Non-thermal dielectric barrier discharge plasma is being developed for a wide range of medical applications, including wound healing, blood coagulation, and malignant cell apoptosis. However, the effect of non-thermal plasma on the vasculature is unclear. Blood vessels are affected during plasma treatment of many tissues and may be an important potential target for clinical plasma therapy. Porcine aortic endothelial cells were treated in vitro with a custom non-thermal plasma device. Low dose plasma (up to 30 s or 4 J cm(-2)) was relatively non-toxic to endothelial cells while treatment at longer exposures (60 s and higher or 8 J cm(-2)) led to cell death. Endothelial cells treated with plasma for 30 s demonstrated twice as much proliferation as untreated cells five days after plasma treatment. Endothelial cell release of fibroblast growth factor-2 (FGF2) peaked 3 h after plasma treatment. The plasma proliferative effect was abrogated by an FGF2 neutralizing antibody, and FGF2 release was blocked by reactive oxygen species scavengers. These data suggest that low dose non-thermal plasma enhances endothelial cell proliferation due to reactive oxygen species mediated FGF2 release. Plasma may be a novel therapy for dose-dependent promotion or inhibition of endothelial cell mediated angiogenesis.

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 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.

Vascularization plays a key role in processes such as wound healing and tissue engineering. Nonthermal 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.

3D human cancer models provide a better platform for drug efficacy studies than conventional 2D culture, since they recapitulate important aspects of the in vivo microenvironment. While biofabrication has advanced model creation, bioprinting generally involves extruding individual cells in a bioink and then waiting for these cells to self-assemble into a hierarchical 3D tissue. This selfassembly is time consuming and requires complex cellular interactions with other cell types, extracellular matrix components, and growth factors. We therefore investigated if we could directly bioprint pre-formed 3D spheroids in alginate-based bioinks to create a model tissue that could be used almost immediately. Human breast epithelial cell lines were bioprinted as individual cells or as preformed spheroids, either in monoculture or co-culture with vascular endothelial cells. While individual breast cells only spontaneously formed spheroids in Matrigel-based bioink, pre-formed breast spheroids maintained their viability, architecture, and function after bioprinting. Bioprinted breast spheroids were more resistant to paclitaxel than individually printed breast cells; however, this effect was abrogated by endothelial cell co-culture. This study shows that 3D cellular structure bioprinting has potential to create tissue models that quickly replicate the tumor microenvironment.