Numerical and Experimental Assessment of Oxygen Distribution in a Perfusion Bioreactor to Mimic the Osteochondral Niche.

for more than a century a large body of evidence has accumulated in the physiological and pathophysiological literature to support the close relationship between the demand of oxygen by the tissues and the supply of oxygen by the cardiorespiratory system. When the demand for oxygen changes, how is

Beyond the lung: oxygen delivery and tissue oxygenation.

Second wave of COVID-19 pandemic and the surge of mucormycosis: Lessons learnt and future preparedness: Indian Society of Anaesthesiologists (ISA National) Advisory and Position Statement.

Purpose: Tumor hypoxia is a key barrier to successful delivery and activity of anti-cancer agents. To tackle this, we designed hypoxia-responsive Au-PEI-Azo-mPEG nanoparticles (NPs) denoted as APAP NPs for targeted delivery of hypoxia-activated prodrug (HAP), tirapazamine (TPZ) to hypoxic breast cancer cells.Methods: AuNPs were first synthesized. And then, were coated with polyethylene imine (PEI) by EDC-NHS chemistry. To realize NP biocompatibility and self-activating potential, a hypoxia-cleavable mPEG-AZO linker shell was coupled to the Au-PEI core. The hypoxia-responsible behavior of nanoparticles was analyzed under 21% O2 (normoxia) and 1% O2 (hypoxia) condition in 2D cell culture as well as MDA-MB-231 and MCF-7 spheroids as reliable biomimetics of tumor hypoxia.Results: APAP NPs elicited comparable cytotoxicity upon MDA-MB-231 cancer cells lowering TPZ IC50 to 7.46 µg/mL after 24 h. And were capable of enhanced ROS generation (P<0.001), and reduced mitochondrial membrane potential under hypoxia condition compared to the control (P<0.0001). Further, these NPs induced widespread apoptosis in both 2D and 3D cancer cell culture (P<0.0001), significantly reduced cell adhesion density (P<0.01), increased cell uptake by ~100 folds under hypoxia condition, and destroyed large MCF-7 spheroids by 72 h. Conclusion: Together, APAP@TPZ as biocompatible, and multi-stage activating platforms afford deepened penetration of HAP to hypoxic tumor core, where PEG detachment and TPZ bioreduction into its active form promote selective and effective eradication of hypoxic breast cancer microtumors.

Modeling the photodynamic effect in 2D versus 3D cell culture under normoxic and hypoxic conditions

Low oxygen tensions experienced in various pathological and physiological conditions are a major stimulus for angiogenesis. Hypoxic conditions play a critical role in regulating cellular behaviour including migration, proliferation and differentiation. This study introduces the use of a microfluidic device that allows for the control of oxygen tension for the study of different three-dimensional (3D) cell cultures for various applications. The device has a central 3D gel region acting as an external cellular matrix, flanked by media channels. On each side, there is a peripheral gas channel through which suitable gas mixtures are supplied to establish a uniform oxygen tension or gradient within the device. The effects of various parameters, such as gas and media flow rates, device thickness, and diffusion coefficients of oxygen were examined using numerical simulations to determine the characteristics of the microfluidic device. A polycarbonate (PC) film with a low oxygen diffusion coefficient was embedded in the device in proximity above the channels to prevent oxygen diffusion from the incubator environment into the polydimethylsiloxane (PDMS) device. The oxygen tension in the device was then validated experimentally using a ruthenium-coated (Ru-coated) oxygen-sensing glass cover slip which confirmed the establishment of low uniform oxygen tensions (<3%) or an oxygen gradient across the gel region. To demonstrate the utility of the microfluidic device for cellular experiments under hypoxic conditions, migratory studies of MDA-MB-231 human breast cancer cells were performed. The microfluidic device allowed for imaging cellular migration with high-resolution, exhibiting an enhanced migration in hypoxia in comparison to normoxia. This microfluidic device presents itself as a promising platform for the investigation of cellular behaviour in a 3D gel scaffold under varying hypoxic conditions.

