Traumatic spinal cord injury (SCI) is a devastating condition for which there is no effective clinical treatment. Neuroprotective molecules that minimize tissue loss have shown promising results; however systemic delivery may limit in vivo benefits due to short systemic half-life and minimal passage across the blood-spinal cord barrier. To overcome these limitations, an injectable intrathecal delivery vehicle comprised of hyaluronan and methylcellulose (HAMC) was developed, and previously demonstrated to be safe and biocompatible intrathecally. Here, HAMC was determined to persist in the intrathecal space for between 4-7 d in vivo, indicating it as an optimal delivery system for neuroprotective agents to reduce tissue degeneration after SCI. HAMC was then investigated as an in vivo delivery system for two neuroprotective proteins: erythropoietin (EPO) and fibroblast growth factor 2 (FGF2). Both proteins demonstrated a diffusive release profile in vitro and maintained significant bioactivity during release. When EPO was delivered intrathecally with HAMC to the injured spinal cord, reduced cavitation in the tissue and significantly improved neuron counts were observed relative to the conventional delivery strategies of intraperitoneal and intrathecal bolus. When FGF2 was delivered intrathecally from HAMC, therapeutic concentrations penetrated into the injured spinal cord tissue for up to 6 h. Poly(ethylene glycol) modification of FGF2 significantly increased the amount of protein that diffused into the tissue when delivered similarly. Because FGF2 is a known angiogenic agent, dynamic computed tomography was developed for small animal serial assessment of spinal cord hemodynamics. Following SCI and treatment with FGF2 from HAMC, moderate improvement of spinal cord blood flow and a reduction in permeability were observed up to 7 d post-injury, suggesting that early delivery of neuroprotective agents can have lasting effects on tissue recovery. Importantly, the entirety of this work demonstrates that HAMC is an effective short-term delivery system for neuroprotective agents by improving tissue outcomes following traumatic SCI.

Traumatic spinal cord injury (SCI) is a devastating condition for which there is no effective clinical treatment. Neuroprotective molecules that minimize tissue loss have shown promising results; however systemic delivery may limit in vivo benefits due to short systemic half-life and minimal passage across the blood-spinal cord barrier. To overcome these limitations, an injectable intrathecal delivery vehicle comprised of hyaluronan and methylcellulose (HAMC) was developed, and previously demonstrated to be safe and biocompatible intrathecally. Here, HAMC was determined to persist in the intrathecal space for between 4-7 d in vivo, indicating it as an optimal delivery system for neuroprotective agents to reduce tissue degeneration after SCI. HAMC was then investigated as an in vivo delivery system for two neuroprotective proteins: erythropoietin (EPO) and fibroblast growth factor 2 (FGF2). Both proteins demonstrated a diffusive release profile in vitro and maintained significant bioactivity during release. When EPO was delivered intrathecally with HAMC to the injured spinal cord, reduced cavitation in the tissue and significantly improved neuron counts were observed relative to the conventional delivery strategies of intraperitoneal and intrathecal bolus. When FGF2 was delivered intrathecally from HAMC, therapeutic concentrations penetrated into the injured spinal cord tissue for up to 6 h. Poly(ethylene glycol) modification of FGF2 significantly increased the amount of protein that diffused into the tissue when delivered similarly. Because FGF2 is a known angiogenic agent, dynamic computed tomography was developed for small animal serial assessment of spinal cord hemodynamics. Following SCI and treatment with FGF2 from HAMC, moderate improvement of spinal cord blood flow and a reduction in permeability were observed up to 7 d post-injury, suggesting that early delivery of neuroprotective agents can have lasting effects on tissue recovery. Importantly, the entirety of this work demonstrates that HAMC is an effective short-term delivery system for neuroprotective agents by improving tissue outcomes following traumatic SCI.

Rupture of the anterior cruciate ligament (ACL) of the knee leads to chronic joint instability and reduced range of motion while the long term results are marred by a high prevalence of degenerative joint disease especially osteoarthritis. Bundles of collagen threads have been widely investigated for the repair of torn ACL, but are limited by insufficient tissue ingrowth to repopulate and completely regenerate these grafts. We have developed a novel in vitro method of characterizing fiber-based thread matrices by probing their ability to promote tissue ingrowth from a wound margin as a measure of their ability to promote repopulation and regeneration. This method is useful in the optimization of thread scaffolds, and is sensitive enough to distinguish between subtle variations in biopolymer chemistry and organization. Furthermore, this method was used to characterize the effects of crosslinking on the cell outgrowth and correlated the findings with the mechanical properties of collagen threads. The results suggest that crosslinking is required to achieve sufficient mechanical properties for high stress applications such as ACL replacement, but regardless of technique, crosslinking attenuated the cell outgrowth properties of the threads. To improve the regenerative capacity of these scaffolds, novel fibrin microthread matrices were developed with a similar morphology to collagen threads and sufficient mechanical strength to be incorporated in composite thread scaffold systems. These fibrin microthreads were loaded with FGF-2, a potent mitogen and chemotactic agent that works synergistically with fibrin in regulating cell signaling and gene expression. Increases in fibroblast migration and proliferation in FGF-2-loaded fibrin threads were successfully demonstrated with the concomitant promotion of oriented, aligned, spindle-like fibroblast morphology. These results suggest that fibrin-FGF-2 microthreads have distinct advantages as a biomaterial for the rapid regeneration of injured tissues such as the ACL.

