Alisa Morss Clyne

Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture.

Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.

Superparamagnetic iron oxide nanoparticles change endothelial cell morphology and mechanics via reactive oxygen species formation

Superparamagnetic iron oxide nanoparticles are used in various medical applications including magnetic resonance imaging, magnetic hyperthermia, and targeted drug and gene delivery. When used in vivo, these nanoparticles interact with endothelial cells lining all blood vessels, therefore it is crucial to understand endothelial cell functional changes and toxicity upon nanoparticle exposure. We incubated porcine aortic endothelial cells with varying concentrations of bare iron oxide nanoparticles (20-40 nm), and measured cellular reactive oxygen species (ROS) formation, morphology and cytoskeletal organization, death, and elastic modulus. Intracellular ROS increased more than 800% after 3 h of nanoparticle exposure (0.5 mg mL(-1)). Endothelial cells elongated to more than twice their initial length by 12 h, and actin stress fibers formed within the cells. This change in the actin cytoskeleton increased cell elastic modulus by 50%. When ROS formation was blocked using scavengers, initial cell morphology and the actin cytoskeleton remained intact, and cell viability increased. These studies suggest that iron oxide nanoparticles induce ROS formation, which disrupts the actin cytoskeleton and alters endothelial cell morphology and mechanics. If ROS formation is decreased using ROS inhibitors, either as a component of the nanoparticle coating or by systemic administration, higher nanoparticle concentrations might be used with greater efficacy and diminished side effects.

Non-thermal dielectric barrier discharge plasma induces angiogenesis through reactive oxygen species.

Vascularization plays a key role in processes such as wound healing and tissue engineering. Non-thermal plasma, which primarily produces reactive oxygen species (ROS), has recently emerged as an efficient tool in medical applications including blood coagulation, sterilization and malignant cell apoptosis. Liquids and porcine aortic endothelial cells were treated with a non-thermal dielectric barrier discharge plasma in vitro. Plasma treatment of phosphate-buffered saline (PBS) and serum-free medium increased ROS concentration in a dose-dependent manner, with a higher concentration observed in serum-free medium compared with PBS. Species concentration inside cells peaked 1 h after treatment, followed by a decrease 3 h post treatment. Endothelial cells treated with a plasma dose of 4.2 J cm–2 had 1.7 times more cells than untreated samples 5 days after plasma treatment. The 4.2 J cm–2 plasma dose increased two-dimensional migration distance by 40 per cent compared with untreated control, while the number of cells that migrated through a three-dimensional collagen gel increased by 15 per cent. Tube formation was also enhanced by plasma treatment, with tube lengths in plasma-treated samples measuring 2.6 times longer than control samples. A fibroblast growth factor-2 (FGF-2) neutralizing antibody and ROS scavengers abrogated these angiogenic effects. These data indicate that plasma enhanced proliferation, migration and tube formation is due to FGF-2 release induced by plasma-produced ROS. Non-thermal plasma may be used as a potential tool for applying ROS in precise doses to enhance vascularization.

Non-thermal dielectric barrier discharge plasma treatment of endothelial cells

Non-thermal dielectric barrier discharge plasma is now being widely developed for various medical applications such as skin sterilization, blood coagulation, induction of apoptosis in malignant tissues, and wound healing among others. In this paper, we investigate the toxicity of non-thermal plasma treatment on endothelial cells, which line all blood contacting surfaces in the body. Our initial results indicate that low power non-thermal plasma is relatively non-toxic to endothelial cells at short exposure times up to 30 s, while non-thermal plasma treatment at longer exposure times is cytotoxic. Non-thermal plasma at shorter exposure times may induce proliferation in the cells.

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm2 shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.

Glycated collagen alters endothelial cell actin alignment and nitric oxide release in response to fluid shear stress

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm(2) shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell-matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes.

The role of printing parameters and scaffold biopolymer properties in the efficacy of a new hybrid nano-bioprinting system

We created a hybrid nano-bioprinting system, which combines the initial patterning capabilities of direct cell writing with the active patterning capabilities of superparamagnetic nanoparticles. Biofabrication conditions, including printing parameters and scaffold biopolymer properties, may affect cell viability, nanoparticle manipulation and patterning capabilities. Nanoparticles were printed under varied conditions either in the biopolymer or loaded inside cells. Cell viability, alginate viscosity, nanoparticle movement and printing resolution were measured. We now show that while nanoparticles decreased cell viability, nozzle size had no significant effect. High printing pressure decreased cell viability, but viability loss was not accentuated by nanoparticles. High nanoparticle concentrations increased alginate viscosity at higher alginate concentrations. Nanoparticle velocity in response to a magnetic field was a function of nanoparticle diameter and scaffold viscosity, which agreed with a mathematical model of nanoparticle movement. Finally, the nano-bioprinting system resolution and patterning precision were not affected by nanoparticles in the prepolymer solution. These data suggest that nanoparticle incorporation in solid freeform fabrication does not change biofabrication parameters unless high nanoparticle concentrations are used. Future work includes developing vascularized tissue engineering constructs using the nano-bioprinting system.

