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- Nanoengineered tissue scaffolds for regenerative medicine in neural cell systems
- Tiryaki, Volkan Mujdat
- Electronic Theses & Dissertations
Central nervous system (CNS) injuries present one of the most challenging problems. Regeneration in the mammal CNS is often limited because the injured axons cannot regenerate beyond the lesion. Implantation of a scaffolding material is one of the possible approaches to this problem. Recent implantations by our collaborative research group using electrospun polyamide nanofibrillar scaffolds have shown promising results in vitro and in vivo. The physical properties of the tissue scaffolds have...
Show moreCentral nervous system (CNS) injuries present one of the most challenging problems. Regeneration in the mammal CNS is often limited because the injured axons cannot regenerate beyond the lesion. Implantation of a scaffolding material is one of the possible approaches to this problem. Recent implantations by our collaborative research group using electrospun polyamide nanofibrillar scaffolds have shown promising results in vitro and in vivo. The physical properties of the tissue scaffolds have been neglected for many years, and it has only recently been recognized that significant aspects include nanophysical properties such as nanopatterning, surface roughness, local elasticity, surface polarity, surface charge, and growth factor presentation as well as the better-known biochemical cues.The properties of: surface polarity, surface roughness, local elasticity and local work of adhesion were investigated in this thesis. The physical and nanophysical properties of the cell culture environments were evaluated using contact angle and atomic force microscopy (AFM) measurements. A new capability, scanning probe recognition microscopy (SPRM), was also used to characterize the surface roughness of nanofibrillar scaffolds. The corresponding morphological and protein expression responses of rat model cerebral cortical astrocytes to the polyamide nanofibrillar scaffolds versus comparative culture surfaces were investigated by AFM and immunocytochemistry. Astrocyte morphological responses were imaged using AFM and phalloidin staining for F-actin. Activation of the corresponding Rho GTPase regulators was investigated using immunolabeling with Cdc42, Rac1, and RhoA. The results supported the hypothesis that the extracellular environment can trigger preferential activation of members of the Rho GTPase family, with demonstrable morphological consequences for cerebral cortical astrocytes. Astrocytes have a special role in the formation of the glial scar in response to traumatic injury. The glial scar biomechanically and biochemically blocks axon regeneration, resulting in paralysis. Astrocytes involved in glial scar formation become reactive, with development of specific morphologies and inhibitory protein expressions. Dibutyryl cyclic adenosine monophosphate (dBcAMP) was used to induce astrocyte reactivity. The directive importance of nanophysical properties for the morphological and protein expression responses of dBcAMP-stimulated cerebral cortical astrocytes was investigated by immunocytochemistry, Western blotting, and AFM. Nanofibrillar scaffold properties were shown to reduce immunoreactivity responses, while PLL Aclar properties were shown to induce responses reminiscent of glial scar formation. Comparison of the responses for dBcAMP-treated reactive-like and untreated astrocytes indicated that the most influential directive nanophysical cues may differ in wound-healing versus untreated situations.Finally, a new cell shape index (CSI) analysis system was developed using volumetric AFM height images of cells cultured on different substrates. The new CSI revealed quantitative cell spreading information not included in the conventional CSI. The system includes a floating feature selection algorithm for cell segmentation that uses a total of 28 different textural features derived from two models: the gray level co-occurance matrix and local statistics texture features. The quantitative morphometry of untreated and dBcAMP-treated cerebral cortical astrocytes was investigated using the new and conventional CSI, and the results showed that quantitative astrocyte spreading and stellation behavior was induced by variations in nanophysical properties.
- Biomaterial and genetic tools to influence neuronal network formation, excitability, and maturity at the electrode interface
- Setien-Grafals, Monica B.
- Electronic Theses & Dissertations
Understanding brain function remains a grand challenge of our time. Likewise, when neurodegeneration occurs, repair efforts are limited due to the highly heterogeneous and interconnected nature of the cerebral cortex. The drive to better understand normal brain function and pathological states has intensified demand for new technologies which can interrogate the nervous system with enhanced spatiotemporal resolution. Implanted brain electrodes are being used and developed to provide a deeper...
Show moreUnderstanding brain function remains a grand challenge of our time. Likewise, when neurodegeneration occurs, repair efforts are limited due to the highly heterogeneous and interconnected nature of the cerebral cortex. The drive to better understand normal brain function and pathological states has intensified demand for new technologies which can interrogate the nervous system with enhanced spatiotemporal resolution. Implanted brain electrodes are being used and developed to provide a deeper understanding for neurological injury and neurodegeneration. However, issues with biological integration come into play and potentially interfere with signal stability over time. Here, this work provides innovative tools that can be used to interface and control the tissue-electrode interface. In particular, we are interested in exploring surface chemistries, genetic tools, and electrode materials which favor neural regeneration around implanted electrodes. The research presented in this dissertation describes the exploration of biomaterial and genetic tools for interfacing the tissue-electrode interface: (1) characterization of surface chemistries presented to differentiating neural progenitors, and an understanding of the conditions which promote neurite outgrowth and electrophysiological maturation, (2) a blue-light inducible gene expression system, which could potentially be used to control gene expression at the implanted electrode interface, and (3) testing the impacts of "next-generation" electrode materials, such as diamond, as candidates for neural interfacing. Chapter 2 uncovers the study of various common substrates and their effects on rat neural progenitor cells, which can be used to create unique morphologies. Chapter 3 explores the use of an optogenetic system from a bacterial transcription factor (EL222) that allows for blue light-dependent transcriptional activation. Here, we validated the use of EL222 for spatial patterning of fluorescent reporter genes and developed stable expression in HEK293 cells, which can be used long-term for developing approaches for light-driven regeneration of neural circuitry. Chapter 4 reveals material and genetic factors that can affect cell structure and function. Here, we report the results of an initial characterization of the biocompatibility of the novel diamond-based materials, including conductive boron-doped polycrystalline diamond (BDD) and insulating polycrystalline diamond (PCD). The results presented will inform the transfer of the novel diamond substrate materials to sensing applications in the in vivo environment, where we expect to leverage the positive performance characteristics of the diamond materials displayed in vitro. Taken together, these chapters offer significant development of material and biological tools and that will help manage and mitigate challenges presented at the tissue-electrode interface. Future directions aim at exploring synergistic effects of electrode material and optogenetic control for controlling excitability and identity of cells at the interface, effectively bridging the divide between electronics and tissue.