An investigation into the cytotoxic effects of microbubbles and their constituents on osteosarcoma and bone marrow stromal cells.

BACKGROUND: Ultrasound-responsive microbubbles offer a means of achieving minimally invasive, localised drug delivery in applications including regenerative medicine. To facilitate their use, however, it is important to determine any cytotoxic effects they or their constituents may have. The aim of this study was to test the hypothesis that phospholipid-shelled microbubbles are non-toxic to human bone-derived cells at biologically-relevant concentrations. METHODS: Microbubbles were fabricated using combinations of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), polyoxyethylene(40) stearate (PEG40S) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (DSPE-PEG2000). Microbubble size and concentration were measured as a function of time and temperature by optical microscopy. Effects on MG63 osteosarcoma and human bone marrow stromal cells (BMSCs) were measured for up to 72 h by assay for viability, metabolic activity and proliferation. RESULTS: DBPC:DSPE-PEG2000 microbubbles were significantly more stable than DSPC:PEG40S microbubbles under all conditions tested. Serum-containing medium had no detrimental effect on microbubble stability, but storage at 37 °C compared to at 4 °C reduced stability for both preparations, with almost complete dissolution of microbubbles at times ≥24 h. DSPC:PEG40S microbubbles had greater inhibitory effects on cell metabolism and growth than DBPC:DSPE-PEG2000 microbubbles, with PEG40S found to be the principle inhibitory component. These effects were only evident at high microbubble concentrations (≥20% (v/v)) or with prolonged culture (≥24 h). Increasing cell-microbubble contact by inversion culture in a custom-built device had no inhibitory effect on metabolism. CONCLUSIONS: These data indicate that, over a broad range of concentrations and incubation times, DBPC:DSPE-PEG2000 and DSPC:PEG40S microbubbles have little effect on osteoblastic cell viability and growth, and that PEG40S is the principle inhibitory component in the formulations investigated.

Reducing deposition of encrustation in ureteric stents by changing the stent architecture: A microfluidic-based investigation.

Ureteric stents are clinically deployed to retain ureteral patency in the presence of an obstruction of the ureter lumen. Despite the fact that multiple stent designs have been researched in recent years, encrustation and biofilm-associated infections remain significant complications of ureteral stenting, potentially leading to the functional failure of the stent. It has been suggested that "inactive" side-holes of stents may act as anchoring sites for encrusting crystals, as they are associated with low wall shear stress (WSS) levels. Obstruction of side-holes due to encrustation is particularly detrimental to the function of the stent, since holes provide a path for urine to by-pass the occlusion. Therefore, there is an unmet need to develop novel stents to reduce deposition of encrusting particles at side-holes. In this study, we employed a stent-on-chip microfluidic model of the stented and occluded ureter to investigate the effect of stent architecture on WSS distribution and encrustation over its surface. Variations in the stent geometry encompassed (i) the wall thickness and (ii) the shape of side-holes. Stent thickness was varied in the range 0.3-0.7 mm, while streamlined side-holes of triangular shape were evaluated (with a vertex angle in the range 45°-120°). Reducing the thickness of the stent increased WSS and thus reduced the encrustation rate at side-holes. A further improvement in performance was achieved by using side-holes with a triangular shape; notably, a 45° vertex angle showed superior performance compared to other angles investigated, resulting in a significant increase in WSS within "inactive" side-holes. In conclusion, combining the optimal stent thickness (0.3 mm) and hole vertex angle (45°) resulted in a ∼90% reduction in encrustation rate within side-holes, compared to a standard design. If translated to a full-scale ureteric stent, this optimised architecture has the potential for significantly increasing the stent lifetime while reducing clinical complications.

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.

Layered acoustofluidic resonators for the simultaneous optical and acoustic characterisation of cavitation dynamics, microstreaming, and biological effects.

The study of the effects of ultrasound-induced acoustic cavitation on biological structures is an active field in biomedical research. Of particular interest for therapeutic applications is the ability of oscillating microbubbles to promote both cellular and tissue membrane permeabilisation and to improve the distribution of therapeutic agents in tissue through extravasation and convective transport. The mechanisms that underpin the interaction between cavitating agents and tissues are, however, still poorly understood. One challenge is the practical difficulty involved in performing optical microscopy and acoustic emissions monitoring simultaneously in a biologically compatible environment. Here we present and characterise a microfluidic layered acoustic resonator (µLAR) developed for simultaneous ultrasound exposure, acoustic emissions monitoring, and microscopy of biological samples. The µLAR facilitates in vitro ultrasound experiments in which measurements of microbubble dynamics, microstreaming velocity fields, acoustic emissions, and cell-microbubble interactions can be performed simultaneously. The device and analyses presented provide a means of performing mechanistic in vitro studies that may benefit the design of predictable and effective cavitation-based ultrasound treatments.

Magnetic targeting to enhance microbubble delivery in an occluded microarterial bifurcation.

