Normothermic Ex Situ Machine Perfusion of Vascularized Composite Allografts with Oxygen Microcarriers for 12 Hours Using Real-Time Mitochondrial Redox Quantification.

BACKGROUND: Normothermic ex situ perfusion of vascularized composite allografts (VCAs) necessitates high oxygen demand and, thus, increased metabolic activity, which, in turn, requires the use of blood-based perfusion solutions. However, blood-derived perfusates, in turn, constitute an antigenic load. To circumvent this immunogenic problem, we used a perfusate enriched with acellular dextrane oxygen microcarriers to perfuse rat hindlimbs. METHODS: Rat hindlimbs (n = 11) were perfused with either (non-), oxygenated dextrane-enriched Phoxilium, or Phoxilium enriched with dextrane oxygen microcarriers (MO2) for 12 h at 37 °C or stored on ice. Oxygenation of the skeletal muscle was assessed with Raman spectroscopy, tissue pO2-probes, and analysis of the perfusate. Transmission electronic microscopy was utilized to assess the ultrastructure of mitochondria of the skeletal muscle. RESULTS: For all evaluated conditions, ischemia time until perfusion was comparable (22.91 ± 1.64 min; p = 0.1559). After 12 h, limb weight increased significantly by at least 81%, up to 124% in the perfusion groups, and by 27% in the static cold storage (SCS) group. Raman spectroscopy signals of skeletal muscle did not differ substantially among the groups during either perfusion or static cold storage across the duration of the experiment. While the total number of skeletal muscle mitochondria decreased significantly compared to baseline, mitochondrial diameter increased in the perfusion groups and the static cold storage group. CONCLUSION: The use of oxygen microcarriers in ex situ perfusion of VCA with acellular perfusates under normothermic conditions for 12 h facilitates the maintenance of mitochondrial structure, as well as a subsequent recovery of mitochondrial redox status over time, while markers of muscle injury were lower compared to conventional oxygenated acellular perfusates.

Injectable Oxygen Microparticles Boost Radiation-Mediated In Situ Vaccination and Systemic Antitumor Immune Responses.

PURPOSE: The aim of this work was to determine whether intratumoral injections of a liquid oxygen solution are effective at boosting radiation-induced abscopal effects. METHODS AND MATERIALS: A liquid oxygen solution, comprising slow-release polymer-shelled oxygen microparticles, was fabricated and injected intratumorally to locally elevate tumor oxygen levels before and after treatment with radiation therapy. Changes in tumor volume were monitored. In a subset of studies, CD8-positive cells were depleted and the experiments were repeated. Histologic analyses of the tumor tissues were performed to quantify the concentration of infiltrating immune cells. RESULTS: Daily intratumoral injections of oxygen-filled microparticles significantly retarded primary and secondary tumor growth, boosted infiltration of cytotoxic T cells, and improved overall survival when used as an adjuvant to radiation therapy. The findings also demonstrated that efficacy requires both radiation and oxygen, suggesting that they act synergistically to enhance in situ vaccination and systemic antitumor immune responses. CONCLUSIONS: This study demonstrated the potential advantages of intratumoral injections of a liquid oxygen solution as a strategy to boost radiation-induced abscopal effects, and the findings warrant future efforts toward clinical translation of the injectable liquid oxygen solution.

Totally Implantable Oxygen Generator (TIOG) for Hypoxia and Hypoxemia.

Hypoxia and hypoxemia are the conditions when oxygen is depleted from the cell due to, for example, respiratory failure, cancer, etc. While the current therapy brought reasonable clinical outcomes, its systematic nature of oxygen delivery can be compromised by a significant dropout and side effects. This paper presents a totally implantable oxygen generator (TIOG) for localized, highly controllable, real-time, and targeted oxygen delivery. METHODS: The TIOG system, an ultra-low power implantable wireless platform, is built using off-the-shelf components. The TIOG can be remotely operated to enable a tailored oxygen delivery based on electrolysis with a precisely controlled electrical signal (i.e., current level, frequency, and duty cycle). RESULTS: The in vitro experiments demonstrate that the TIOG could deliver oxygen with a rate of 9.27 ± 1.9 µmol/L/min with the pulsed electrical current (800 µA, 600 µs pulse or 6% duty cycle with 10 ms period). The system could also suppress chlorine generation under the safety guideline (5 mg/L). Operating at 433 MHz ISM band, the TIOG could be wirelessly controlled from up to 600 cm distance with a 0%-bit error rate (BER) and 0%-packet error rate (PER). A single charge of the battery could operate the system for up to 3.3 hr, which can be wirelessly recharged for long-term operation. CONCLUSION: The longevity of the TIOG system enables ambulatory oxygen therapy in a much longer-term than current practice.

