3D Hydrogels Containing Interconnected Microchannels of Subcellular Size for Capturing Human Pathogenic Acanthamoeba Castellanii

3D Hydrogels Containing Interconnected Microchannels of Subcellular Size for Capturing Human Pathogenic Acanthamoeba Castellani

Toxicity of functional nano-micro zinc oxide tetrapods: impact of cell culture conditions, cellular age and material properties.

With increasing production and applications of nanostructured zinc oxide, e.g., for biomedical and consumer products, the question of safety is getting more and more important. Different morphologies of zinc oxide structures have been synthesized and accordingly investigated. In this study, we have particularly focused on nano-micro ZnO tetrapods (ZnO-T), because their large scale fabrication has been made possible by a newly introduced flame transport synthesis approach which will probably lead to several new applications. Moreover, ZnO-T provide a completely different morphology then classical spherical ZnO nanoparticles. To get a better understanding of parameters that affect the interactions between ZnO-T and mammalian cells, and thus their biocompatibility, we have examined the impact of cell culture conditions as well as of material properties on cytotoxicity. Our results demonstrate that the cell density of fibroblasts in culture along with their age, i.e., the number of preceding cell divisions, strongly affect the cytotoxic potency of ZnO-T. Concerning the material properties, the toxic potency of ZnO-T is found to be significantly lower than that of spherical ZnO nanoparticles. Furthermore, the morphology of the ZnO-T influenced cellular toxicity in contrast to surface charges modified by UV illumination or O2 treatment and to the material age. Finally, we have observed that direct contact between tetrapods and cells increases their toxicity compared to transwell culture models which allow only an indirect effect via released zinc ions. The results reveal several parameters that can be of importance for the assessment of ZnO-T toxicity in cell cultures and for particle development

SnO₂ inhibits HSV-1 entry into HCE cells.

<p>HCE cells were mock treated or treated with SnO₂ and exposed to HSV-1 at an MOI of 10 for 6 hours. A) After 6 hours of infection cells were washed, permeabilized and incubated with ONPG substrate for quantification of β-galactosidase activity from the viral genome. A dosage dependent decrease in entry was noted in cells as minimal entry occurred. B) X-gal staining of HCE cells. HCE cells grown in a 6-well plated were pretreated with SnO₂ before being challenged with HSV-1 for 6 hours. Cells were washed with PBS, fixed, permeabilized and incubated with X-gal, yielding blue cells. Infected cells were imaged at 10× objective using Zeiss Axiovert microscope. C) The average number of infected cells in SnO₂ treated cells is significantly lower than mock treated cells.</p

SnO₂ Inhibits cell-to-cell spread and plaque formation in HCE cells.

<p>A) Confluent monolayers of HCE cells were infected with HSV-1 (KOS) K26RFP and viral replication and spread were imaged 72 hours post infection. The effect of SnO₂ on viral spread was assayed through the measurement of infected cell clusters and the intensity of RFP emission. B) In conjugation with the infectious spread assay, a plaque assay was performed to evaluate the SnO₂ effect on viral transmission. UV treated SnO₂ was added to cells prior to a 2 hour incubation with HSV-1(KOS). Following the 2-hour absorption phase virus inoculum was removed and cells were overlaid with methylcellulose. 3-days post infection cells were fixed with methanol at room temperature for 20 minutes and strained with crystal violet. Images were taken with a Zeiss Axiovert 200 microscope using a 10× objective.</p

SnO₂ treatment reduces glycoprotein mediated cell-to-cell fusion.

<p>Two populations of cells were generated to determine the effect of SnO₂ treatment on cell fusion. Effector cells were transfected with plasmids gB, gD, gH, gL and T7. Target cells were transfected with gD, receptor Nectin-1 and a luciferase expressing plasmid under the control of a T7 promoter. Target and Effector cells were mixed together at a 1∶1 ratio. Luciferase activity was determined in the presence of firefly luciferase, allowing the measurement of relative light units (RLU). CHO-K1 cells were either mock treated or treated with SnO₂. As a negative control, effector cells lacking gB were mixed with the target cells.</p

Scanning electron microscopy results of SnO₂ nanowires synthesized by flame transport approach.

<p>A)–C): SEM images of SnO₂ nanowires in increasing order of magnifications. D) Energy dispersive X-ray absorption (EDAX) spectrum showing the purity of SnO₂ nanowires. The inset E) in D) is the digital camera image demonstrating the wire type fluffy structures of tin oxide.</p

SnO₂ cytotoxicity determination.

<p>To determine the effect of SnO₂ nanowires on cell viability a cytotoxicity assay was performed. HCE were treated for 24 hours in the presence of SnO₂. Cell viability was evaluated by a chromogenic kit (CellTiter Aqueous96; Promega, Madison, WI, USA) and colorimetric detections were performed using a mircroplate ELISA reader (Spectra Max 190). Results are expressed as 100% wild type (WT) viability where they represent the percent corrected absorbance after subtracting the background absorbance relative to untreated cells (0 µg/ml).</p

A tunable scaffold of microtubular graphite for 3D cell growth

Aerographite (AG) is a novel carbon-based material that exists as a self-supportive 3D network of interconnected hollow microtubules. It can be synthesized in a variety of architectures tailored by the growth conditions. This flexibility in creating structures presents interesting bioengineering possibilities such as the generation of an artificial extracellular matrix. Here we have explored the feasibility and potential of AG as a scaffold for 3D cell growth employing cyclic RGD (cRGD) peptides coupled to poly(ethylene glycol) (PEG) conjugated phospholipids for surface functionalization to promote specific adhesion of fibroblast cells. Successful growth and invasion of the bulk material was followed over a period of 4 days

Phase contrast images of normal human dermal fibroblasts (NHDF) depending on passage numbers and seeding cell density after 24 h treatment with ZnO-T and ZnCl₂.

<p>A, D, G and J: untreated NHDF; B, E, H and K: 0.5 mg/ml ZnO-T; C, F, I and L: 30 µg/ml ZnCl₂. Passage numbers: P11–P15 (A–C and G–I) vs. P22–P26 (D–F and J–L). Seeded cell number: 25000 (A–F) vs. 100000 cells/cm² (G–L); time prior treatment: 48 h; duration of treatment: 24 h. Each symbol represents the mean ± SE of n = 3 independent experiments with fourfold determinations. Additionally experiments were done with three different frozen stocks of NHDF to verify the result.</p