Ultrasound and microbubbles have been shown to accelerate the breakdown of blood clots both in vitro and in vivo. Clinical translation of this technology is still limited, however, in part by inefficient microbubble delivery to the thrombus. This study examines the obstacles to delivery posed by fluid dynamic conditions in occluded vasculature and investigates whether magnetic targeting can improve microbubble delivery. A 2D computational fluid dynamic model of a fully occluded Y-shaped microarterial bifurcation was developed to determine: (i) the fluid dynamic field in the vessel with inlet velocities from 1-100 mm s-1 (corresponding to Reynolds numbers 0.25-25); (ii) the transport dynamics of fibrinolytic drugs; and (iii) the flow behavior of microbubbles with diameters in the clinically-relevant range (0.6-5 µm). In vitro experiments were carried out in a custom-built microfluidic device. The flow field was characterized using tracer particles, and fibrinolytic drug transport was assessed using fluorescence microscopy. Lipid-shelled magnetic microbubbles were fluorescently labelled to determine their spatial distribution within the microvascular model. In both the simulations and experiments, the formation of laminar vortices and an abrupt reduction of fluid velocity were observed in the occluded branch of the bifurcation, severely limiting drug transport towards the occlusion. In the absence of a magnetic field, no microbubbles reached the occlusion, remaining trapped in the first vortex, within 350 µm from the bifurcation center. The number of microbubbles trapped within the vortex decreased as the inlet velocity increased, but was independent of microbubble size. Application of a magnetic field (magnetic flux density of 76 mT, magnetic flux density gradient of 10.90 T m-1 at the centre of the bifurcation) enabled delivery of microbubbles to the occlusion and the number of microbubbles delivered increased with bubble size and with decreasing inlet velocity.

An experimental and computational study of the hydrodynamics of high-velocity water microdrops for interproximal tooth cleaning.

The flow field and local hydrodynamics of high-velocity water microdrops impacting the interproximal (IP) space of typodont teeth were studied experimentally and computationally. Fourteen-day old Streptococcus mutans biofilms in the IP space were treated by a prototype AirFloss delivering 115 µL of water at a maximum exit-velocity of 60 ms(-1) in a 33-ms burst. Using high-speed imaging, footage was generated showing the details of the burst, and demonstrating the removal mechanism of the biofilms. Footage was also generated to characterize the viscoelastic behavior of the biofilms when impacted by an air-only burst, which was compared to the water burst. Image analysis demonstrated the importance of fluid forces on the removal pattern of interdental biofilms. X-ray micro-Computed Tomography (µ-CT) was used to obtain 3D images of the typodont and the IP spaces. Computational Fluid Dynamics (CFD) simulations were performed to study the effect of changing the nozzle position and design on the hydrodynamics within the IP space. Results confirmed our previous data regarding the wall shear stress generated by high-velocity water drops which dictated the efficacy of biofilm detachment. Finally, we showed how CFD models could be used to optimize water drop or burst design towards a more effective biofilm removal performance.

Removal of interproximal dental biofilms by high-velocity water microdrops.

The influence of the impact of a high-velocity water microdrop on the detachment of Streptococcus mutans UA159 biofilms from the interproximal (IP) space of teeth in a training typodont was studied experimentally and computationally. Twelve-day-old S. mutans biofilms in the IP space were exposed to a prototype AirFloss delivering 115 µL water at a maximum exit velocity of 60 m/sec in a 30-msec burst. Using confocal microscopy and image analysis, we obtained quantitative measurements of the percentage removal of biofilms from different locations in the IP space. The 3D geometry of the typodont and the IP spaces was obtained by micro-computed tomography (µ-CT) imaging. We performed computational fluid dynamics (CFD) simulations to calculate the wall shear stress (τw ) distribution caused by the drops on the tooth surface. A qualitative agreement and a quantitative relationship between experiments and simulations were achieved. The wall shear stress (τw ) generated by the prototype AirFloss and its spatial distribution on the teeth surface played a key role in dictating the efficacy of biofilm removal in the IP space.

An artificial model for studying fluid dynamics in the obstructed and stented ureter.

Fluid dynamics in the obstructed and stented ureter represents a non-trivial subject of investigation since, after stent placement, the urine can flow either through the stent lumen or in the extra-luminal space located between the stent wall and the ureteric inner wall. Fluid dynamic investigations can help understanding the phenomena behind stent failure (e.g. stent occlusions due to bacterial colonization and encrustations), which may cause kidney damage due to the associated high pressures generated in the renal pelvis. In this work a microfluidic-based transparent device (ureter model, UM) has been developed to simulate the fluid dynamic environment in a stented ureter. UM geometry has been designed from measurements on pig ureters. Pressure in the renal pelvis compartment has been measured against three variables: fluid viscosity (µ), volumetric flow rate (Q) and level of obstruction (OB%). The measurements allowed a quantification of the critical combination of µ, Q and OB% values which may lead to critical pressure levels in the kidney. Moreover, an example showing the possibility of applying particle image velocimetry (PIV) technology to the developed microfluidic device is provided.

