Agarose hydrogel scaffolds were engineered to stimulate and guide neuronal process extension in three dimensions in vitro. The extracellular matrix (ECM) protein laminin (LN) was covalently coupled to agarose hydrogel using the bifunctional cross-linking reagent 1,19- carbonyldiimidazole (CDI). Compared to unmodified agarose gels, LN-modified agarose gels significantly enhanced neurite extension from three-dimensionally (3D) cultured embryonic day 9 (E9) chick dorsal root ganglia (DRGs), and PC 12 cells. After incubation of DRGs or PC 12 cells with YIGSR peptide or integrin beta1 antibody respectively, the neurite outgrowth promoting effects in LN-modified agarose gels were significantly decreased or abolished. These results indicate that DRG/PC 12 cell neurite outgrowth promoting effect of LN-modified agarose gels involves receptors for YIGSR/integrin beta1 subunits respectively. 1,2-bis(10, 12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC)-based lipid microcylinders were loaded with nerve growth factor (NGF), and embedded into agarose hydrogels. The resulting trophic factor gradients stimulated directional neurite extension from DRGs in agarose hydrogels. A PC 12 cell-based bioassay demonstrated that NGF-loaded lipid microcylinders can release physiologically relevant amounts of NGF for at least 7 days in vitro. Agarose hydrogel scaffolds may find application as biosynthetic 3D bridges that promote regeneration across severed nerve gaps.
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.
Sulfated proteoglycans have inhibitory effects on neurite extension, and the negative charge of the glycosaminoglycan side chains may be involved in the inhibitory process. The main goal of this study is to investigate the effects of charge on three-dimensional neurite extension. Various concentrations of dermatan sulfate (DS), a chondroitin sulfate glycosaminoglycan, and consequently, various degrees of negative charge were presented on three-dimensional agarose hydrogels and the effect of charge on neurite extension from primary neurons was investigated. Dose-response experiments were also performed with the polycationic (positively charged) polysaccharide chitosan covalently coupled to agarose. The amount of DS or chitosan coupled to the agarose gel was quantified via metachromatic dye or Fourier transform infrared spectroscopy methods, respectively. The length of embryonic day 9 (E9) chick dorsal root ganglia neurites extended through charged agarose gels is dependent on the polarity and quantity of ambient charge. The inhibitory effects of the sulfated DS and the enhancing effects of the polycationic chitosan on neurite extension decrease as the amount of DS or chitosan coupled to agarose is decreased. These findings indicate that primary neural process extension is influenced by the polarity of ambient charge in a dose-responsive manner.
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