel bladder model was designed and fabricated with the aim of providing a pre-clinical testing platform for urological stents and catheters. The model is collapsible, has a Young's modulus that is comparable to a biological bladder, and can be actuated on-demand to enable voiding. Moreover, the developed fabrication technique provides versatility to adjust the model's shape, size, and thickness, through a rapid and relatively inexpensive process. When compared to a biological bladder, there is a significant difference in compliance; however, the model exhibits cystometry profiles during priming and voiding that are qualitatively comparable to a biological bladder. The developed bladder model has therefore potential for future usage in urological device testing; however, improvements are required to more closely replicate the architecture and relevant flow metrics of a physiological bladder.
Mitigating infections in implantable urological continence devices: risks, challenges, solutions, and future innovations. A comprehensive literature review.
PURPOSE OF REVIEW: Stress urinary incontinence is a growing issue in ageing men, often following treatment for prostate cancer or bladder outflow obstruction. While implantable urological devices offer relief, infections are a significant concern. These infections can lead to device removal, negating the benefits and impacting patient outcomes. This review explores the risks and factors contributing to these infections and existing strategies to minimize them. These strategies encompass a multifaceted approach that considers patient-specific issues, environmental issues, device design and surgical techniques. However, despite these interventions, there is still a pressing need for further advancements in device infection prevention. RECENT FINDINGS: Faster diagnostics, such as Raman spectroscopy, could enable early detection of infections. Additionally, biocompatible adjuncts like ultrasound-responsive microbubbles hold promise for enhanced drug delivery and biofilm disruption, particularly important as antibiotic resistance rises worldwide. SUMMARY: By combining advancements in diagnostics, device design, and patient-specific surgical techniques, we can create a future where implantable urological devices offer men a significant improvement in quality of life with minimal infection risk.
Contrast-enhanced ultrasound (CEUS) reveals perfusion of human bone fracture in acute-phase healing
Bone fractures are common injuries with reported non-union rates of up to 9%. Current treatments for non-union include surgery, which is expensive and involves significant morbidity. Microbubbles are used clinically in ultrasonography as contrast agents and have been shown to deliver therapeutics to desired locations by noninvasive stimulation of cavitation using extracorporeal ultrasound in preclinical studies. Contrast enhanced ultrasound (CEUS) has been used to determine the cause of established fracture non-unions, though this is not in widespread use clinically. This study aimed to test the hypothesis that peripherally injected microbubbles are detectable in acute fracture sites. Adult patients (18 – 75 yrs) with acute humeral shaft fractures were recruited to undergo CEUS within 28 days of injury. They underwent peripheral injection of SonoVue microbubbles with ultrasound imaging. B-mode and contrast-mode videos were collected and time-intensity curve analysis was used to assess for the presence of microbubbles at the fracture site. Ten patients were recruited, 8 underwent humeral shaft fracture scans with 7 analysed. All fracture sites demonstrated increased contrast signal following injection. The wash-in volume of microbubbles was greater than the wash-out volume in all cases, with a mean difference of 1.4 × 10⁻⁵ (± 1.7 × 10⁻⁵) (p=0.015 Wilcoxon Test). There was a noticeable decrease in PI and Time to Peak (TtP) with age of fracture, although this was not statistically significant (R² = 0.44, p = 0.1; R² = 0.24, p = 0.26, respectively). This study demonstrates that commercially available microbubbles perfuse acute fractures in ultrasonographically detectable quantities using commercially available equipment. John Wiley
Preventing Biofilm Formation and Encrustation on Urinary Implants: (Bio)molecular and Physical Research Approaches
Stents and catheters are used to facilitate urine drainage within the urinary system. When such sterile implants are inserted into the urinary tract, ions, macromolecules and bacteria from urine, blood or underlying tissues accumulate on their surface. We presented a brief but comprehensive overview of future research strategies in the prevention of urinary device encrustation with an emphasis on biodegradability, molecular, microbiological and physical research approaches. The large and strongly associated field of stent coatings and tissue engineering is outlined elsewhere in this book. There is still plenty of room for future investigations in the fields of material science, surface science, and biomedical engineering to improve and create the most effective urinary implants. In an era where material science, robotics and artificial intelligence have undergone great progress, futuristic ideas may become a reality. These ideas include the creation of multifunctional programmable intelligent urinary implants (core and surface) capable to adapt to the complex biological and physiological environment through sensing or by algorithms from artificial intelligence included in the implant. Urinary implants are at the crossroads of several scientific disciplines, and progress will only be achieved if scientists and physicians collaborate using basic and applied scientific approaches.
Investigation of the Acoustic Vaporization Threshold of Lipid-Coated Perfluorobutane Nanodroplets Using Both High-Speed Optical Imaging and Acoustic Methods.