Apart from periodontal ligament fibroblasts, immune cells like macrophages also play an important mediating role in orthodontic tooth movement (OTM). Upon orthodontic force application to malpositioned teeth, macrophages in the periodontal ligament get exposed to both mechanical strain and hypoxic conditions (via a compression of blood vessels). In this study, we assessed the relative impact of orthodontically induced mechanical strain and hypoxic conditions on macrophages for the mediation and regulation of OTM. Macrophages were stimulated with physiological orthodontic compressive forces of 2 g/cm2 for 4 h and 24 h on gas-impermeable or gas-permeable cell culture plates under normoxic or hypoxic cell culture conditions. We quantified expression of genes involved in inflammation (Tnf, Il-6, and Cox-2), extracellular remodelling (Mmp-9), and angiogenesis (Vegf) by RT-qPCR. Furthermore, we analysed HIF-1α, prostaglandin-E2, and VEGF protein expression via immunoblotting or ELISA. Mechanical strain and oxygen supply both differentially affected expression of genes and proteins involved in inflammation and angiogenesis. In this context, we found that HIF-1α protein levels were elevated by combined mechanical strain and hypoxic conditions, whereas gas-permeable plates providing sufficient oxygen supply prevented HIF-1α stabilization at the protein level after pressure application on macrophages. Our results thus indicate that macrophages involved in the mediation of OTM are affected by and respond differently to hypoxic conditions and mechanical compressive strain, which occur concomitantly during OTM, than periodontal ligament fibroblasts (PDLF), thus indicating different roles of these cells in the regulation of OTM at the cellular-molecular level. We further observed that contrary to PDLF HIF-1α stabilization in macrophages is rather induced via the decreased oxygen supply associated with OTM than via mechanotransduction by mechanical strain.

Remarkable advances in three-dimensional (3D) cell cultures and organ-on-a-chip technologies have opened the door to recapitulate complex aspects of human physiology, pathology, and drug responses in vitro. The challenges regarding oxygen delivery, throughput, assay multiplexing, and experimental complexity are addressed to ensure that perfused 3D cell culture organ-on-a-chip models become a routine research tool adopted by academic and industrial stakeholders. To move the field forward, we present a throughput-scalable organ-on-a-chip insert system that requires a single tube to operate 48 statistically independent 3D cell culture organ models. Then, we introduce in-well perfusion to circumvent the loss of cell signaling and drug metabolites in otherwise one-way flow of perfusate. Further, to augment the relevancy of 3D cell culture models in vitro, we tackle the problem of oxygen transport by blood using, for the first time, a breathable hemoglobin analog to improve delivery of respiratory gases to cells, because in vivo approximately 98% of oxygen delivery to cells takes place via reversible binding to hemoglobin. Next, we show that improved oxygenation shifts cellular metabolic pathways toward oxidative phosphorylation that contributes to the maintenance of differentiated liver phenotypes in vitro. Lastly, we demonstrate that the activity of cytochrome P450 family of drug metabolizing enzymes is increased and prolonged in primary human hepatocytes cultured in 3D compared to two-dimensional (2D) cell culture gold standard with important ramifications for drug metabolism, drug-drug interactions and pharmacokinetic studies in vitro.

Hypoxia switches the metabolism of tumor cells and induces drug resistance. Currently, no therapeutic exists that effectively and specifically targets hypoxic cells in tumors. Development of such therapeutics critically depends on the availability of in vitro models that accurately recapitulate hypoxia as found in the tumor microenvironment. Here, we report on the design and validation of an easy-to-fabricate tumor-on-a-chip microfluidic platform that robustly emulates the hypoxic tumor microenvironment. The tumor-on-a-chip model consists of a central chamber for 3D tumor cell culture and two side channels for medium perfusion. The microfluidic device is fabricated from polydimethylsiloxane (PDMS), and oxygen diffusion in the device is blocked by an embedded sheet of polymethyl methacrylate (PMMA). Hypoxia was confirmed using oxygen-sensitive probes and the effect on the 3D tumor cell culture investigated by a pH-sensitive dual-labeled fluorescent dextran and a fluorescently labeled glucose analogue. In contrast to control devices without PMMA, PMMA-containing devices gave rise to decreases in oxygen and pH levels as well as an increased consumption of glucose after two days of culture, indicating a rapid metabolic switch of the tumor cells under hypoxic conditions towards increased glycolysis. This platform will open new avenues for testing anti-cancer therapies targeting hypoxic areas.