Antithrombin III (ATIII) replacement therapy in patients with sepsis and/or postsurgical complications: a controlled double-blind, randomized, multicenter study.

BACKGROUND: ATIII is decreased in sepsis and/or shock and its baseline value correlates with mortality. The efficacy of ATIII therapy on mortality was assessed in a selected group of patients admitted to the intensive care unit (ICU) in a double-blind, randomized, multicenter study. METHODS: 120 patients admitted to the ICU with an ATIII concentration < 70% were randomized to receive ATIII (total dose 24000 units) or placebo treatment for 5 days; 56 patients had septic shock. RESULTS: ATIII concentrations in the treated group remained constant throughout the treatment period (range 97-102%). The Kaplan-Meier analysis showed no difference in overall survival between the two groups: 50 and 46% for ATIII and placebo, respectively. Septic shock and hemodynamic support were unbalanced in the two groups at admission. Therefore the Cox analysis was carried out after adjusting for these two variables. Treatment with ATIII decreases the risk of death with an odds ratio (OR) of 0.56. Of the covariates analyzed, septic shock and the baseline multiple organ failure score were negatively associated with survival and plasma activity level was positively associated with survival with an OR of 0.97 for each 1% increase in the ATIII plasma concentration at baseline. CONCLUSIONS: The results of ATIII treatment in this population of patients suggests that replacement therapy reduces mortality in the subgroup of septic shock patients only.

["ARCHIDIA": a system for patients' data collection and computerized filing. Part I. Methodology]. / Sistema di raccolta dati paziente e archiviazione computerizzata "ARCHIDIA". Parte I--Metodologia.

This report describes a computer based program of patient clinical data collection: the ARCHIDIA system. The project relies on descriptive analysis of clinical events according to well defined methodological criteria. This allows the formulation of a concise diagnosis which is, at the same time, exhaustive of all essential information. Two are the basis principles of this methodology: To define, as accurately as possible, the logical steps necessary to elaborate the diagnosis, that is construed by a sequence of codes. To define all the conditions that must be followed so to use any code in a controlled and independent way. These criteria were derived from literature. The major claim of the system is likely to be the introduction of a "common language" between different ICUs. Uniformed diagnostic and clinical criteria are the main source of large data collection for descriptive, analytic and prospective studies. After a one year pilot study performed by 4 ICUs, ARCHIDIA was used, in 1991, by 20 centers from the area of Milan, Pavia, Como, Varese (70% of total) and 4148 patient data were collected. A descriptive analysis will be reported in the following paper.

["ARCHIDIA": a system for patients' data collection and computerized filing. Part II. General description of a caseload]. / Sistema di raccolta dati paziente e archiviazione computerizzata "ARCHIDIA". Parte II--Descrizione generale della casistica.

OBJECTIVE: To describe a population of patients admitted in ICU in an homogeneous urban area by means of a computed system. EXPERIMENTAL DESIGN: Observational study. SETTING: 20 general intensive care units of general and university hospitals. PATIENTS: Patients admitted in ICU from 1-1-1991 to 31-12-1991. 3 centers collected patients only for 6 months, starting on 1-6-1991. MEASUREMENTS: For each patient demographic data, hospitalization data, outcome, diagnosis and diagnostic procedures used during hospitalization according to defined criteria previously described, were collected. Data have been collected on PC using dedicated software. RESULTS: All centers concluded data collection, none abandoned the study. General characteristics of 4148 valuable patients were reported. Age was 52.9 years, SAPS 12.4 and mortality 21.7%. The patients spent 8.7 days in ICU and, when transferred to a general ward, the following hospitalization was 21.5 days. CONCLUSIONS: Data collection demonstrated the project feasibility. It realizes a continue up to date system inside each unit and allows the use of a "common language" and homogeneous methodology between centers.

Reliability of the vestibulo-ocular reflex as an index of the effects of hypnotic drugs on the central nervous system.

The vestibulo-ocular reflex was tested after the administration of step doses of thiopentone or propanidid in 171 unpremedicated patients. The dose of thiopentone (3 mg kg-1) required to induce loss of nystagmus also abolished the response to verbal command. The dose required to inhibit the reflex in 95% of patients was 7.22 mg kg-1 and prevented adrenergic responses to nociceptive stimulation. The dose of propanidid required to induce loss of nystagmus was greater than that necessary to abolish response to verbal command. The reflex inhibition rate increased proportionally to the dose up to 8 mg kg-1; larger doses exerted a facilitatory effect and reduced the reflex inhibition rate. The vestibulo-ocular reflex is a reliable and sensitive means of demonstrating the different effects exerted by step doses of hypnotic drugs.

The treatment of hydrogen sulfide intoxication: oxygen versus nitrites.

A case of severe H2S intoxication, treated with oxygen, respiratory support and thiopental cerebral protection, is presented. The usual antidotal treatments using nitrites or oxygen are discussed, examining the risks of nitrite use and the efficacy of oxygen. The successful outcome of the case presented suggests that H2S poisoning be treated with oxygen and vigorous organ supportive care.