A combination of ultrahigh-speed optical imaging (5 × 106 frames/s), B-mode ultrasound and passive cavitation detection was used to study the vaporization process and determine both the acoustic droplet vaporization (ADV) and inertial cavitation (IC) thresholds of phospholipid-coated perfluorobutane nanodroplets (PFB NDs, diameter = 237 ± 16 nm). PFB NDs have not previously been studied with ultrahigh-speed imaging and were observed to form individual microbubbles (1-10 μm) within two to three cycles and subsequently larger bubble clusters (10-50 μm). The ADV and IC thresholds did not statistically significantly differ and decreased with increasing pulse length (20-20,000 cycles), pulse repetition frequency (1-100 Hz), concentration (108-1010 NDs/mL), temperature (20°C-45°C) and decreasing frequency (1.5-0.5 MHz). Overall, the results indicate that at frequencies of 0.5, 1.0 and 1.5 MHz, PFB NDs can be vaporized at moderate peak negative pressures (<2.0 MPa), pulse lengths and pulse repetition frequencies. This finding is encouraging for the use of PFB NDs as cavitation agents, as these conditions are comparable to those required to achieve therapeutic effects with microbubbles, unlike those reported for higher-boiling-point NDs. The differences between the optically and acoustically determined ADV thresholds, however, suggest that application-specific thresholds should be defined according to the biological/therapeutic effect of interest.
Synthesis and characterization of liposomes encapsulating silver nanoprisms obtained by millifluidic-based production for drug delivery
Silver nanoprisms (SNPs) have attracted significant attention due to their surface plasmon resonance behaviour, which is strongly dependent on their size and shape. The enhanced light absorption and scattering capacity of SNPs, make them a promising candidate system for non-invasive imaging and drug delivery in nanoparticle-assisted diagnostics and therapy. However, systemic administration of silver nanoparticles (AgNPs) at high concentrations may result in toxic side-effects, arising from non-targeted bio-distribution. These drawbacks could be mitigated by employing liposomes as carriers for AgNPs. However, there is a lack of systematic studies on production and subsequent physico-chemical characterisation of liposomal systems encapsulating SNPs. The present study therefore investigated the synthesis of liposomes encapsulating SNPs (Lipo/SNPs) using a continuous-flow millimetre-scale reactor, whereby liposome formation was governed by a solvent exchange mechanism. An aqueous phase and an ethanolic lipid phase were conveyed through two separate inlet channels, and subsequently travelled through a serpentine-shaped channel where mixing between the two phases took place. The synthesis process was optimised by varying both liposome formulation and the operating fluidic parameters, including the ratio between inlet flow rates (or flow rate ratio) and the total flow rate. The obtained Lipo/SNPs were characterised for their size and electrostatic charge, using a dynamic light scattering apparatus. Liposome morphology and encapsulation efficiency of SNPs within liposomes were determined by transmission electron microscopy (TEM) imaging. The synthesised negatively charged Lipo/SNP samples were found to have an average size of ∼150 nm (size dispersity < 0.3). The AgNPs encapsulation efficiency was equal to 77.48%, with mostly single SNPs encapsulated in liposomes. By using a multiangle TEM imaging approach, quasi-3D images were obtained, further confirming the encapsulation of nanoparticles within liposomes. Overall, the formulation and production technique developed in the present study has potential to contribute towards mitigating challenges associated with AgNP-mediated drug delivery and diagnostics.
The interplay between bacterial biofilms, encrustation, and wall shear stress in ureteral stents: a review across scales.
Ureteral stents are hollow tubes that are inserted into the ureter to maintain the flow of urine from the kidney to the bladder. However, the use of these indwelling stents is associated with potential complications. Biofilm, an organized consortium of bacterial species embedded within a self-producing extracellular matrix, can attach to the outer and inner surfaces of ureteral stents. Furthermore, encrustation - defined as the buildup of mineral deposits on the stent surface - can occur independently or in parallel with biofilm formation. Both phenomena can cause stent obstruction, which can lead to obstructive pyelonephritis and make stent removal difficult. Understanding the influence of flow on the development of biofilm and encrustation and the impact of small mechanical environmental changes (e.g., wall shear stress distribution) is key to improve the long-term performance of stents. Identifying the optimal stent properties to prevent early bacterial attachment and/or crystal deposition and their growth, would represent a breakthrough in reducing biofilm-/encrustation-associated complications. This review identifies the most prevalent bacterial strains and crystal types associated with ureteral stents, and the process of their association with the stent surface, which often depends on patient comorbidities, stent material, and indwelling time. Furthermore, we focus on the often-overlooked role of fluid dynamics on biofilm and encrustation development in ureteral stents, across a range of physical scales (i.e., from micro- to macro-scale) with the aim of providing a knowledge base to inform the development of safer and more effective ureteral stents.