Citrate-stabilized gold nanoparticles as negative controls for measurements of neurite outgrowth.

Gold nanoparticles (AuNPs) are promising candidates for medical diagnostics and therapeutics, due to their chemical stability, optical properties, and ease of functionalization. Citrate-stabilized reference materials also have potential as negative controls in toxicology studies of other nanoparticles. Here we examine the impact of 30 nm particles on the in vitro development of rat-cortex neural progenitor cells (NPCs), which mimic aspects of the developing neurological environment. AuNPs dispersed in a low serum culture medium initially agglomerated, but then remained stable during a three day incubation period, and agglomerated only slightly during a ten day incubation period, as determined by dynamic light scattering. Transmission electron microscopy indicated the presence of individual nanoparticles at all time points examined. Fixed cells were cross-sectioned by ion milling and imaged by scanning electronmicroscopy and helium-ion microscopy to evaluate particle incorporation. Individual nanoparticles could be resolved inside cross-sectioned cells. AuNPs were incubated with developing NPCs for ten days at concentrations of 0.5 µg/mL Au, 0.1 µg/mL Au, or 0.05 µg/mL Au. Adenosine triphosphate levels, as determined by bioluminescence measurements sensitive to low cell numbers, were not affected by AuNPs and the particles did not interfere with the assay. Multiple endpoints of neurite outgrowth were not altered by AuNPs, in particular, total neurite outgrowth per cell, a sensitive measure of neuronal development. Slide-level comparisons demonstrated the consistent response of NPCs to gold nanoparticles and a positive control chemical, neuroactive lithium. These results indicate that 30 nm citrate-stabilized AuNPs could serve as negative-control reference materials for in vitro measurements of neurite outgrowth.

Three-dimensional hydrogel constructs for exposing cells to nanoparticles.

In evaluating nanoparticle risks to human health, there is often a disconnect between results obtained from in vitro toxicology studies and those from in vivo activity, prompting the need for improved methods to rapidly assess the hazards of engineered nanomaterials. In vitro studies of nanoparticle toxicology often rely on high doses and short exposure periods due to the difficulty of maintaining monolayer cell cultures over extended time periods as well as the difficulty of maintaining nanoparticle dispersions within the culture environment. In this work, tissue-engineered constructs are investigated as a platform for providing doses of nanoparticles over different exposure periods to cells within a three-dimensional environment that can be tuned to mimic in vivo conditions. Uptake of quantum dots (QDs) by model neural cells was first investigated in a high-dose exposure scenario, resulting in a strong concentration-dependent uptake of carboxyl-functionalised QDs. Poly(ethylene glycol) hydrogel scaffolds with varying mesh sizes were then investigated for their ability to support cell survival and proliferation. Cells were co-encapsulated with carboxyl-functionalised poly(ethylene glycol)-coated QDs at a lower dose than is typical for monolayer cultures. Although the QDs leach from the hydrogel within 24 h, they are also incorporated by cells within the scaffold, enabling the use of these constructs in future studies of cell behaviour and function.

Neurite outgrowth and differentiation of rat cortex progenitor cells are sensitive to lithium chloride at non-cytotoxic exposures.

Neuron-specific in vitro screening strategies have the potential to accelerate the evaluation of chemicals for neurotoxicity. We examined neurite outgrowth as a measure of neuronal response with a commercially available rat cortex progenitor cell model, where cells were exposed to a chemical during a period of cell differentiation. In control cultures, the fraction of beta-III-tubulin positive neurons and their neurite length increased significantly with time, indicating differentiation of the progenitor cells. Expression of glial fibrillary acidic protein, an astrocyte marker, also increased significantly with time. By seeding progenitor cells at varying densities, we demonstrated that neurite length was influenced by cell-cell spacing. After ten days, cultures seeded at densities of 1000 cells/mm(2) or lower had significantly shorter neurites than cultures seeded at densities of 1250 cells/mm(2) or higher. Progenitor cells were exposed to lithium, a neuroactive chemical with diverse modes of action. Cultures exposed to 30 mmol/L or 10 mmol/L lithium chloride (LiCl) had significantly lower metabolic activity than control cultures, as reported by adenosine triphosphate content, and no neurons were observed after ten days of exposure. Cultures exposed to 3 mmol/L, 1 mmol/L, or 0.3 mmol/L LiCl, which encompass lithium's therapeutic range, had metabolic activity similar to control cultures. These cultures exhibited concentration-dependent decreases in neurite outgrowth after ten days of LiCl exposure. Neurite outgrowth results were relatively robust, regardless of the evaluation methodology. This work demonstrates that measurement of neurite outgrowth in differentiating progenitor cell cultures can be a sensitive endpoint for neuronal response under non-cytotoxic exposure conditions.

