A Laminin and Nerve Growth Factor-Laden Three-Dimensional Scaffold for Enhanced Neurite Extension

Abstract

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.

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

“…LMTs-A drug delivery system using LMTs of 1,2-bis (tricosa-10,12-diyomoyl)-sn-3-phosphocholine (DC8,9PC; Avanti Polar Lipids, Inc., Alabster, AL) was prepared by ethanol deposition method, and used for slow-release of NGF, as described in other studies [30,42]. The LMTs had an average length of 45±20 μm ( Figure 1).…”

“…NGF has been shown to stimulate neurite outgrowth and promote the survival of sensory ganglia, including those which give rise to spinal sensory nerves and sciatic nerves [23][24][25]. Due to their potent functions, LN-1 (an isoform of LN) and NGF have been used successfully for neurite extension in vitro and for nerve regeneration in vivo [26][27][28][29][30]. Hence, in this study we have used LN-1 and NGF to promote sciatic nerve regeneration across a 20 mm nerve gap in rats.…”

“…Various proteins and glycosaminoglycans can be covalently linked to the agarose gels through functional groups on its polysaccharide chains. 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].…”

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

“…Additionally, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine based lipid micro-cylinders were loaded with nerve growth factor (NGF), and embedded into agarose hydrogels which resulted in a directional neurite extension from DRGs in agarose hydrogels due to the presence of trophic factor gradients. The NGF-loaded lipid micro-cylinders released physiologically relevant amounts of NGF for at least 7 days in vitro based on a PC12 cell-based bioassay confirming that the agarose hydrogel scaffolds can be used as biosynthetic 3D bridges capable of promoting regeneration across severed nerve gaps [14].…”

Bioactive Nanomaterials for Neural Engineering

“…Collagen, fi bronectin, and laminin are examples of some major components of the ECM that have been used for this purpose (Yu and Bellamkonda 2003 ;Armstrong et al 2007 ;Koh et al 2010 ). Laminin, in particular, has been used most frequently for surface modifi cation or ECM mimicking purposes among others due to its ability to improve neurite extension and provide Schwann cell adhesion, proliferation, and migration (Yu and Bellamkonda 2003 ;Silva et al 2004 ;Yu et al 1999 ;Itoh et al 2001 ;Rangappa et al 2000 ;Rutkowski et al 2004 ;Toba et al 2001 ;Matsumoto et al 2000 ;Koh et al 2010 ;Bellamkonda et al 1995 ). Collagen and fi bronectin also have regenerative capacity in terms of Schwann cell adhesion, proliferation, and neurite outgrowth improvement; however, outcomes have shown to be signifi cantly lower than that of laminin in terms of regeneration (Yu and Bellamkonda 2003 ;Armstrong et al 2007 ;Koh et al 2010 ).…”