A simplified implementation of edge detection in MATLAB is faster and more sensitive than fast fourier transform for actin fiber alignment quantification.

Fiber alignment plays a critical role in the structure and function of cells and tissues. While fiber alignment quantification is important to experimental analysis and several different methods for quantifying fiber alignment exist, many studies focus on qualitative rather than quantitative analysis perhaps due to the complexity of current fiber alignment methods. Speed and sensitivity were compared in edge detection and fast Fourier transform (FFT) for measuring actin fiber alignment in cells exposed to shear stress. While edge detection using matrix multiplication was consistently more sensitive than FFT, image processing time was significantly longer. However, when MATLAB functions were used to implement edge detection, MATLABs efficient element-by-element calculations and fast filtering techniques reduced computation cost 100 times compared to the matrix multiplication edge detection method. The new computation time was comparable to the FFT method, and MATLAB edge detection produced well-distributed fiber angle distributions that statistically distinguished aligned and unaligned fibers in half as many sample images. When the FFT sensitivity was improved by dividing images into smaller subsections, processing time grew larger than the time required for MATLAB edge detection. Implementation of edge detection in MATLAB is simpler, faster, and more sensitive than FFT for fiber alignment quantification.

Thermal Processing of Tissue Engineering Scaffolds

Tissue engineering requires complex three-dimensional scaffolds that mimic natural extracellular matrix function. A wide variety of techniques have been developed to create both fibrous and porous scaffolds out of polymers, ceramics, metals, and composite materials. Existing techniques include fiber bonding, electrospinning, emulsion freeze drying, solvent casting/particulate leaching, gas foaming/particulate leaching, high pressure processing, and thermally induced phase separation. Critical scaffold properties, including pore size, porosity, pore interconnectivity, and mechanical integrity, are determined by thermal processing parameters in many of these techniques. In this review, each tissue engineering scaffold preparation method is discussed, including recent advancements as well as advantages and disadvantages of the technique, with a particular emphasis placed on thermal parameters. Improvements on these existing techniques, as well as new thermal processing methods for tissue engineering scaffolds, will be needed to provide tissue engineers with finer control over tissue and organ development.

Elevated Fibroblast Growth Factor-2 Increases Tumor Necrosis Factor-α Induced Endothelial Cell Death in High Glucose

Glucose and tumor necrosis factor‐α (TNFα) concentrations are elevated in diabetes. Both of these factors correlate with diabetic vasculopathy and endothelial cell apoptosis, yet their combined effects have not been measured. We have previously shown that the angiogenic growth factor fibroblast growth factor‐2 (FGF‐2), which is generally protective against endothelial cell death, is similarly elevated in high glucose conditions. We therefore investigated the effect of TNFα on endothelial cell death under normal and elevated glucose conditions, with a particular focus on FGF‐2. Porcine aortic endothelial cells were cultured in 5 and 30 mM glucose and stimulated with TNFα, together with FGF‐2 or a neutralizing FGF‐2 antibody. Cell death was measured via cell counts or an annexin apoptotic assay, and cell cycle phase was determined by propidium iodide labeling. TNFα‐induced endothelial cell death increased for cells in high glucose, and cell death was enhanced with increasing FGF‐2 exposure and negated by a neutralizing FGF‐2 antibody. Endothelial cells were most susceptible to TNFα‐induced cell death when stimulated with FGF‐2 18 h prior to TNFα, corresponding to cell entry into S phase of the proliferative cycle. The FGF‐2 associated increase in TNFα‐induced cell death was negated by blocking cell entry into S phase. Endothelial cell release of FGF‐2 in high glucose leads to cell cycle progression, which makes cells more susceptible to TNFα‐induced cell death. These data suggest that growth factor outcomes in high glucose depend on secondary mediators such as cytokines and stimulation cell cycle timing. J. Cell. Physiol. 217: 86–92, 2008.