Integrated phosphorescence-based photonic biosensor (iPOB) for monitoring oxygen levels in 3D cell culture systems

Engineering Human Tumor-specific Cytotoxic T Cells to Function in a Hypoxic Environment

In many cases, alumina scales are assumed to grow predominantly by oxygen diffusion, but some authors have found that the growth can be controlled by aluminium diffusion. These mechanisms can be modified by active elements. The problem with alumina is that there is a lack of data about self-diffusion coefficients, and, due to the stoichiometry of alumina, diffusion data correspond to an extrinsic diffusion mechanism so that it is not possible to compare oxygen and aluminium diffusion coefficients. In order to obtain information about the alumina scale growth mechanism, oxygen (18O) and aluminium (26Al) self-diffusion coefficients in Al2O3 were determined in the same materials and in the same experimental conditions, thus allowing a direct comparison. For both isotopes, bulk and sub-boundary diffusion coefficients were determined in single crystals of undoped alumina. Grain-boundary diffusion coefficients have been computed only for oxygen diffusion in polycrystals. Oxygen diffusion has been also studied for yttria-doped α-alumina in the lattice, sub-boundaries and grain boundaries. Oxygen and aluminium bulk diffusion coefficients are of the same order of magnitude. In the sub-boundaries, aluminium diffusion is slightly faster than oxygen diffusion. Yttria doping induces a slight increase of the oxygen bulk diffusion, but decreases the grain-boundary diffusion coefficients on account of segregation phenomena. These results are compared with the oxidation constants of alumina former alloys (alloys which develop an alumina scale by oxidation). It appears that neither lattice self-diffusion nor grain boundary self-diffusion can explain the growth rate of alumina scales. Such a situation is compared to the case of Cr2O3.

Angiogenesis is an essential process in adulthood during wound healing and restoration of blood flow to injured tissues. Angiogenesis is regulated by a very sensitive interplay of growth factors and inhibitors; their imbalance can lead to very diverse diseases. Excessive angiogenesis is involved in malignant, diabetic retinopathy and inflammatory disorders (eg, rheumatoid arthritis, psoriasis, atherosclerosis). Conversely, insufficient angiogenesis may underlie conditions such as ischemic heart diseases, stroke, hypertension, and diabetes. Hence, during the evolution of these degenerative pathologies, inadequate blood vessel growth and insufficient microvascular density leads to poor circulation and tissue suffering or, ultimately, necrosis and death.1 In several physiological conditions, such as muscular exercise training and detraining, acclimatization to altitude, and aging, an adaptation of the microvascular network structure and function to new conditions has been reported.2,3 Interestingly, there is a close link between cerebral angiogenesis and learning; during cognitive decline in relation to senescence or degenerative cerebral diseases, microvascular density is decreased in specific cerebral areas. Specifically, there is a striking relationship between the capillary density, the cerebral tissue blood flow, the local glucose use, and other measures of neuronal signaling, such as the NA+/K+ ATPase (reviewed in Reference 4). Therefore, microvascular plasticity, defined as the ability of the arteriole and capillary network to adapt to the metabolic local conditions by proangiogenesis or antiangiogenesis processes, likely plays a key role in many tissue homeostatic processes. In hypertension, the role of microcirculation (arteriolar and capillary) is of particular importance in increasing periph-eral resistance …

Guest Editorial: Oxygen balance – What’s that?

Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.