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Characterization and Analysis of Highly Hydrated, Three Dimensional Cell-Matrix Constructs

Neural Tissue Engineering involves the use of three-dimensional biomaterial scaffolds for transplantation of neural tissue or for induction of regeneration of severed peripheral or central nerves. There is a paucity of information regarding the optimization parameters for the design of 3D constructs from the mechanical, morphological, or charge perspective such that neurite extension is maximized. In addition, cytoskeletal organization and response of neurons in 3D matrices to various stimuli is not well understood. Our laboratory has been using a combination of electron, force, light, video and confocal microscopy to address these issues. Custom surface rendering image reconstruction techniques were used to reconstruct the three-dimensional structure of neurons extending processes in 3D as visualized by confocal microscopy. We have previously reported that low concentration gels of hydroxyethylated agarose are capable of supported neurite extension from a variety of neural cells in vitro. Agarose hydrogels were fabricated in concentrations ranging from 0.75%-3.0% (w/v). These highly hydrated gels were prepared for electron microscopy using a variety of sample preparation protocols including freeze drying, and critical point drying and their morphological structure and pore radius was computed using scanning electron microscopy.