Field of Interest
Engineers have historically served as the link between scientific theory and technological application. As a student engineer, I am deeply interested in applying those theories learned in the classroom to real life models for experimentation and implementation.
My interests in chemistry, physics, and molecular biology have lead me towards my current plan of study as an undergraduate at UConn with a dual de gree in Chemical Engineering and Biophysics. I have also always been attracted to scientific research and I am fortunate that UConn has made research as an undergraduate a possibility. I fell into Dr. Akiko Nishiyama’s neurobiology lab at the end of my freshman year and have been working there since. My father is a neurologist and likewise neurology and neurobiology have always been a part of my life to some extent. It is only until recently that I have been able to pursue on my own those concepts that intrigued me as a child.
Personally, I feel that there is no other area of research that pulls from so many other scientific disciplines, such as biology, chemistry, physics, medicine, engineering, and philosophy. To a young and hungry mind neurobiology is an academic delight. For me, the real intrigue of neurobiology research lies in this multidisciplinary nature. A single research problem in neuroscience could be approached from any of these perspectives. In this sense I find engineering and neurobiology to be very similar. Both fields require a concrete foundation in several areas of science combined with the ability to draw on any or all of these sources to solve the problem at hand.
As vague as my future is at this stage of my life, I am quite certain that I want to pursue a Ph.D. in some area of biotechnology, particularly if it involves neuroscience. As a University Scholar, I feel I would be better prepared for advanced studies in these fields by the coursework and research I hope to add to my current undergraduate curriculum.
Project Proposal
Spinal Cord Regeneration Through Biodegradable Polylactic Acid Foams:
Analyzing the Regenerative Capacity of NG2 Cells In Vivo
Introduction:
To this day, trauma to the central nervous system (CNS) remains an irrevocable injury. Nerve damage in the CNS cannot be repaired by the body alone and requires external assistance for functional recovery (Friedman 2002). This is a marked difference when compared to the peripheral nervous system (PNS). Injured neurons in the PNS are capable of regeneration and synaptic reformation over small gaps across a lesion site (Battison 2000). In the CNS however, axonal regeneration is severely limited. Since CNS neurons have been shown to possess the inherent capacity to regenerate outside the body, it is suspected that the hostile CNS lesion environment is the main reason for these regenerative discrepancies (Grimpe 2002). In order for recovery to be made in the CNS after injury, it is necessary to overcome the inhibitory effects of the lesion scar and motivate damaged neurons to re-grow.
Spinal cord injury (SCI) is one of the most studied insults to the CNS. Approximately 10,000 Americans are victim to SCI each year. The results are lifelong nerve damage and functional loss below the level of injury (Kirshblum 1998). Even with all the research efforts to provide an efficient therapeutic strategy, there has been little progress in SCI repair (Xiang S 2005). The current procedures used today are mainly focused on preventing further injury from scar tissue and encourage only modest recovery (Friedman 2002). It is imperative that novel approaches to SCI repair are explored so that these difficulties can be overcome.
My goal as a University Scholar is to develop a unique model for SCI repair. This strategy will take advantage of previously described polymer scaffolds as a support structure for a unique cell type, the NG2 cell, that has shown promise for promoting neuron outgrowth in vitro. This cell-seeded scaffold will be transplanted into an injured rat spinal cord and its regenerative capacity will be characterized.
NG2 Cells:
The NG2 protein is a chondroitin sulfate proteoglycan present on the surface of a certain population of non-neuronal cells in the CNS. These NG2 cells have been shown to differentiate into oligodendrocytes in vitro (Nishiyama 1999; Wilson 2003) and in vivo (Watanabe 2002), and therefore have been termed oligodendrocyte progenitor cells (OPCs). Mature oligodendrocytes are responsible for myelinating CNS axons and thus these cells and their progenitors have received much attention from groups working to cure demyelinating diseases such as multiple sclerosis (Nishiyama 2002).
Preliminary research on NG2 cells focused around the NG2 protein itself. Experiments have shown NG2 to be inhibitory to growing neurons in vitro. There is also a large upregulation of NG2 protein in the hostile CNS lesion scar as well. From these tests, NG2 has been classified as an inhibitory molecule to neuron growth. Many researchers have extended this association of apparent negative effects of NG2 on neurons to NG2 cells as well. However, no direct in vivo evidence for this conclusion has been shown (Nishiyama 2002).
