title: Injectable poly (ethylene glycol) hydrogels for spinal cord injury repair
Creators: name: Venters, Douglas
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: Biomaterial, hydrogels, microfluidics, microspheres, in vitro, biomedical engineering, spinal cord injury, neurons, astrocytes, stem cells.

description: Poly (Ethylene Glycol) (PEG) hydrogels are becoming more ubiquitous in tissue engineering applications due to their inherent biocompatibility, ability to replicate the mechanical environment of soft tissues and their capacity for a diverse range of modifications which make them useful in numerous biological environments. However, spinal cord injury repair is a branch of tissue engineering that is currently provides very limited options on biomaterial strategies to promote recovery and therefore could benefit from an increased focus on developing new materials. This body of work describes the physico-chemical characterisation of hydrogels based on 4-armed PEG-maleimide (PEG-4MAL) which incorporate matrix metalloprotease sensitive peptide cross-linkers to impart degradability, and further functionalisation with integrin-recognisable peptide ligands. Formulations of the hydrogels were created to include the fibronectin derived RGD peptide sequence or the laminin derived IKVAV sequence, and additional variants were formed by altering proportions of the degradable VPM peptide cross-linker in substitution for non-degradable PEG-dithiol, which was found to enable tuneability of the hydrogel degradation rate. Degradation of the gels in type 1 collagenase could be tuned to range from hours to months and potentially longer by altering the cross-linker character. Young’s moduli in the order of hundreds of pascals to low kilopascal range were achieved to make appropriate substrates that replicate the stiffness of the spinal cord. The gelation properties and swelling behaviour were also characterised and the release profiles of nerve growth factor and brain derived neurotrophic factor from the PEG-4MAL hydrogels were evaluated, demonstrating their capability to be loaded with the neurotrophins and rapidly release them into aqueous environments. PEG-4MAL hydrogels were used to investigate the 3D behaviour of several cell types derived from neural tissues, to include cortical astrocytes, spinal cord dissociated neurons and dorsal root ganglia explants suspended within the hydrogels, along with additional experiments conducted using mesenchymal stem cells. Each of the cell types analysed confirmed the capability of the RGD-tethered hydrogels to promote cellular adhesion and migration in 3D. Neurite outgrowth from DRGs was promoted in RGD bound gels and further encouraged by the incorporation of nerve growth factor into the hydrogel. Spinal cord neurons displayed extensive neuritogenesis within each of the PEG-4MAL gels with the highest neurite densities observe in RGD-tethered hydrogels. A microfluidics-based system was devised for creating hydrogel microspheres to expedite the implantation of the material into spinal cord injury in vivo models. These devices enabled the reliable production of microspheres with a high throughput and narrow size dispersity. Biocompatibility was also measured using mesenchymal stem cells and high levels of viability were retained after 7 days culture on the hydrogel microspheres. MSCs grown on the RGD functionalised microspheres were observed to adhere to their surfaces and conform to the topography presented by the microspheres. Finally, the hydrogels were evaluated in pilot in vivo studies using rat contusion models of spinal cord injury. Hydrogel microspheres were injected into the injury site of the contused spinal cord and development of the resulting cellular response was observed after 7 weeks. Histological analysis revealed a degree of astrocyte infiltration into the contusion cavities filled with RGD microspheres along with some deposition of laminin around them. The microsphere injected spinal cords also displayed evidence of reduced astrocyte reactivity. Modest evidence of axonal presence within the cavities was also observed. This has laid the groundwork for future studies of a larger scale to fully elucidate the potential of the hydrogels as a therapy for spinal cord injury.