Microporous water with high gas solubilities.

Liquids with permanent microporosity can absorb larger quantities of gas molecules than conventional solvents1, providing new opportunities for liquid-phase gas storage, transport and reactivity. Current approaches to designing porous liquids rely on sterically bulky solvent molecules or surface ligands and, thus, are not amenable to many important solvents, including water2-4. Here we report a generalizable thermodynamic strategy to preserve permanent microporosity and impart high gas solubilities to liquid water. Specifically, we show how the external and internal surface chemistry of microporous zeolite and metal-organic framework (MOF) nanocrystals can be tailored to promote the formation of stable dispersions in water while maintaining dry networks of micropores that are accessible to gas molecules. As a result of their permanent microporosity, these aqueous fluids can concentrate gases, including oxygen (O2) and carbon dioxide (CO2), to much higher densities than are found in typical aqueous environments. When these fluids are oxygenated, record-high capacities of O2 can be delivered to hypoxic red blood cells, highlighting one potential application of this new class of microporous liquids for physiological gas transport.

A microfluidic device for real-time on-demand intravenous oxygen delivery.

SignificanceThe treatment of hypoxemia that is refractory to the current standard of care is time-sensitive and requires skilled caregivers and use of specialized equipment (e.g., extracorporeal membrane oxygenation). Most patients experiencing refractory hypoxemia will suffer organ dysfunction, and death is common in this cohort. Here, we describe a new strategy to stabilize and support patients using a microfluidic device that administers oxygen gas directly to the bloodstream in real time and on demand using a process that we call sequential shear-induced bubble breakup. If successful, the described technology may help to avoid or decrease the incidence of ventilator-related lung injury from refractory hypoxemia.

Hyperbaric polymer microcapsules for tunable oxygen delivery.

Over the past decade, there have been many attempts to engineer systems capable of delivering oxygen to overcome the effects of both systemic and local hypoxia that occurs as a result of traumatic injury, cell transplantation, or tumor growth, among many others. Despite progress in this field, which has led to a new class of oxygen-generating biomaterials, most reported techniques lack the tunability necessary for independent control over the oxygen flux (volume per unit time) and the duration of delivery, both of which are key parameters for overcoming tissue hypoxia of varying etiologies. Here, we show that these critical parameters can be effectively manipulated using hyperbarically-loaded polymeric microcapsules (PMC). PMCs are micron-sized particles with hollow cores and polymeric shells. We show that oxygen delivery through PMCs is dependent on its permeability through the polymeric shell, the shell thickness, and the pressure gradient across the shell. We also demonstrate that incorporating an intermediate oil layer between the polymeric shell and the gas core prevents rapid outgassing by effectively lowering the resultant pressure gradient across the polymeric membrane following depressurization.

Safety of inhaled hydrogen gas in healthy mice.