Recently, much work has been done to characterize NG2 cells and their role in the mature and immature CNS. Dr. Nishiyama has been intimately involved in this entire process and her findings have raised some interesting questions. For one, Dr. Nishiyama has found that NG2 cells actually promote neurite extension in vitro. Cultured neurons grew along the processes and cell body of NG2 cells and were encouraged to extend longer axons when compared to controls. Electron microscopy in vivo also revealed that NG2 cells and neurons formed physical membrane-to-membrane contacts suggesting similar NG2-neuronal interactions could be present (in publication). To extend these findings, it must be shown that NG2 cells exhibit the same growth promoting behavior in vivo as in vitro. I hope to demonstrate this characteristic of NG2 cells in the injured spinal cord with the objective of encouraging SCI repair. This is the main goal of my University Scholar proposal.
Polymer Scaffolds and Polylactic Acid:
Tissue engineering has recently emerged as a powerful field of research. The purpose of tissue engineering is to encourage cells to grow into functional tissue by guiding this growth with a three dimensional structural support and supporting cell growth with chemical cues. The engineered tissue is then transplanted into a host to replace lost or damaged cells with the intent of regaining functional activity of the injured tissue or organ (Zhang 2005).
The techniques of tissue engineering have been applied to several disciplines, including neuroscience. Much work has been performed to establish a polymer scaffold capable of guiding regenerating neurons across a lesion site. The most successful of these polymer strategies have been those implemented in the peripheral nervous system (PNS). Scaffolds in the PNS offer a clinical strategy for overcoming PNS nerve injury. However, similar nerve guidance scaffolds have been not as successful in the CNS (Hudson 2000). Unique models must be developed and tested for the CNS if recovery is to be accomplished.
As a University Scholar, I will design such a lesion model in the mouse spinal cord. This model will take advantage of a previously describe polylactic acid macroporous scaffold that will be seeded with a high density of NG2 cells on the surface and throughout the highly porous three dimensional internal structure. Polylactic acid (PLA) is a biodegradable polymer made up of lactic acid subunits. PLA slowly hydrolyzes over time in vivo releasing lactic acid which safely enters the metabolic cycles of nearby cells. It has been approved by the FDA as a biomaterial in surgical procedures along with many other uses (Friedman 2002). For these reasons, PLA is a strong polymer candidate for an in vivo tissue.
Simple three dimensional macroporous foams have been created out of PLA using a simple solvent-casting/particle-leaching procedure (Mikos 2000). In this method, PLA is dissolved in an appropriate solvent and poured over a mold filled with fine particles of salt. The solvent is allowed to evaporate and the PLA hardens encapsulating the salt. This composite is then washed in water for an extended period of time. The water dissolves the salt but not the PLA leaving interconnected internal pores within the polymer. When all the salt has been dissolved, cells can be grown on these polymer constructs and then transplanted into various lesion sites to witness neuron ingrowth. For my project, I will seed this scaffold with NG2 cells. My preliminary work has shown that NG2 cells are capable of growing on two-dimensional PLA films while extending branched processes indicative of a healthy culture.
Once the scaffold has been successfully seeded throughout, it will be transplanted into a spinal cord hemisection in the mouse. In this procedure, half of the spinal cord is removed leaving a gap of neural tissue 2-3 mm long that is isolated on one side of the cord. This gap will be replaced with a similar shaped section of polymer foam seeded with NG2 cells. The hemisection provides many locations of spinal cord contact with the scaffold so as to allow many points of nerve innervation. Progress of neuronal regeneration as compared to controls will be tracked, as well as direct visualization of NG2/neuron interactions in vivo. For the first time, I will be able to show whether or not Dr. Nishiyama’s in vitro experiments of NG2 cells promoting neuronal growth are representative of NG2 cells in vivo. Further, I will develop and refine a novel model for repairing spinal cord injury that, if successful, will open up many more avenues for SCI regenerative research.
Project Summary:
In order to accomplish this University Scholar project, I will work closely with Dr. Nishiyama to successfully harvest and seed NG2 cells for transplantation as well as performing the actual surgeries and data collection. I will also work with Dr. Richard Parnas from Chemical Engineering to refine the three-dimensional polymer scaffold to optimize its characteristics for cell growth and neuronal support. I plan to successfully develop such a scaffold for in vitro use by the end of this spring and to focus the remainder of my University Scholar research on implementing and analyzing the transplant in vivo. If time permits, I will try to maximize the regenerative capacity of the model to facilitate the greatest functional recovery possible.
My University Scholar project will also double as my honors thesis for Chemical Engineering. Funding for any needed materials, as well as access to a laboratory, will be provided by Dr. Nishiyama. I have been approved to work with the mice needed for this experiment and the protocol briefly described above has also been approved (#A04-185).
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