Analysis of ISCD-NIST survey for bone health.

In 2007, the National Institute of Standards and Technology and the International Society for Clinical Densitometry designed a survey to prioritize 7 research and standardization action items to improve accuracy and cross-comparability of dual-energy X-ray absorptiometry (DXA) measurements of bone mineral density. In this article, we analyze the 1074 survey responses as one means to define consensus priorities of the community studying bone health and to determine possible correlations between prioritization and demographic data, including geographic location, years of experience practicing DXA, and medical specialty. We find that the distribution of ranks from all respondents is such that we can conclude with statistical confidence that there are perceived distinctions between the relative merits of the 7 action items. Applying a standard vote-counting rule to the data, we determine a complete ranking of the action items. We observe that a consistent ranking of each action item across all demographic subcategories is hard to achieve. When we arrange the 7 action items into 4 groups, however, we can determine a reasonably consistent prioritization. The group containing the development of standard reference databases and phantoms receives the highest priority. In addition, we report consistent themes that emerge from the free-response portion of the survey.

Biocompatibility of atomic layer-deposited alumina thin films.

Presented in this paper is a study of the biocompatibility of an atomic layer-deposited (ALD) alumina (Al2O3) thin film and an ALD hydrophobic coating on standard glass cover slips. The pure ALD alumina coating exhibited a water contact angle of 55 degrees +/- 5 degrees attributed, in part, to a high concentration of -OH groups on the surface. In contrast, the hydrophobic coating (tridecafluoro-1,1,2,2-tetrahydro-octyl-methyl-bis(dimethylamino)silane) had a water contact angle of 108 degrees +/- 2 degrees. Observations using differential interference contrast microscopy on human coronary artery smooth muscle cells showed normal cell proliferation on both the ALD alumina and hydrophobic coatings when compared to cells grown on control substrates. These observations suggested good biocompatibility over a period of 7 days in vitro. Using a colorimetric assay technique to assess cell viability, the cellular response between the three substrates can be differentiated to show that the ALD alumina coating is more biocompatible and that the hydrophobic coating is less biocompatible when compared to the control. These results suggest that patterning a substrate with hydrophilic and hydrophobic groups can control cell growth. This patterning can further enhance the known advantages of ALD alumina, such as conformality and excellent dielectric properties for bio-micro electro mechanical systems (Bio-MEMS) in sensors, actuators, and microfluidics devices.

A uniaxial bioMEMS device for quantitative force-displacement measurements.

There is a need for experimental techniques that allow the simultaneous imaging of cellular cystoskeletal components with quantitative force measurements on single cells. A bioMEMS device has been developed for the application of strain to a single cell while simultaneously quantifying its force response. The prototype device presented here allows the mechanical study of a single, adherent cell in vitro. The device works in a fashion similar to a displacement-controlled uniaxial tensile machine. The device is calibrated using an AFM cantilever and shows excellent agreement with the calculated spring constant. The device is demonstrated on a single fibroblast. The force response of the cell is seen to be linear until the onset of de-adhesion with the de-adhesion from the cell platform occurring at a force of approximately 1500 nN.

Standards and measurements for assessing bone health-workshop report co-sponsored by the International Society for Clinical Densitometry (ISCD) and the National Institute of Standards and Technology (NIST).

This article reports and discusses the results of the recent ISCD-NIST Workshop on Standards and Measurements for Assessing Bone Health. The purpose of the workshop was to assess the status of efforts to standardize and compare results from dual-energy X-ray absorptiometry (DXA) scans, and then to identify and prioritize ongoing measurement and standards needs.