Anisotropic scaffolds facilitate enhanced neurite extension in vitro

Abstract

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.

Differences between the effect of anisotropic and isotropic laminin and nerve growth factor presenting scaffolds on nerve regeneration across long peripheral nerve gaps

“…First, thermoreversible SeaPrep ® agarose hydrogel (BMA, Rockland, Rockland, ME) was covalently coupled with ECM protein LN-1 using photochemical conjugation technique reported previously [38]. Briefly, LN-1 (BD Biosciences, Bedford, MA) was first conjugated to a bifunctional photochemical crosslinker, Sulfo-SANPAH (Sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate) (PIERCE, Rockford, IL) through the amine groups on the LN-1 molecule.…”

“…For example, LN-1 protein or fragments of LN-1 can be covalently coupled to SeaPrep ® agarose gels to further enhance their ability to support neurite extension [30]. Photochemical conjugation can be used to photoimmobilize gradients of LN-1 in agarose gels to promote enhanced neurite extension in vitro [38].…”

“…However, we hypothesized that for longer nerve gaps, 15-mm or more in rodents or over 80 mm in humans, isotropic scaffolds might not be able to match the performance of nerve autogratfts. Anisotropic agarose hydrogel scaffolds, with gradients of LN-1, have been shown to promote enhanced neurite extension from chick dorsal root ganglia (DRG) in vitro, as compared to isotropic scaffolds [38]. Similarly, gradients of NGF can initiate turning of neurites towards the NGF-source [44], and guide the neurite growth [28,45].…”

Long-Term Survival and Integration of Transplanted Engineered Nervous Tissue Constructs Promotes Peripheral Nerve Regeneration

“…[5][6][7][8][9][10][11][12][13][14] Anisotropic properties are important to direct axon growth, and are typically achieved via gradients (e.g., neurotrophic or extracellular matrix), longitudinally aligned fibers, or tailored porosity. [15][16][17][18] In practice, axonal guidance cues and anisotropy are typically provided by a choreographed organization of Schwann cells (SCs), either delivered or endogenous, which form aligned regenerative columns (i.e., Bands of Bungner). These columnar-aligned SCs provide neurotrophic support and contact guidance to direct axonal regeneration toward target tissue, with axonal functional maturation occurring after elimination of extraneous or erroneous connections.…”

Processing and Templating of Bioactive-Loaded Polymeric Neural Architectures: Challenges and Innovative Strategies

“…The scaffolds were fabricated via photochemical coupling approach wherein LN-1 was immobilized onto three-dimensional (3D) agarose scaffolds in gradients of differing slopes. A significant increase in the Dorsal root ganglia (DRG) neurite extension rates were observed in case of anisotropic scaffolds as compared to their isotropic analogues proving the importance and potential of built-in directional cues for guided tissue or nerve regeneration [7]. In line with the functionalized scaffold properties developed above, Crompton et al, 2007, described the potential of Polylysine-functionalized chitosan as an in vitro substrate, scaffold for cortical cells, and for neural tissue engineering as an injectable scaffold.…”