Date of Award
Doctor of Philosophy (PhD)
Regeneration of lost synaptic connections following spinal cord injury (SCI) is limited due to local ischemia, cell death, and an excitotoxic environment, which leads to the development of an inhibitory glial scar surrounding a cystic cavity. Myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs) are major inhibitors to axon growth inhibition found within the glial scar and limit functional recovery. The NEP1-40 peptide competitively binds the Nogo receptor and partially blocks inhibition from MAIs, while chondroitinase ABC (ChABC) enzymatically digests CSPGs, which are upregulated at the site of injury. The first part of this work develops drug delivery systems which provide sustained delivery of both NEP1-40 and ChABC. In vitro studies showed that the combination of ChABC and NEP1-40 increased neurite extension compared to either treatment alone when dissociated embryonic dorsal root ganglia were seeded onto inhibitory substrates containing both MAIs and CSPGs. Furthermore, the ability to provide sustained delivery of biologically active ChABC and NEP1-40 from biomaterial scaffolds was achieved by loading ChABC into lipid microtubes and NEP1-40 into poly (lactic-co-glycolic acid) (PLGA) microspheres, obviating the need for invasive intrathecal pumps or catheters. Fibrin scaffolds embedded with the drug delivery systems (PLGA microspheres and lipid microtubes) were capable of releasing active ChABC for up to one week and active NEP1-40 for over two weeks in vitro. In addition, the loaded drug delivery systems in fibrin scaffolds decreased CSPG deposition and development of a glial scar, while also increasing axon growth after spinal cord injury in vivo. Therefore, the sustained, local delivery of ChABC and NEP1-40 within the injured spinal cord may block both myelin and CSPG-associated inhibition and allow for improved axon growth.
The second part of this work looked to improve upon previously established therapies using a combination strategy. A variety of single therapy interventions provide small improvements in functional recovery after SCI but are limited due to the multitude of obstacles limiting recovery. Therefore, a multifactorial therapeutic option that combines several single therapies may provide a better chance of improving recovery. To this end, fibrin scaffolds were modified to provide sustained delivery of neurotrophic factors, the sustained delivery of anti-inhibitory molecules, and encapsulation of embryonic stem cell-derived progenitor motor neurons (pMNs). The efficacy of the scaffolds, prior to transplantation, was established by validating pMN viability, migration, and extension of processes was unaffected by culture within scaffolds with sustained delivery of anti-inhibitory molecules. The combination scaffolds were then transplanted into a rat sub-acute SCI model. The anti-inhibitory molecules were capable of removing proteoglycans within the glial scar when embedded in fibrin scaffolds without pMNs included. While pMNs incorporated into fibrin scaffolds without anti-inhibitory molecules showed significant cell survival, differentiation into neuronal cell types, axonal extension in the transplant area, and the ability to integrate into the host tissue, the combination of pMNs with anti-inhibitory molecules led to decreased cell survival and increased inflammation in the lesion site. Thus combination therapies maintain therapeutic potential for treatment of SCI but further work is needed to improve cell survival and limit inflammation.
Donald Elbert, James Huettner, Vitaly Klyachko, Paul Stein, Matthew Wood