Full Length ArticleZnO nanowire florets promote the growth of human neurons
Graphical abstract
Introduction
Nanowires are 1-dimensional cylindrical nanostructures that are typically manufactured from inorganic, conducting or semiconducting materials [1]. Nanowires typically have a diameter below 100 nm, a length in the micrometre range and have unique physical properties, such as high aspect ratios and large surface areas, which are not observed in bulk materials [2]. There is a great deal of interest in the application of ZnO nanowires to cell biology due to the ease of synthesis in varying geometries and morphologies. Their size makes them ideal for interactions with cells, which have a typical cell membrane width of approximately 10 nm and a cell diameter of approximately 10 µm. In recent years, the interaction of nanowires with living cells has been reviewed extensively for biological applications [3], [4], [5], [6], [7], [8]. Specifically, nanowires have been used for cell guidance and spatial patterning [9,10], electrical recording [11], [12], [13], [14], transfection [15], mechanical sensing [16] and biomolecule transport [17]. In particular, ZnO nanowires have demonstrated a potential to be used on microelectrode arrays for measuring electrical signals from neurons since their 3D structure can reduce the electrical impedance of the electrodes [18].
Despite previous use of ZnO nanowires for biological applications there are conflicting reports of cytotoxicity under different experimental conditions. The interaction between nanowires and cells can vary considerably depending on application and parameters such as the cell type, nanowire material, geometry, morphology and the fabrication process [3].
ZnO nanowires can easily be grown via a low temperature (below 100 °C) hydrothermal synthesis [19] with a low cost and high scalability on various substrates [20], [21], [22]. The morphology and geometry of nanowires can be controlled by varying both of the hydrothermal growth parameters (e.g. precursor concentrations, growth time) and through the area and spacing of the seed layers available, via photolithographic patterning [23], [24], [25].
To date, no study has been reported on interaction of ZnO nanowires with human neurons. In this article, we assess the biocompatibility of ZnO nanowires of various morphologies with human neurons that were derived from the human teratocarcinoma cell line (hNT). hNT neurons express a variety of characteristic neuronal markers [26], exhibit spontaneous electrical activity [27], and have been validated as an alternative to primary human neurons [28]. hNT neurons have been used in cell patterning applications [29] and have been used in clinical applications as a cell transplantation treatment to aid in recovery following stroke [30]. In addition, hNTs raise few ethical concerns as neurons are derived from an stem cell line and provide an accessible way to produce large quantities of human neurons.
Section snippets
ZnO nanowire fabrication
ZnO nanowire samples were fabricated in a pattern comprised 8 arrays nanowire florets with inter-floret gaps (G) varying from 5 µm to 100 µm. The floret diameter was 15 µm and was similar to the average size of a neuron soma. Fig. 1A and B shows brightfield images of a single ZnO nanowire sample and one of the nanowire arrays, respectively. The nanowire fabrication process is depicted in Fig. 1C and is described below. The nanowires were formed on 7 × 7 mm2 silicon substrates with a 100 nm
Results
In this section, we describe the growth of hNT neurons on ZnO nanowire samples. First, we characterised the morphology of ZnO nanowires that were hydrothermally grown under various conditions. Next, we evaluated whether neuron growth was promoted on individual nanowire florets. Then, we evaluated the influence of both the array geometry and the nanowire morphology on neuron growth. Finally, we evaluated whether the neurons within the nanowire arrays were functionally viable by examining their
Discussion
In this article, we have described how vertical ZnO nanowires either promote or inhibit the growth of human hNT neurons depending on their morphology. Using a hydrothermal growth method developed by Maddah et al. [23], combined with photolithography to generate spatially patterned nanowire florets, we were able to examine the growth of neurons over a wide range of nanowire morphologies. Unlike previous literature, we report either an increase or a decrease in neuron growth that was modulated by
Conclusions
In this article we have demonstrated both the promotion and inhibition of hNT neuron growth on ZnO nanowires. It was found that neuron growth was promoted when ZnO nanowires were less than 500 nm in length with a density of greater than 350 nanowires per µm2, however, as the nanowires grew longer, they became less dense and supported less extensive neuron growth. The promotion of neuron growth was attributed to the nanowire covered surfaces presenting a favourable surface roughness. In
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We acknowledge funding support from The Royal Society of New Zealand Marsden fund (UOA1510/3709273).
