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Title: Neurite Extension in 3-dimensional space: engineering platform to study neurite guidance
Keywords: guidance, neural tissue engineering, microsphere, hydrogel, neurite extension, scaffold
Issue Date: 12-Dec-2011
Citation: CHEN WENHUI (2011-12-12). Neurite Extension in 3-dimensional space: engineering platform to study neurite guidance. ScholarBank@NUS Repository.
Abstract: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres, with properties such as injectibility and more efficient drug delivery properties, have important benefits for neural tissue engineering but have not been well studied. Thus, PHBV microspheres? suitability as neural tissue engineering scaffolds was investigated using PC12 cells, cortical neurons (CN), and neural progenitor cells (NPC) to cover a variety of neuronal types for different applications. Microspheres were fabricated using an emulsion-solvent-evaporation technique. DNA quantification, cell viability assays, and immunofluorescent staining were carried out. PC12 cultures on PHBV microspheres showed growth trends comparable to 2D controls. This was further verified by cell spread staining. Also, CN expressed components of the signalling pathway on PHBV microspheres, and had greater axon-dendrite segregation (4.1 times for axon stains and 2.3 times for dendrite stains) than on coverslips. NPC were also found to differentiate into neurons on the microspheres. These results indicate that PHBV microspheres are suitable scaffolds for neural tissue engineering. However, neurons on microspheres (neuron-microspheres) were isolated compared to well-integrated neural tissue. It was found that neurites bridged the microspheres but these connections were fragile. Thus, neuron-microspheres were encapsulated in a laminin-collagen hydrogel to promote and protect the bridging formations. Bridges were found across the continuous surface between microspheres in contact (surface bridges), and in the gel space between microspheres (suspended bridges). This neuron-microsphere-hydrogel construct increased the proportion of bridge-forming neurites by 31% as compared to neuron-microspheres alone. Furthermore, the neuron-microsphere-hydrogel was found to increase the proportion of suspended bridges by 3.5 times. The surface bridges were subsequently verified to form from neurites extending across continuous surfaces, and thus packing microspheres closer to generate more continuous surfaces increased surface bridging by 70%. However, seeding cells into the gel space did not increase the proportion of suspended bridges but this still increased the overall proportion of bridges by 21%. Images of neurites in the gel-space suggested these suspended bridges could have formed instead from neurites extending out of microsphere surfaces, into the gel-space, then onto other microspheres. Neurites in the gel-space resembled a key step here. Varying gel-space permissiveness increased such gel-space neurites by 36% which then increased bridging by 54%, verifying the hypothesis. Reducing microsphere permissiveness further increased bridging by 30%. Permissiveness-difference between the gel-space and microsphere was found to be driving bridging. There were also 8 times more non-bridging gel-space neurites when cells were seeded into the gel-space instead, demonstrating how permissiveness-difference guided neurites away from microspheres. Surface bridging involves neurites on microsphere surfaces while suspended bridging involves neurites in gel-space. This seemingly competing relationship was investigated and it was found that they could be enhanced simultaneously, although the latter can be further increased (46%) by reducing the former. This study showed PHBV microspheres' suitability for neural tissue engineering, improved bridging between microspheres, elucidated bridging mechanisms, further justified bridging study, introduced new perspectives such the need to minimise scaffold permissiveness instead, and uncovered injectible guidance cues in the form of continuous surfaces and permissiveness difference that are different from common preformed ones.
Appears in Collections:Ph.D Theses (Open)

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