Aragonite crystalline matrix as an instructive microenvironment for neural development.

The ability to mimic cell-matrix interactions in a way that closely resembles the natural environment is of a great importance for both basic neuroscience and for fabrication of potent scaffolding materials for nervous tissue engineering. Such scaffolding materials should not only facilitate cell attachment but also create a microenvironment that provides essential developmental and survival cues. We previously found that porous aragonite crystalline matrices of marine origin are an adequate and active biomaterial that promotes neural cell growth and tissue development. Here we studied the mechanism underlying these neural cell-material interactions, focusing on the three-dimensional (3D) surface architecture and matrix activity of these scaffolds. We introduced a new cloning technique of the hydrozoan Millepora dichotoma, through which calcein or (45)Ca(2+) were incorporated into the organism's growing skeleton and neuronal cells could then be cultured on the labelled matrices. Herein, we describe the role of matrix 3D architecture on neural cell type composition and survival in culture, and report for the first time on the capacity of neurons and astrocytes to exploit calcium ions from the supporting biomatrix. We found that hippocampal cells growing on the prelabelled aragonite lattice took up aragonite-derived Ca(2+), and even enhanced this uptake when extracellular calcium ions were chelated by EGTA. When the aragonite-derived Ca(2+) uptake was omitted by culturing the cells on coral skeletons coated with gold, cell survival was reduced but not arrested, suggesting a role for matrix architecture in neural survival. In addition, we found that the effects of scaffold architecture and chemistry on cell survival were more profound for neurons than for astrocytes. We submit that translocation of calcium from the biomaterial to the cells activates a variety of membrane-bound signalling molecules and leads to the subsequent cell behaviour. This kind of cell-material interaction possesses great potential for fabricating advanced biomaterials for neural tissue-engineering applications.

Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.

Identifying scaffolds supporting in vitro reconstruction of active neuronal tissues in their 3-dimensional (3D) conformation is a major challenge in tissue engineering. We have previously shown that aragonite coral exoskeletons support the development of neuronal tissue from hippocampal neurons and astrocytes. Here we show for the first time that the porous aragonite skeleton obtained from bio-fabricated hydrozoan Millepora dichotoma supports the spontaneous organization of dissociated hippocampal cells into highly interconnected 3D ganglion-like tissue formations. The ganglion-like cell spheres expanded hundreds of microns across and included hundreds to thousands of astrocytes and mature neurons, most of them having only cell-cell and no cell-surface interactions. The spheres were linked to the surface directly or through a neck of cells and were interconnected through thick bundles of dendrites, varicosity-bearing axons, and astrocytic processes. Thus, M. dichotoma exoskeleton is a novel scaffold with the unprecedented ability to support a highly ordered organization of neuronal tissue. This unexpected organization opens new opportunities for neuronal tissue regeneration, because the spheres resemble in vivo nervous tissue having high volume of cells associated primarily through cell-cell rather than cell-matrix interactions.

Aragonite crystalline biomatrices support astrocytic tissue formation in vitro and in vivo.

Astrocytes play a pivotal role in the development and function of the central nervous system by regulating synaptic activity and supporting and guiding growing axons. It is therefore a central therapeutic and scientific challenge to develop means to control astrocytic survival and growth. We cultured primary hippocampal astrocytes on a crystalline three-dimensional (3D) aragonite biomatrix prepared from the exoskeleton of the coral Porites lutea. Such culturing led to the formation of astrocytic tissue-like 3D structures in which the cells had a higher survival rate than astrocytes grown in conventional cell culture. Within the pore void areas, multiple layers of astrocytic processes formed concave sheet structures that had no physical contact with the surface. The astrocytes attached to the crystalline perpendicular edges of the crystalline template surface extended processes in 3D and expressed glial fibrillary acidic protein. The astrocytes also expressed gap junctions and developed partly synchronized cytosolic Ca2+ oscillations. Preliminary in vivo models showed that astrocytic networks were also developed when the matrices were implanted into cortical areas of postnatal rat brains. Hence, we suggest that the biomatrix is a biocompatible supportive scaffold for astrocytes and may be exploited in applications for neuronal tissue restoration in injured or diseased central nervous system.

Compact self-wiring in cultured neural networks.

We present a novel approach for patterning cultured neural networks in which a particular geometry is achieved via anchoring of cell clusters (tens of cells/each) at specific positions. In addition, compact connections among pairs of clusters occur spontaneously through a single non-adherent straight bundle composed of axons and dendrites. The anchors that stabilize the cell clusters are either poly-D-lysine, a strong adhesive substrate, or carbon nanotubes. Square, triangular and circular structures of connectivity were successfully realized. Monitoring the dynamics of the forming networks in real time revealed that the self-assembly process is mainly driven by the ability of the neuronal cell clusters to move away from each other while continuously stretching a neurite bundle in between. Using the presented technique, we achieved networks with wiring regions which are made exclusively of neuronal processes unbound to the surface. The resulted network patterns are very stable and can be maintained for as long as 11 weeks. The approach can be used to build advanced neuro-chips for bio-sensing applications (e.g. drug and toxin detection) where the structure, stability and reproducibility of the networks are of great relevance.