References (46)
- et al.
Bionanoelectronics with 1D materials
Mater. Today
(2009) - et al.
Nanoelectronics-biology frontier: from nanoscopic probes for action potential recording in live cells to three-dimensional cyborg tissues
Nano Today
(2013) - et al.
Synthesis, characterization, and applications of ZnO nanowires
J. Nanomater.
(2012) - et al.
First human hNT neurons patterned on parylene-C/silicon dioxide substrates: combining an accessible cell line and robust patterning technology for the study of the pathological adult human brain
J. Neurosci. Methods
(2010) - et al.
Clonal human (hNT) neuron grafts for stroke therapy
Am. J. Pathol.
(2002) Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro
Dev. Biol.
(1984)- et al.
Turning teratocarcinoma cells into neurons: rapid differentiation of NT-2 cells in floating spheres
Dev. Brain Res.
(2003) - et al.
Enrichment of differentiated hNT neurons and subsequent analysis using flow-cytometry and xCELLigence sensing
J. Neurosci. Methods
(2014) - et al.
ZnO nanowire arrays as substrates for cell proliferation and differentiation
Mater. Sci. Eng. C
(2012) - et al.
Influence of nanoscale surface roughness on neural cell attachment on silicon
Nanomed. Nanotechnol. Biol. Med.
(2005)
The control of cell adhesion and viability by zinc oxide nanorods
Biomaterials
Contributions of surface topography and cytotoxicity to the macrophage response to zinc oxide nanorods
Biomaterials
Semiconductor nanowire: what's next?
Nano Lett.
Interactions between semiconductor nanowires and living cells
J. Phys. Condens. Matter
Exploring arrays of vertical one-dimensional nanostructures for cellular investigations
Nanotechnology
Interfacing inorganic nanowire arrays and living cells for cellular function analysis
Small
Towards a better prediction of cell settling on nanostructure arrays—simple means to complicated ends
Adv. Funct. Mater.
Nanowire-based nanoelectronic devices in the life sciences
MRS Bull.
From immobilized cells to motile cells on a bed-of-nails: effects of vertical nanowire array density on cell behaviour
Sci. Rep.
Rectifying and sorting of regenerating axons by free-standing nanowire patterns: a highway for nerve fibers
Langmuir
Fine-tuning the degree of stem cell polarization and alignment on ordered arrays of high-aspect-ratio nanopillars
ACS Nano
Nanowire-based electrode for acute in vivo neural recordings in the brain
PLoS One
Vertical nanowire electrode arrays as a scalable platform for intracellular interfacing to neuronal circuits
Nat. Nanotechnol.
Cited by (5)
Advanced theragnostics for the central nervous system (CNS) and neurological disorders using functional inorganic nanomaterials
2023, Advanced Drug Delivery ReviewsNanostructured gold electrodes promote neural maturation and network connectivity
2021, BiomaterialsCitation Excerpt :In general terms, vertical nanoscale structures efficiently support the growth of a diversity of mammalian cells, including neural cells [38]. For example, Raos and colleagues demonstrated that human neurons derived from a human teratocarcinoma cell line (hNT) were able to properly grow and remain functionally viable on individual florets composed of vertically-arranged ZnO NWs which were below 500 nm long [39]. Moreover, rat neuroblasts were successfully cultured on single vertical platinum NWs (1 μm in length; 50 nm in diameter) used for the detection of neuronal activity [40].
On the interaction between 1d materials and living cells
2020, Journal of Functional Biomaterials