The purpose of this work was to determine the safety of inhaled hydrogen gas in healthy animals. Female mice were exposed to medical air with or without hydrogen gas (concentration 2.4%) for 72 hours (n = 25 mice/group). Mice underwent a standardized and validated neurobehavioral examination, SmithKline Beecham, Harwell, Imperial College, Royal London Hospital, Phenotype Assessment (SHIRPA) protocol, prior to and following the exposure period. Blood was withdrawn for serologic evaluation and all major organ tissues were evaluated histologically. The average hydrogen concentration within the chamber was 2.27%. Following exposure, there was no significant change in body weight in either group. Similarly, there was no significant change in the total SHIRPA score, although hydrogen-treated mice exhibited significantly lower spontaneous locomotor activity (P < 0.0001) in a subset of the test; all other aspects of the mouse neurologic exam were normal in hydrogen-treated animals. Brain histopathology was also normal in all mice, as was the histology of all other major organs. There were no significant differences in complete blood count, serum chemistry, or arterial blood gases between control and hydrogen-treated mice (P > 0.05 for all). Hydrogen gas did not appear to cause significant adverse effects when administered to healthy mice for 72 hours, with the possible exception of decreased spontaneous locomotor activity. The study was approved by the Institutional Animal Care and Use Committee at Boston Children's Hospital, USA (approved number 18-01-3536) on January 25, 2018.

Perioperatively Inhaled Hydrogen Gas Diminishes Neurologic Injury Following Experimental Circulatory Arrest in Swine.

This study used a swine model of mildly hypothermic prolonged circulatory arrest and found that the addition of 2.4% inhaled hydrogen gas to inspiratory gases during and after the ischemic insult significantly decreased neurologic and renal injury compared with controls. With proper precautions, inhalational hydrogen may be administered safely through conventional ventilators and may represent a complementary therapy that can be easily incorporated into current workflows. In the future, inhaled hydrogen may diminish the sequelae of ischemia that occurs in congenital heart surgery, cardiac arrest, extracorporeal life-support events, acute myocardial infarction, stroke, and organ transplantation.

Changes in tissue oxygen tension, venous saturation, and Fick-based assessments of cardiac output during hyperoxia.

BACKGROUND: Hyperoxemia (arterial oxygen tension >100 mm Hg) may occur in critically ill patients and have effects on mixed venous saturation (SvO2 ) and on Fick-based estimates of cardiac output (CO). We investigated the effect of hyperoxemia on SvO2 and on assessments of CO using the Fick equation. METHODS: Yorkshire swine (n = 14) were anesthetized, intubated, and paralyzed for instrumentation. SvO2 (co-oximetry) and tissue oxygen tension (tPO2 , implantable electrodes) in brain and myocardium were measured during systematic manipulation of arterial oxygen tension (PaO2 ) using graded hyperoxia (fraction of inspired oxygen 0.21 → 0.8). Secondarily, oxygen- and carbon dioxide-based estimates of CO (FickO2 and FickCO2 , respectively) were compared with measurements from a flow probe placed on the aortic root. RESULTS: Independent of changes in measured oxygen delivery, cerebral and myocardial tPO2 increased in proportion to PaO2 , as did SvO2 (P < 0.001 for all). Based on mixed model analysis, each 100 mm Hg increase in PaO2 resulted in a 4.8 ± 0.9% increase in SvO2 under the conditions tested. Because neither measured oxygen consumption, arterial oxyhemoglobin saturation or cardiac output varied significantly during hyperoxia, changes in SvO2 resulted in successively increasing errors in FickO2 during hyperoxia (34% during normoxia, 72% during FiO2 0.8). FickCO2 lacked the progressively worsening errors present in FickO2 , but correlated poorly with CO. CONCLUSION: SvO2 acutely changes following changes in PaO2 even absent changes in measured DO2 . This may lead to errors in FickO2 estimates of CI. Further work is necessary to understand the impact of this phenomenon in disease states.

Tunable Nonlinear Acoustic Reporters Using Micro- and Nanosized Air Bubbles with Porous Polymeric Hard Shells.

The ability to tailor acoustic cavitation of contrast agents is pivotal for ultrasound applications in enhanced imaging, drug delivery, and cancer therapy, etc. A biopolymer-based system of microbubbles and nanobubbles was developed as acoustic reporters that consist of extremely porous hard shells. Despite the existence of an incompressible shell, these porous contrast agents exhibited strong nonlinear acoustic response under very low acoustic pressure, e.g, harmonics, characteristic of free gas bubbles. The large air/water surface area within the transmural capillaries are believed to facilitate oscillation of the inner gas core. Furthermore, the acoustic cavitation can be tailored by variation in polymer structures. This synthetically based platform offers insight for the rational design of advanced acoustic biomaterials.

