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"A Novel Multifunctional Microsphere Scaffold to Deliver Neural Precursors for Traumatic Brain Injury Repair"

by
Nolan Skop
Biomedical Engineering Program
B.S. 2007, University of Connecticut


Thesis Advisors:
Steven Levison, Ph.D.
Professor, Neurology & Neurosciences Dept. (NJMS)
Chirag Gandhi, M.D.
Associate Professor, Neurological Surgery Dept. (NJMS)
Cheul Cho, Ph.D.
Assistant Professor, Biomedical Engineering Dept. (NJIT)

Wednesday, November 6, 2013
1:00 P.M., MSB C-600


Abstract

Advances in scientific research have improved the possibility of functional recovery from traumatic brain injuries (TBI). Regenerating the central nervous system (CNS) has become more feasible with the growing field of stem cell therapies. Stem cells have the potential to replace or regenerate neurons, which lack the capability of undergoing mitosis, and are destroyed during severe head injuries.

Still several problems exist that limit the utility of these cells; namely the source of neural precursors (NPs) for such therapies and the harsh environment produced as a consequence of head injuries. The majority of cells in the embryonic ventricular zone (VZ) are primitive cells (known as radial glia). They extend their processes across the width of the developing central nervous system ultimately generating the pyramidal neurons and glia of the neocortex during development. . Transplanting radial glia with a survival factor, fibroblast growth factor-2 (FGF-2), may increase their chances of engrafting. When present, FGF-2 prevents their differentiation and less differentiated cells require fewer trophic factors, further increasing their potential to repopulate a damaged brain.

Previous studies have shown that only a few percent of the transplanted NPs survive. This may be due to the harsh inflammatory environment created by the injury. Furthermore cell transplantation without a tissue-engineered matrix does not provide the cells with a supporting substrate to survive. Therefore, the goal of this thesis, was to fabricate a novel biomaterial scaffold that would improve the engraftment and survival of NPs transplanted into the brain after a TBI.

To create a vehicle for cell transplantation, a 3% chitosan solution was electrosprayed into a coagulation bath to generate microspheres (range: 30-100Ám, mean: 64Ám). Heparin, which binds FGF-2 with high affinity while retaining its biological activity, was covalently cross-linked to the chitosan scaffolds using genipin. At 1 g/mL approximately 80% of the FGF-2 bound to the scaffold. The MTT assay and microscopic analysis revealed that the scaffold containing tethered FGF-2 sustained survival and growth of NPs compared to standard culture conditions. Fetal rat NPs plated onto this multifunctional film proliferated and remained multipotent for at least 3 days without providing soluble FGF-2. Moreover, they remained less mature and more highly proliferative than cells maintained on fibronectin-coated substrates in culture medium supplemented with soluble FGF-2.

NPs from the postnatal subventricular zone (SVZ) were compared to NPs from the VZ of fetal rats. Upon differentiation in vitro the VZ cells generated a greater number of neurons than SVZ cells. Furthermore, differentiated VZ cells generated pyramidal neurons positive for laminar cortical neuron markers Reelin, Cutl1, Tle4 and FoxP2. By contrast, SVZ cells generated interneurons that expressed calretinin and calbindin and very few cells that expressed pyramidal neuron markers. Accordingly VZ cells were selected for subsequent experiments to test their capacity to regenerate the neocortex after a concussive brain injury.

Microspheres, containing adherent VZ cells, that expressed green fluorescent protein (GFP), were injected subacutely (7 days post injury) into the lesion cavity of adult rats that had previously sustained controlled cortical impact (CCI) injuries. Three days after transplantation, the GFP+ cells were positive for the stem cell/progenitor markers Nestin and Brain Lipid Binding Protein (BLBP) as well as for the proliferative marker Ki67. At 2 weeks post-transplantation, the GFP+ cells showed a reduction in stem cell/progenitor markers compared to 3 days, having acquired doublecortin (DCX), Vimentin, and Oligodendrocyte Lineage Transcription Factor 2 (Olig2), markers indicative of maturation towards neuronal, astrocytic and oligodendrocytic lineages respectively.
Altogether, these results demonstrate that seeding VZ cells on a chitosan-based multifunctional microsphere scaffold is an effective method for transplanting NPs to replace lost neurons after TBI. We have developed a new transplantation paradigm to treat TBI injuries with the objective of reforming a proliferative ventricular zone, ultimately leading to regeneration of a laminated neocortex, de novo.


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