Neurons establish a complex network of synapses that transfer signals in the brain. The ability to re-grow these networks after damage would be an important breakthrough for the treatment of neurodegenerative diseases such as Parkinson’s and Alzheimer’s. However, scientists have struggled to develop a way of culturing neuronal networks that could be applied in vivo. Growth of functional murine neuronal networks in culture relies on protein coats on an extracellular matrix to help the cells attach and develop as a culture. However, these foreign proteins are usually not biocompatible and therefore not suitable for in vivo applications.
But neurogeneration may not depend on protein coats much longer. Researchers from University College London (UCL) have reported that their new nanodiamond (ND) monolayer coatings might be a suitable alternative to protein coatings when it comes to neuronal cell culture (1). The team of researchers—led by Richard Jackman, a professor in the department of electronic and electrical engineering and the London Center for Nanotechnology at UCL—hypothesized that small, nanometer-sized diamonds would be easier for the cells to use to attach to a substrate.
Diamonds are biocompatible in vivo because their simple carbon composition does not interact negatively with natural biological structures. They are also known for their mechanical and electrical properties, which make them a promising material for the development of electrical interfaces with neurons in mice and potentially even in humans.
The new technique developed by Jackman’s team can deposit ND monolayers onto different substrates to support the formation of function neuronal networks without the need for protein coated extracellular matrices. The approach uses dispersion of primary particles in solution to generate individual ND particles that can attach to a substrate through Van der Waals forces (the attractive or repulsive forces between molecules). Previously prepared solutions of NDs were agitated by sonication, which enabled the NDs to attach to a variety of substrates and scaffolds used in 3-D cell cultures. Jackman told BioTechniques that the NDs layered in this way onto substrates successfully because the ratio of surface area (which is relatively large) to the bulk volume of NDs (which is relatively small) gives rise to modified surface chemical behavior. This behavior facilitates attraction of NDs to the substrate. Notably, the ratio is thrown off when using larger diamonds, which take up more surface area and do not generate the same chemical interactions. The topological effects of the substrate surface created by the size and radius of the NDs also affect their successful attachment.
The researchers tested their process by layering NDs onto four different substrates: glass, mechanically polished polycrystalline diamond, nonocrystalline diamond, and silicon. All four substrates were treated with monodispersed NDs through an ultra sonication bath to form monolayers of NDs on the various substrate surfaces. “We wanted to determine whether the [growth] process was purely dependent on diamond properties, or influenced by NDs on different types of substrate,” said Jackman.
Embryonic mouse hippocampal neurons were seeded onto the ND layered surfaces and left to develop for 12 days. The researchers used atomic force microscopy to analyze the physical nature of NDs and X-ray photoelectron spectroscopy to determine the chemical nature of the surface with the NDs. These analyses revealed that neural cells successfully attached to all ND coated substrate materials. “[This revealed that] substrate was not important, which is a good result as it clearly shows the versatility of NDs on different materials,” said Jackman.
The researchers also evaluated the formation of successful electrical impulse connections between the neurons. The team used confocal microscopy of immunostained neural cultures on the four substrates to show successful neuronal connectivity after five days in culture. According to Jackman, it was important to test for connectivity since cells may have attached and undergone initial outgrowth, but did not necessarily form functional connections. According to Jackman, the neurons did form function connections, indicating that the NDs were successful in encouraging the attachment and subsequent growth of neuronal networks.
The team will next explore how varying the size of NDs affects attachment, and investigate different methods for ND attachment to substrates prior to cell growth to ensure long-term stability. Jackman’s laboratory has partnered with the European Union's DREAMS project to create retinal implants for patients with impaired vision.
“In the long term the technique may offer a biocompatible method for attaching neuronal cells to implantable devices for repair of the nervous system,” said Jackman.
Funding for this research was provided by the UK Engineering and Physical Sciences Research Council and the EU Framework Six STREP program (DREAMS). The paper, “The use of nanodiamond monolayer coatings to promote the formation of functional neuronal networks” was published online ahead of print Dec. 24, 2009 in Biomaterials.
1. Chen YC, D.C. Lee, C.Y. Hsiao, Y.F. Chung, H.C. Chen, J.P. Thomas, et al. 2009. The effect of ultra-nanocrystalline diamond films on the proliferation and differentiation of neural stem cells. Biomater. 30:3428-3435.