Intravenous Amiodarone and Sotalol Impair Contractility and Cardiac Output, but Procainamide Does Not: A Langendorff Study.

INTRODUCTION: Direct comparison of the effects of antiarrhythmic agents on myocardial performance may be useful in choosing between medications in critically ill patients. Studies directly comparing multiple antiarrhythmic medications are lacking. The use of an experimental heart preparation permits examination of myocardial performance under constant loading conditions. METHODS: Hearts of Sprague Dawley rats (n = 35, 402-507 g) were explanted and cannulated in working heart model with fixed preload and afterload. Each heart was then exposed to a 3-hour infusion of procainamide (20 µg/kg/min), esmolol (100 or 200 µg/kg/min), amiodarone (10 or 20 mg/kg/d), sotalol (80 mg/m2/d), or placebo infusions (n = 5 per dose). Cardiac output, contractility (dP/dTmax), diastolic performance (dP/dTmin), and heart rate were compared between groups over time by linear mixed modeling. RESULTS: Compared with placebo, sotalol decreased contractility by an average of 24% ( P < .001) over the infusion period, as did amiodarone (low dose by 13%, P = .029; high dose by 14%, P = .013). Compared with placebo, mean cardiac output was significantly lower in animals treated with sotalol (by 22%, P = .016) and esmolol 200 µg/kg/min (by 23%, P = .012). Over time, amiodarone decreased cardiac output (20 mg/kg/d, ß = -89 [-144, -33] µL/min2 decrease, P = .002) and also worsened diastolic function, decreasing dP/dTmin by ∼18% and 22% ( P = .032 and P = .011, low and high doses, respectively). Procainamide did not have a significant effect on any measures of systolic or diastolic performance. CONCLUSIONS: In isolated hearts, amiodarone and sotalol depressed myocardial contractility, cardiac output, and diastolic function. However, procainamide did not negatively affect myocardial performance and represents a favorable agent in settings of therapeutic equivalence.

Tunable Polymer Microcapsules for Controlled Release of Therapeutic Gases.

Encapsulation and delivery of oxygen, carbon dioxide, and other therapeutic gases, using polymeric microcapsules (PMCs) is an emerging strategy to deliver gas as an injectable therapeutic. The gas cargo is stored within the PMC core and its release is mediated by the physiochemical properties of the capsule shell. Although use of PMCs for the rapid delivery of gases has been well described, methods which tune the material properties of PMCs for sustained release of gas are lacking. In this work, we describe a simple method for the high-yield production of gas-in-oil-filled PMCs with tunable sizes and core gas content from preformed polymers using the sequential phase separation and self-emulsification of emulsion-based templates. We demonstrate that prolonged gas release occurs from gas-in-oil PMCs loaded with oxygen and carbon dioxide gas, each of which could have significant clinical applications.

Injectable Oxygen: Interfacing Materials Chemistry with Resuscitative Science.

Intravascular oxygen delivery holds great potential to treat numerous hypoxic conditions and emergencies, including pulmonary disorders, hypoxic tumors, hemorrhagic shock, stroke, cardiac arrest and so on. Tremendous effort has been made in the past to find material solutions for the development of intravenous oxygen carriers and have ranged from blood substitutes to microbubbles with limited success. This paper highlights previous and recent progress in perfluorocarbon-emulsions and microbubbles as intravenous gas carriers, including concerns over their long-term stability, in vivo safety profiles, and oxygen transport efficacy. Their use as potential resuscitative therapeutics for treating various types of cardiac arrest is also discussed.

A Device for the Quantification of Oxygen Consumption and Caloric Expenditure in the Neonatal Range.

