Axonal Regeneration Supported by Oligo[poly(ethylene glycol)fumarate] Cell-Loaded Hydrogel Scaffolds in the Transected Rat Spinal Cord
Madigan, Nicolas Niall
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Spinal cord injury results in complete tissue destruction and irreversible loss of neurologic function below the level of the lesion in 40% of patients in Ireland. Tissue engineering using polymer scaffolds offers potential to rebuild neural tissue through the injury site and to re-establish functional connections. An introductory review highlights current tissue engineering strategies and novel therapeutic approaches to axonal regeneration. Given the patient morbidity associated with respiratory compromise, the discrete tracts in the spinal cord conveying innervation for breathing represent an important and achievable therapeutic target. A variety of naturally derived and synthetic biomaterial polymers have been developed for placement in the injured spinal cord. Axonal growth is seen to be supported by inherent properties of the selected polymer, the architecture of the scaffold, permissive microstructures such as pores, grooves or polymer fibres, and surface modifications to provide improved adherence and growth directionality. Structural support of axonal regeneration is combined with integrated polymeric and cellular delivery systems for therapeutic drugs and for neurotrophic molecules. This thesis proposes that cell-seeded hydrogel polymer scaffolds in a thoracic cord transection model allow for separate and controlled manipulation of the architecture, surface properties, and the molecular and cellular micro-environment of the regenerating spinal cord. The ability to control these variables with precision may enable the scaffold implant to be informative about individual facets of the repair process. A novel hydrogel, oligo[poly(ethylene glycol)fumarate] (OPF) has been developed, integrating chemical modification for positive surface charge as a substrate for axon growth. OPF scaffolds are loaded with either Schwann cells or mesenchymal stem cells, derived from the bone marrow of transgenic rats with expression of the enhanced green fluorescent protein (eGFP-MSCs). The capacity of each cell type to influence the regenerating environment is compared. Control scaffolds contain extracellular matrix only. Chapter 2 describes the isolation of rat eGFP-MSCs and their characterization as stems cells capable of phenotypic differentiation to mesenchymal lineages. The isolation and characterization of Schwann cells from neonatal rat pups is also described. OPF polymer synthesis, scaffold fabrication, scaffold cell loading with eGFP-MSCs and Schwann cells, thoracic spinal cord transection surgery and scaffold implantation in rats and postoperative outcomes are shown. Gross pathology of spinal cord specimens demonstrates scaffold integration and alignment. In Chapter 3, the architecture of tissue formed after 4 weeks in response to the implantation of each scaffold type is examined initially by means of a general histopathology overview. Detailed immunohistochemistry and stereology approaches are then applied to the model. An analysis of the cell types that are contributing to separate structural and functional compartments within scaffold channels is done using antibodies to glial fibrillary acid protein (GFAP), S-100, vimentin, and neuroglycan-2. Image analysis quantifies the proportional area occupied by each cell type. Established astrocytosis is seen in a peripheral channel compartment, involved in producing boundaries which may organize axon growth. A structurally separate channel core contains immature astrocytes, Schwann cells, eGFP-MSCs, blood vessels and regenerating axons. Schwann cells double stain with GFAP and S-100 antibodies and are seen to populate each scaffold type equally, demonstrating migration into the scaffold from the animal. eGFP-MSCs are shown to be distributed in close association to blood vessels, in keeping with their function as pericytes. We propose that the tissue formed in MSC scaffold channels is granulation tissue. The distribution of inflammatory leukocytes, T-cells and microglia is detailed. Microglial cells dominate the channel core area, whereas leukocyte infiltrate is diffuse. Image analysis provides evidence of T-cell immunomodulation in the MSC group. Quantification of axonal counts demonstrates regeneration is augmented by the presence of Schwann cells in implanted scaffolds. MSCs placed in scaffolds do not support axon growth to any extent. Axon regeneration is analysed in relationship to the developing channel vasculature. Methods of unbiased stereology provide insight into physiologic parameters of blood vessels in scaffold channels, derived from estimations of volume fraction, length density, and surface density. Mean vessel diameter and cross sectional area for each channel type are calculated. Whereas Schwann cell channels have high numbers of small, densely packed vessels, infrequent and large vessels dominate the structure of MSC scaffold channels. Significant correlations between axon counts and vessel length and surface density are shown. Axon number is also shown to statistically correlate with decreasing vessel diameter, implicating the importance of blood flow rate in channels. Radial diffusion distances in vessels correlated significantly to axon number as a hyperbolic function. In Chapter 4 the development of a retroviral library for gene delivery of neurotrophic factors to Schwann cells and MSCs is described. Retroviral expression plasmids, encoding the cDNA transcripts for human neurotrophin 3 (NT-3), brain derived neurotropic factor (BDNF), and glial derived neurotrophic factor (GDNF), were constructed by molecular cloning. Neurotrophin genes were cloned into the pLXSN backbone, which has been modified to contain an internal ribosomal entry site (IRES) for bicistronic expression of eGFP in target cells. DNA sequence accuracy and eGFP-neurotrophin co-expression were verified prior to the development of GP+E86 packaging cell lines for NT-3, GDNF and BDNF retrovirus production. Neurotrophins are secreted at physiologic levels from target cells following viral infection. Stimulation of neurite outgrowth from dorsal root ganglia is shown in response to conditioned media from target cells infected with NT-3 retrovirus. Stably transduced Schwann cell and MSC lines have been made following retroviral gene transfer and cell selection for use in OPF+ scaffolds.