The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold

Abstract

Understanding neural cell differentiation and neurite extension in three-dimensional scaffolds is critical for neural tissue engineering. This study explores the structure-function relationship between a 3D hydrogel scaffold and neural cell process extension and examines the role of ambient charge on neurite extension in 3D scaffolds. A range of agarose hydrogel concentrations was used to generate varied gel physical structures and the corresponding neurite extension was examined. Agarose gel concentration and the corresponding pore radius are important physical properties that influence neural cell function. The average pore radii of the gels were determined while the gel was in the hydrated state and in two different dehydrated states. As the gel concentration was increased, the average pore radius decreased exponentially. Similarly, the length of neurites extended by E9 chick DRGs cultured in agarose gels depends on gel concentration. The polycationic polysaccharide chitosan and the polyanionic polysaccharide alginate were used to incorporate charge into the 3D hydrogel scaffold, and neural cell response to charge was studied. Chitosan and alginate were covalently bound to the agarose hydrogel backbone using the bi-functional coupling agent 1,1'-carbonyldiimidazole. DRGs cultured in chitosan-coupled agarose gel exhibited a significant increase in neurite length compared to the unmodified agarose control. Conversely, the alginate-coupled agarose gels significantly inhibited neurite extension. This study demonstrates a strong, correlation between the ability of sensory ganglia to extend neurites in 3D gels and the hydrogel pore radius. In addition, our results demonstrate that charged biopolymers influence neurite extension in a polarity dependent manner.

Collagen-Dependent Neurite Outgrowth and Response to Dynamic Deformation in Three-Dimensional Neuronal Cultures

“…For example, the effects of agarose concentration on DRG neurite outgrowth revealed matrix stiffness and pore size differentially influence the rate and degree of neurite extension, with maximal neurite outgrowth occurring in low concentration (<1.00%) gels. 5,7,16 However, low concentration hydrogels have been shown to be unsuitable for the survival and neurite outgrowth of cortical neurons in the present study as well as previous work, 47 underscoring that different intrinsic mechanisms exist between different neuronal sub-types, and thus engineered systems must be optimized for a particular neuronal population. DRG neurite outgrowth was studied in collagen matrices of varying concentrations, and hence stiffness, finding that neurite extension was maximized in lower (0.6 mg/mL) rather than higher (2 mg/mL) concentration gels.…”

“…20,27,30,43 Models consisting of neurons distributed throughout a 3-D matrix material have previously been developed. 6,7,46,47,60 Dorsal root ganglia (DRG) extend neurites through hydrogel matrices in a manner dependent on the physical properties (e.g., agarose pore size 16 and stiffness 5 ), ligand concentration (e.g., collagen 59 and RGD peptides in fibrin 51 ), and substrate geometry. 62 Embryonic cortical neurons have been plated within 3-D matrices of collagen and various hydrogels (e.g., poly[N-(2-hydroxypropyl)-methacrylamide], 60 poly(acrylate), 46 and agarose 47 ).…”

Polymer Based Tissue Engineering Strategies for Neural Regeneration

“…These materials are easily obtained and can be cross-linked to form three dimensional scaffolds. [57][58][59][60][61][62] Alginate scaffolds are formed by calcium cross-linking and can be degraded by calcium chelation, while agarose forms a gel based on its thermodynamic properties above a certain temperature. Both polysaccharides must undergo extensive purification to prevent immune responses after implantation.…”

Exploitation of physical and chemical constraints for three-dimensional microtissue construction in microfluidics

“…Other groups have developed biosensors with cells cultured in a 3D-polymer matrix, including acrylamide derivatives, agarose, and collagen. [216][217][218] Neural progenitor cells entrapped within the collagen matrices forming 3D microspheres can give rise to neuronal progeny that are responsive to environmental toxicants. 218 This development can be further expanded to other cell types such as the immune cells and primary hepatocytes.…”