BACKGROUND: The accurate measurement of oxygen consumption (VO2) and energy expenditure (EE) may be helpful to optimize the treatment of critically ill patients. However, current techniques are limited in their ability to accurately quantify these end points in infants due to a low VO2, low tidal volume, and rapid respiratory rate. This study describes and validates a new device intended to perform in this size range. METHODS: We created a customized device that quantifies inspiratory volume using a pneumotachometer and concentrations of oxygen and carbon dioxide gas in the inspiratory and expiratory limbs. We created a customized algorithm to achieve precise time alignment of these measures, incorporating bias flow and compliance factors. The device was validated in 3 ways. First, we infused a certified gas mixture (50% oxygen/50% carbon dioxide) into an artificial lung circuit, comparing measured with simulated VO2 and carbon dioxide production (VCO2) within a matrix of varying tidal volume (4-20 mL), respiratory rate (20-80 bpm), and fraction of inspired oxygen (0.21-0.8). Second, VO2, VCO2, and EE were measured in Sprague Dawley rats under mechanical ventilation and were compared to simultaneous Douglas bag collections. Third, the device was studied on n = 14 intubated, spontaneously breathing neonates and infants, comparing measured values to Douglas measurements. In all cases, we assessed for difference between the device and reference standard by linear regression and Bland-Altman analysis. RESULTS: In vitro, the mean ± standard deviation difference between the measured and reference standard VO2 was +0.04 ± 1.10 (95% limits of agreement, -2.11 to +2.20) mL/min and VCO2 was +0.26 ± 0.31 (-0.36 to +0.89) mL/min; differences were similar at each respiratory rate and tidal volume measured, but higher at fraction of inspired oxygen of 0.8 than at 0.7 or lower. In rodents, the mean difference was -0.20 ± 0.55 (-1.28 to +0.89) mL/min for VO2, +0.16 ± 0.25 (-0.32 to +0.65) mL/min for VCO2, and -0.84 ± 3.29 (-7.30 to +5.61) kcal/d for EE. In infants, the mean VO2 was 9.0 ± 2.5 mL/kg/min by Douglas method and was accurately measured by the device (bias, +0.22 ± 0.87 [-1.49 to +1.93] mL/kg/min). The average VCO2 was 8.1 ± 2.3 mL/kg/min, and the device exhibited a bias of +0.33 ± 0.82 (-1.27 to +1.94) mL/kg/min. Mean bias was +2.56% ± 11.60% of the reading for VO2 and +4.25% ± 11.20% of the reading for VCO2; among 56 replicates, 6 measurements fell outside of the 20% error range, and no patient had >1 of 4 replicates with a >20% error in either VO2 or VCO2. CONCLUSIONS: This device can measure VO2, VCO2, and EE with sufficient accuracy for clinical decision-making within the neonatal and pediatric size range, including in the setting of tachypnea or hyperoxia.

Interfacial Nanoprecipitation toward Stable and Responsive Microbubbles and Their Use as a Resuscitative Fluid.

A new approach has been developed to prepare stable microbubbles (MBs) by interfacial nanoprecipitation of bioabsorbable polymers at air/liquid interfaces. This facile method offers robust control over the morphology and chemophysical properties of MBs by simple chemical modifications. This approach is amenable to large-scale manufacturing, and is useful to develop functional MBs for advanced biomedical applications. To demonstrate this, a MB-based intravenous oxygen carrier was created that undergoes pH-triggered self-elimination. Intravenous injection of previous MBs increased the risk of pulmonary vascular obstruction. However, we show, for the first time, that our current design is superior, as they 1) yielded no evidence of acute risks in rodents, and 2) improved the survival in a disease model of asphyxial cardiac arrest (from 0 to 100 %), a condition that affects more than 100 000 in-hospital patients, and carries a mortality of about 90 %.

Responsive monitoring of mitochondrial redox states in heart muscle predicts impending cardiac arrest.

