Cell instructive biointerfaces are versatile tools to mimic a natural cellular environment and control cell fate in vitro. The particular interest lies in combining information gained from surface and interface analysis tools with biological analysis to explore and understand fundamental processes such as neuronal stem cell differentiation at the biointerface. A major challenge in biointerface design is to mimic and study the complex interactions of the natural processes in the extracellular matrix (ECM) with artificially designed surfaces and interfaces. In the past, peptide surfaces have been used as ECM mimics, however, more research is required in this field to tune the properties of peptide surface to modulate the outcome of stem cell fate. The present work aims to address this challenge by designing new synthetic peptide surfaces with well controlled composition and functionality able to impart have control over the differentiation of neuronal stem cells with the ultimate goal to relate surface properties and stem cell response to understand and control how neuronal networks function. Compositionally well-defined surfaces of two short laminin peptide sequences, Arg-Gly-Asp (RGD) and Ile-Lys-Val-Ala-Val (IKVAV) were prepared of various ratios via the “grafting from” stepwise approach. The surface modification was confirmed with surface analysis techniques to indicate successful peptide functionalization. The neural progenitor stem cells (NPSCs) were set up from embryonic rat hippocampi (E18). Immunocytochemistry (ICC) observed cell viability and differentiation to specific NPSCs lineages for βIII-Tubulin and GFAP. The surface characterizing techniques of WCA, AFM, ToF-SIMS, and XPS validated the successful orthogonal functionalization of controllable peptides composition surfaces with the increase of RGD composition a relative decrease in the IKVAV composition was observed. The increase in the normalized total ion fragments of RGD in the ToF-SIMS measurements can be related linearly to the % area coverage of neurons versus astrocytes observed on the controllable peptide composition surfaces. Well-defined peptide surfaces were designed and successfully demonstrated that the amount of RGD peptide composition present on the surface influences cell adhesion, proliferation and differentiation to a desirable cell fate or controllable cell population (i.e. neurons and astrocytes). Recent technological developments demonstrate a shift from two-dimensional (2D) surfaces to three-dimensional (3D) surfaces, so we used the designed versatile 2D surfaces as the template to design comparable 3D surfaces to examine the biological response of NPSCs to both microenvironments. This study proposes the design of novel 3D nanoparticles (NPs) made of gold and surface-conjugated with differentiation-inducing peptides. The NPs-peptides will guide stem cell differentiation to neurons that self-aggregate around the NPs by cell-cell contacts to form neurospheres. The neural stem cells will establish 3D structures biomimicking the cytoarchitecture of the brain. Successively, they could be used as an alternative 3D in vitro model for neurotoxicity testing of drugs and chemicals. Size-controllable NPs will be surface-conjugated with RGDC and IKVAVC(19) peptides via the SPPS “grafting to” approach. Two different sizes of NPs were characterized as AuNPs and SiO2@AuNPs characterization and validated by TEM, DLS, -potential, EDX; and the surface functionalization on NPs was successfully confirmed by UV/vis spectroscopy and -potential. The NPSCs were set up from E18 rat hippocampi and cell viability and differentiation to specific NSPCs lineages was stained for βIII-Tubulin and GFAP. AuNPs and SiO2@AuNPs peptide immobilized surfaces supported cell adhesion, proliferation, and survival. The confocal light scanning microscope (CLSM) images indicate that the RGDC functionalized AuNPs and SiO2@AuNPs surfaces induced a preferential differentiation towards a neurons cell fate and the IKVAVC(19) functionalized surfaces of AuNPs and SiO2@AuNPs favored an astrocytes cell. The cellular uptake of functionalized AuNPs by the cells in the neurospheres was observed via TEM micrographs, whilst the micrographs of functionalized SiO2@AuNPs surfaces suggest that they were not taken-up into the cells. Hence, indicating that there is a difference in the cellular uptake mechanism for the functionalized AuNPs and SiO2@AuNPs, which might be due to the agglomeration of the nanoparticles than the individual size of the nanoparticles. In conclusion, the relation between the 2D and 3D surfaces may provide new insight in understanding how surface properties affect the regulation of physiological relevance in directing neural cell differentiation, which will be essential to understand how neural networks function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:757578 |
| Date | January 2018 |
| Creators | Dhowre, Hala Shakib |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/53315/ |
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