Gene therapy techniques delivering exogenous angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), are currently being investigated as potential treatments for ischemia resulting from a variety of conditions, such as peripheral artery disease (PAD) and chronic wounds. Despite these intense efforts, a viable clinical option to promote therapeutic neovascularization remains elusive. Electroporation is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues including skin and muscle. This study investigated intradermal injection of plasmids encoding angiogenic growth factors with electroporation as a novel therapeutic approach to increase perfusion in areas of ischemia. Two common animal models of ischemia were employed: a skin flap model, used to study wound healing, and a hindlimb ischemia model, used to investigate potential therapies for PAD. In the skin flap model, delivery of plasmid VEGF with electroporation significantly increased VEGF expression for 5 days after delivery compared to injection of the plasmid alone. While the increase in VEGF expression was short-term, it significantly increased expression of the downstream angiogenic growth factor endothelial nitric oxide synthase, as well as perfusion and healing in the distal area of the skin flap. To facilitate the translation of electroporation to the clinic, a novel electrode configuration was previously designed for cutaneous delivery of plasmids to a large surface area. The design of the Multielectrode Array allows for delivery to a large surface area without the need to increase the applied voltage. Conditions for plasmid delivery with this electrode were optimized and it was then utilized to deliver plasmid FGF-2 (pFGF) to the hindlimb ischemia model. FGF-2 expression, perfusion, and angiogenesis were assessed. FGF-2 expression was significantly higher for 10 days after treatment with pFGF with electroporation compared to injection of pFGF alone. This increase in FGF-2 expression induced a significant increase in perfusion and angiogenesis in the ischemic limb. The research presented here suggests intradermal injection of plasmids encoding angiogenic factors by electroporation is a novel potential therapeutic approach to increase perfusion to areas of ischemia and promote wound healing.

A mixed self assembling monolayer (mSAM) chip with attached heparin was developed to analyze heparin-protein interactions using a Reichert Inc, SR7000, surface plasmon resonance (SPR) instrument. The heparin was attached via streptavidin-biotin linkage where the streptavidin was covalently coupled to the mSAM and biotinylated heparin bound to it. These chips were then used to quantify the interactions of fibroblast growth factor-2 (FGF-2) with the surface bound heparin. Kinetic rate constants of association and disassociation were calculated. The association data of FGF-2 with heparin was fit to a single compartment, well-mixed model as the data did not exhibit mass transfer limitations. The results suggested that rebinding was prevalent and observed disassociation rates differed significantly in the presence of competing soluble heparin during disassociation. Our results indicate that the Reichert instrument and mSAM chips can be used to analyze heparin-protein interactions but that a careful protocol, outlined in this thesis, should be followed to obtain optimal data. / Master of Science

New biomarkers are needed to improve knowledge about pathophysiology, in order to provide earlier correct diagnosis and to follow disease progression of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). The aim of this thesis was to find new biomarkers for these diseases. First, increased serum levels and unchanged levels in postmortal spinal cord of vascular endothelial growth factor (VEGF) were demonstrated. VEGF was not detected in cerebrospinal fluid (CSF) in ALS. Second, increased levels of fibroblast growth factor 2 were found in the CSF and serum of ALS patients. Both studies used enzyme-linked immunoassays. Third, a proteomics method for CSF analysis was explored, based on tryptic digestion and subsequent separation and detection of the peptides by on-line liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. ALS-specific patterns were observed. Four out of five samples were correctly assigned, but no single protein biomarker could be identified. Fourth, [11C](L)-deprenyl-D2 (DED) positron emission tomography (PET) demonstrated increased retention in the pons and white matter in ALS. DED binds to monoamino oxidase B, which in the brain is primarily located in astrocytes. Thus evidence was provided that astrocytosis may be detected in vivo in ALS. Fifth, normal [11C]-PIB binding in five nondemented patients with PD was reported, in contrast to previous findings of increased retention in Alzheimer's disease reflecting amyloid aggregation. Finally, the combined use of fluorodeoxyglucose and L-[β 11C]-DOPA PET for the differential diagnosis of parkinsonian syndromes was evaluated. PET provided support for the clinical diagnosis in 62 out of 75 patients, and served to exclude suspected diagnoses in another five patients.

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 103 pages. Includes vita. Includes bibliographical references.