Assessing the adequacy of oxygen delivery to tissues is vital, particularly in the fields of intensive care medicine and surgery. As oxygen delivery to a cell becomes deficient, changes in mitochondrial redox state precede changes in cellular function. We describe a technique for the continuous monitoring of the mitochondrial redox state on the epicardial surface using resonance Raman spectroscopy. We quantify the reduced fraction of specific electron transport chain cytochromes, a metric we name the resonance Raman reduced mitochondrial ratio (3RMR). As oxygen deficiency worsens, heme moieties within the electron transport chain become progressively more reduced, leading to an increase in 3RMR. Myocardial 3RMR increased from baseline values of 18.1 ± 5.9 to 44.0 ± 16.9% (P = 0.0039) after inferior vena cava occlusion in rodents (n = 8). To demonstrate the diagnostic power of this measurement, 3RMR was continuously measured in rodents (n = 31) ventilated with 5 to 8% inspired oxygen for 30 min. A 3RMR value exceeding 40% at 10 min predicted subsequent cardiac arrest with 95% sensitivity and 100% specificity [area under the curve (AUC), 0.98], outperforming all current measures, including contractility (AUC, 0.51) and ejection fraction (AUC, 0.39). 3RMR correlated with indices of intracellular redox state and energy production. This technique may permit the real-time identification of critical defects in organ-specific oxygen delivery.

Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation.

After birth cardiomyocytes undergo terminal differentiation, characterized by binucleation and centrosome disassembly, rendering the heart unable to regenerate. Yet, it has been suggested that newborn mammals regenerate their hearts after apical resection by cardiomyocyte proliferation. Thus, we tested the hypothesis that apical resection either inhibits, delays, or reverses cardiomyocyte centrosome disassembly and binucleation. Our data show that apical resection rather transiently accelerates centrosome disassembly as well as the rate of binucleation. Consistent with the nearly 2-fold increased rate of binucleation there was a nearly 2-fold increase in the number of cardiomyocytes in mitosis indicating that the majority of injury-induced cardiomyocyte cell cycle activity results in binucleation, not proliferation. Concurrently, cardiomyocytes undergoing cytokinesis from embryonic hearts exhibited midbody formation consistent with successful abscission, whereas those from 3 day-old cardiomyocytes after apical resection exhibited midbody formation consistent with abscission failure. Lastly, injured hearts failed to fully regenerate as evidenced by persistent scarring and reduced wall motion. Collectively, these data suggest that should a regenerative program exist in the newborn mammalian heart, it is quickly curtailed by developmental mechanisms that render cardiomyocytes post-mitotic.

Hemodynamic Effects of Lipid-Based Oxygen Microbubbles via Rapid Intravenous Injection in Rodents.

PURPOSE: Low oxygen levels, or hypoxemia, is a common cause of morbidity and mortality in critically ill patients. Hypoxemia is typically addressed by increasing the fraction of inspired oxygen, the use of mechanical ventilation, or more invasive measures. Recently, the injection of oxygen gas directly into the bloodstream by packaging it within lipid-based oxygen microbubbles (LOMs) has been explored. The purpose of this work is to examine the acute hemodynamic effects of intravenous injections of LOMs. METHODS: LOMs composed of 1,2-distearoyl-sn-glycero-3-phosphocoline and cholesterol were manufactured using a process of shear homogenization under an oxygen headspace. A 5 mL aliquot of either PlasmaLyte A, or low (37%) or high (55%) concentration LOMs (n = 10 per group) was injected over a 1 min period into Sprague Dawley rats instrumented for measurement of cardiac index and pulmonary (PVR) and systemic (SVR) vascular resistance during a 60 min observation period. Hemodynamics were compared between groups by linear mixed modeling. RESULTS: Approximately 1011 LOMs with mean diameter 3.77 ± 1.19 µm were injected over the 1 min period. Relative to controls, rodents treated with high concentration LOMs exhibited a higher pulmonary artery pressure (20 ± 0.4 mmHg vs 18 ± 0.4 mmHg, P < 0.001) and higher PVR (0.31 ± 0.01 vs 0.23 ± 0.01 mmHg/mL*min*kg, P < 0.001. Despite a stable cardiac index (62.2 ± 3.5 vs 62.3 ± 3.4 mL/min*kg, P < 0.001), mean arterial blood pressure decreased significantly in LOM-treated animals (46 ± 2 vs 60 ± 2 mmHg, P < 0.001) due to a decrease in SVR. Injections with aged LOM emulsions (>48 h since manufacture) resulted in a higher incidence of hemodynamic collapse during the observation period (P = 0.02). CONCLUSIONS: LOMs may be injected in quantities sufficient to deliver clinically meaningful volumes of oxygen but cause significant decrements in blood pressure and elevations in PVR.