Adult central nervous system tissue does not retain the ability to regenerate and restore functional tissue lost to disease or trauma. The peripheral nervous system only has the capacity to regenerate when tissue damage is minor. Most in vitro research investigating the neurobiological mechanisms relevant for enhancing nerve regeneration has focused on culture of neuronal cells on a 2D surface under static conditions. We have performed studies enabling development of an advanced in vitro culture model based on hollow fiber-based bioreactors to allow high density neuronal cell networking with directed axonal outgrowth.
The model neuronal-like PC12 cell line was initially used to compare neurite outgrowth after nerve growth factor stimulation between cultures under either static or dynamic conditions with 2D or 3D configurations. High density PC12 cell cultures with extensive neurite outgrowth in three dimensional collagen gels were only possible under the dynamically perfused conditions of a hollow fiber-based bioreactor. Analysis of neurite networking within cultures demonstrated enhanced active synapsin I+ synaptic vesicle clustering among PC12 cells cultured within the 3D dynamic bioreactor compared to cells cultured statically on a 2D surface. We further used two different hollow fiber-based bioreactor designs to investigate primary mouse neural stem cell differentiation within different injectable extracellular matrix hydrogel scaffolds cultured under dynamic conditions. HyStem, a cross-linked hyaluronan hydrogel, allowed structure formation with improved neuronal differentiation compared to collagen and Matrigel hydrogels.
We have made further developments in order to create a new hollow fiber-based bioreactor device for controlling directed axonal growth. Excimer laser modification was utilized to fabricate hollow fiber scaffolds allowing control over axonal outgrowth from neurons within a 3D space. Incorporation of these scaffolds into a novel hollow fiber-based bioreactor design will produce a device for high density neuronal tissue formation with axonal outgrowth in a 3D configuration. Such a device will provide an advanced research tool for more accurate evaluation of neurobiological events and development of therapeutic strategies useful for enhancing nerve regeneration.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-11102008-160024 |
| Date | 28 January 2009 |
| Creators | Brayfield, Candace A |
| Contributors | John P. Leonard, Jorg C. Gerlach, Tracy X. Cui, Kacey G. Marra |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-11102008-160024/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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