Spinal cord injury (SCI) affects approximately 10,000 individuals in the United States every year. SCI occurs most commonly in young adults, leaving them seriously disabled for the remainder of their lives. Apart from paralysis, patients of SCI suffer from additional disabilities including bladder, bowel and sexual dysfunction, and neuropathic pain syndromes. Several potentially useful therapeutic strategies have emerged over the last decade. These include delivery of neurotrophic factors and other therapeutic proteins, synthetic scaffolds and bridges, and use of stem cells to promote neuronal regeneration and functional recovery1. However, current strategies have yet to resolve the surgical difficulties of implantation, and none have seen the crossing of axons across their “bridge”. A new method in which the scaffold can be injected, therefore providing a minimally invasive surgical technique, is desirable. The scaffold should also be multifunctional, in that is should provide not only a surface for cellular growth, but also a protected environment that adapts to the growing axons allowing them to grow into the scaffold, through it and back out in order to reconnect with their target cells.

It is proposed that the use of a novel injectable, multifunctional scaffold, which upon injection to the spinal cord would provide not only mechanical support to the injury site but also provide a local sustained release of a combination of therapeutic proteins as well. The scaffold will therefore provide a protected area for the regeneration of injured axons by creating polymeric-cellular bridge. The proposed scaffold is made from a thermally responsive, injectable polymer, poly (N-isopropyl)-graft-poly (ethylene glycol) (PNIPAAm-PEG), combined with neural precursor cells. Below its LCST, typically around 29-32C, the polymer forms a miscible solution with water, but above its LCST, it becomes hydrophobic, separating from water and forming a semi porous gel. This makes PNIPAAm-PEG an ideal candidate since it provides not only a minimally invasive surgical technique, but also a scaffold that is space filling. Since the polymeric scaffold is semiporous it can easily incorporate cell growth and can be engineered to match the mechanical properties of the native neuronal tissue. This is of great importance in the design, since as many failed attempts prior have shown, mechanical mismatch is a critical parameter in implant design. The incorporation of neural precursor cells provides a matrix of necessary growth factors, extra cellular matrix, and other support molecules for axon growth. The included precursor cells not only sprout axons themselves, but in the process also stimulate the injured host axons to grow.

In order to provide a protected environment for axon growth, growth promoting trophic facts must be present and the limited contact with growth inhibitory molecules is necessary. Since the direct injection of these trophic factors, such as brain derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), and vascular endothelial growth factor (VEGF), lead to flooding of the local tissue, which does not support growth, the inclusion of these trophic factors into the scaffold is proposed. Since the polymeric scaffold is hydrophilic it sufficiently traps the trophic factors within its matrix after injection, slowly releasing them to the local tissue. This therefore provides a mechanism to not only draw axons into the scaffold (where the trophic factors are localized) but the prolonged release allows a motivation for axons to exit the scaffold to the local tissue. Further control of the release can be achieved by incorporating the trophic factors within biodegradable microparticles. Microparticles can be made via a simple emulsion technology using poly-(lactic acid) (PLA), which has been shown to be biocompatible in other biomaterial applications.

It is therefore hypothesized that simultaneous, sustained, localized delivery of multiple proteins into the CNS along with an injectable polymeric-cellular scaffold creates a synergistic therapeutic effect by synchronously modulating different biochemical pathways. Engineering this injectable hydrogel and cellular based scaffold to mimic the host tissues will lead to a novel platform technology with applications in treatment of SCI and other tissue engineering applications. Specifically, the aims of this project were to:

1. Synthesize and characterize microparticle drug delivery system.
2. Synthesize, characterize and mechanically optimize the scaffold system and asses the effect of the addition of microparticles on the scaffold.
3. Characterize the in vitro cellular scaffold system.
4. Characterize the in vivo cellular scaffold system in a rodent model of spinal cord injury.