The design of nanostructured scaffolds has gathered a significant amount of interest over the last few years in nanomedicine and tissue engineering fields. Within the design of such scaffolds, the materials undergoing a serious amount of research are hydrogels, as they can produce realistic tissue constructs resembling living tissues.
A team of Researchers from Spain and Italy have now created a new series of 3D hydrogel scaffolds for neuronal growth using a combination of aqueous graphene dispersions and acrylamide synthesized by in situ radical polymerization.
One area in which the design of new scaffolds in tissue engineering research has focused on is in the adaptation of the micro-environment of 3D tissue-scaffolds in order to regulate neural cell adhesion or tissue growth in neurological applications. 3D scaffolds have been designed to incorporate essential features for implantable prosthesis, such as optimizing the biocompatibility, stability and homogeneity at the microscale, conductivity and controlled mechanical properties.
Whilst acrylamide hydrogels have been synthesized previously for scaffold applications, they have commonly suffered with biocompatibility issues–the most important property for implantable scaffolds. To combat this the Researchers have created a series of graphene-polyacrylamide hydrogels which support the growth of living primary neurons.
The Researchers prepared an aqueous dispersion of graphene by exfoliating graphite with melamine using a ball-milling method (Retsch PM100 planetary mill) and dispersing the mixture in water. The poorly exfoliated graphite was precipitated from solution and the melamine was removed through washing steps.
The Researchers also prepared a polyacrylamide hydrogel using acrylamide, a N,N′- methylenebisacrylamide (MBA) crosslinker and a potassium peroxodisulfate (KPS) initiator. The Researchers homogenized the solution through a combination of mixing and sonication methods until polymerization took place. Unreacted monomers and initiator molecules were dialyzed to create a pure hydrogel.
A similar process was then followed to combine the polyacrylamide hydrogel with the graphene dispersion to create the graphene-hydrogel nanocomposite. The Researchers trialed graphene dispersion ranging between 0.05 and 2mgmL–1. Primary hippocampal cultures were adhered to the scaffold and cultured for 8-10 days.
The Researchers also used a combination of scanning electron microscopy (SEM, PHILIPS XL30 and FEI QUANTA 250), Raman spectroscopy (InVia Renishaw), compressive tests (Mecmesin Multitest 2.5-i dynamic mechanical analyzer), immunofuorescence confocal microscopy (Leica Microsystems GmbH) and calcium imaging experiments (IMAGO CCD camera; Till Photonics) to characterize the graphene-polyacrylamide hydrogels. The main focus of the characterization was focused upon the graphene contributions to the hydrogel network.
The incorporation of graphene into the hydrogels brought a remarkable and noticeable improvement to the properties of the hybrid hydrogel. By varying the amount of graphene incorporated into these materials, it allowed the Researchers to study the role that graphene plays in these materials.
The Researchers discovered that the graphene becomes an intrinsic component of the polymer network and not just an embedded nanomaterial. The hydrogel with a concentration of only 0.2 mgmL–1 was used as the support to grow the brain cells and develop synaptic activity.
Hippocampal neurons and astrocytes were efficiently developed on the hydrogel scaffolds, but only on the scaffold which contained graphene (pure polyacrylamide did not promote growth). In addition to the growth, the cultured cell networks produced active synaptic networks, which was observed directly by imaging techniques.
The observation of the neuronal networks with the graphene incorporated networks is the most significant finding in this research.
The Researchers measured the compressive moduli of the scaffolds and the values were much higher for the scaffolds containing graphene. The Researchers also found no obvious fatigue behavior in the scaffolds, even with the scaffolds containing the highest concentration of graphene.
It was deduced that the mechanical properties of the scaffold are not critical for neuronal growth in the presence of graphene, but more so that graphene plays a critical intrinsic role for the development of neurons.
The Researchers have showcased that graphene plays a significant role in helping to improve the biocompatibility of polyacrylamide hydrogels, enabling neuronal adhesion to occur. The hybrid scaffolds produced by the Researchers also have the potential to help engineer the neural interface of brain devices in the future.
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“Graphene Improves the Biocompatibility of Polyacrylamide Hydrogels: 3D Polymeric Scaffolds for Neuronal Growth”- Martin C., et al, Scientific Reports, 2017, DOI:10.1038/s41598-017-11359-x