Anisotropic scaffolds of agarose hydrogels containing gradients of laminin-1 (LN-1) and nerve growth factor (NGF) molecules were used to promote sciatic nerve regeneration across a challenging 20 mm nerve gap in rats.Step and continuous gradient anisotropic scaffolds were fabricated and characterized and regeneration was compared to that in isotropic scaffolds with uniform concentrations of LN-1 and NGF and sciatic nerve grafts harvested from syngenic rats. Polysulfone tubular guidance channels were used to present the agarose based scaffolds to the nerve stumps. Fourmonths after implantation, regenerating axons were observed in animals implanted with anisotropic scaffolds with gradients of both LN-1 and NGF molecules and nerve grafts, but not in animals with isotropic scaffold implants. Also, the scaffolds with gradient of either LN-1 or NGF, with the other component being uniformly distributed in the scaffold, did not elicit axonal regeneration. The total number of myelinated axons was similar for the anisotropic scaffold and the nerve graft conditions, with the anisotropic scaffolds having a higher density of axons than the nerve grafts. Axonal diameter distribution was similar for the anisotropic scaffolds and the nerve grafts. The nerve grafts and anisotropic scaffolds resulted in better functional outcome compared to isotropic scaffolds as measured by the relative gastrocnemius muscle weight (RGMW). Additionally the state of neuromuscular junctions as assessed by pre-and post-synaptic staining revealed that both the anistropic scaffolds performed as well as nerve grafts.

Tissue engineering (TE) techniques to enhance nerve regeneration following nerve damage have had limited success in matching the performance of autografts across short nerve gaps (< 10 mm). For regeneration over longer nerve gaps, TE techniques have been less successful than autografts. Most engineered scaffolds do not present directional cues to the regenerating nerves. In our efforts to design a TE scaffold to replace the autograft, we hypothesize that anisotropic hydrogel scaffolds with gradients of a growth-promoting glycoprotein, laminin-1 (LN-1), may promote directional neurite extension and enhance regeneration. In this study we report the engineering of three-dimensional (3D) agarose scaffolds with photoimmobilized gradients of LN-1 of differing slopes. Dorsal root ganglia (DRG) from chicken embryos were cultured in the agarose scaffolds and their neurite extension rate was determined. DRG neurite extension rates were significantly higher in the anisotropic scaffolds, with a maximal growth rate in an anisotropic scaffold twice that of the maximal growth rate in isotropic scaffolds of LN-1. We suggest that these anisotropic scaffolds, presenting an optimal gradient of LN-1, may significantly impact nerve regeneration. Such anisotropic scaffolds may represent a new generation of tissue engineered materials with built-in directional cues for guided tissue or nerve regeneration.

Human neural progenitor cells differentiated from human embryonic stem cells offer a potential cell source for studying neurodegenerative diseases and for drug screening assays. Previously, we demonstrated that human neural progenitors could be maintained in a proliferative state with the addition of leukemia inhibitory factor and basic fibroblast growth factor. Here we demonstrate that 96 hours after removal of basic fibroblast growth factor the neural progenitor cell culture was significantly altered and cell replication halted. 14 days after the removal of basic fibroblast growth factor, most cells expressed MAP2 and TUJ1, markers characterizing a post-mitotic neuronal phenotype as well as neural developmental markers Cdh2 and Gbx2. Real-time PCR was performed to determine the ionotrophic receptor subunit expression profile. Differentiated neural progenitors express subunits of glutamatergic, GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium and calcium channel subunits were also expressed. Functionally, virtually all the NP cells tested under whole-cell voltage clamp exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited tetrodotoxin-sensitive, voltage-dependent sodium channel current. Action potentials could also be elicited by current injection under whole-cell current clamp in a minority of cells. These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and has the capability to produce excitable cells that can generate action potentials, a landmark characteristic of a neuronal phenotype. This is the first report of an efficient and simple means of generating human neuronal cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.