Hydrogel as a tool for vascular endothelial growth factor gradual release in peripheral nerve regeneration.

The aim of this study was to obtain an hydrogel for the controlled delivery of Vascular Endothelial Growth Factor (VEGF-A165). Hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. The advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications. Agarose/Gelatin (A/GL) hydrogel was crosslinked with genepin. Rheological measurements show that A/GL hydrogel can be injected through a syringe into the inner cavity of a nerve guide. In vitro assays performed to evaluate adhesion, proliferation, viability and migration – using a fibroblast cell line (NIH3T3), Neonatal Olfactory Bulb Ensheathing Cells (NOBEC) and a Schwann cell line (RT4-D6P2T) - show that hydrogel allows cell adhesion, proliferation, viability and migration. VEGF-A165 is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. VEGF is known to have a positive effect on Schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibres. The hydrogel preparation protocol was optimised to be functionally efficient at physiological conditions, allowing the loading of VEGF without the risk of its denaturation. Different amounts of VEGF-A165 (50-100-200 ng/ml) were incorporated in the A/GL hydrogel and the releasing rate in vitro up to 65 days was quantified by ELISA . Released VEGF bioactivity was validated in Human Umbilical Vein Endothelial Cells (HUVEC) and in NOBEC by evaluating phosphorylation of VEGF-Receptor 2 (VEGFR2), akt and erk1-2. Moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of VEGF, and an increased neurite outgrowth was quantitatively assessed. These results demonstrate that VEGF can be successfully incorporated and bioactively released from A/GL hydrogel inducing VEGFR2 activation and neurite outgrowth. In vivo test are ongoing on adult rats: one centimeter lesions on medial nerve were performed, followed by implantation of Porous Poly--caprolactone tubes filled with A/GL hydrogel containing VEGF-A165. Functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration.