Scaffolds biofunctionalized with polyelectrolyte multilayers for regeneration of the central nervous system

This thesis focuses on the biofunctionalization of polymer scaffold surfaces using the Layer-by-Layer (LbL) deposition technique, where different cues (physical, chemical and biological) could be incorporated to improve cell-scaffold interactions. Cellular responses such as cell adhesion, cell migration, neurite outgrowth could be effectively controlled using this technique. The long-term goal of the study was to develop biomaterials for central nervous system (CNS) regeneration, able to restore neuronal function following neurodegeneration or trauma by providing complex synthetic micro-environments for both endogeneous and transplanted cells as tissue engineering scaffolds. In the first part of the thesis, LbL deposited heparin and poly-L-lysine (PLL) polyelectrolytes were studied on 2D polycaprolactone surfaces, where the deposition process was monitored in situ and the properties of the resulting polyelectrolyte mutilayers (PEMs) were characterized and confirmed by various techniques. Cell adhesion and neurite outgrowth were then studied on heparin and PLL terminating PEM surfaces and was compared to unmodifed PCL surfaces. In addition, brain-derived neurotrophic factor (BDNF) was then adsorbed onto PEM surfaces to provide further neurotrophic support to cultured cells. These combined chemical and biological cues were then evaluated in terms of neurite length and mRNA expression of BDNF receptor genes and regeneration associated genes. For the second part of the thesis, the LbL deposition technique was extended to biofunctionalize electrospun nanofibres of complex 3D structure. The effects of the combined chemical (cell adhesive / non-adhesive), physical (random/aligned 3D nanofibre substrates) and biological (adsorbed/immobilized BDNF) features on neurite extension and gene expression were studied. The results from these studies therefore offer strategies for LbL functionalized scaffolds to achieve CNS regeneration. In this chapter, electrospinning conditions were explored to obtain optimal nanofibre properties (e.g. diameter, uniformity and alignment). Additionaly, both physically adsorbed and immobilized BDNF of different concentrations on heparin and PLL surfaces were employed to provide localized neurotrophic support to cultured cells, and the combined cues on neurite length and gene expression were evaluated. In the third part of the thesis, hollow PEM fibres were investigated as a variation on complex 3D scaffolds. The hollow PEMs either maintained the electrospun nanofibre morphology or were used as short hollow fibres incorporation with a self-assembling tripeptide hydrogel, which is not reported in the literature. To prepare these two types of PEM scaffolds, electrospun nanofibre PCL and microfibre silica templates were used respectively. The sol-gel and electrospinning conditions for the latter were studied and optimized. Moreover, short silica microfibres with controlled length were achieved in this work via sonication-induced scission. The self-assembly of tripeptide hydrogels catalyzed by enzymes adsorbed on these short PEM fibres was studied and provides an insight into the application of injectable hydrogel composites for in vivo purposes. Since nervous system function essentially operates via complex ion transport events, similar to an electrical circuit, bioactive and electrically conductive graphene-PEM coatings on electrospun nanofibres were also investigated. Electrical conductivity of the fibres was achieved by incorporation of graphene into the PEM coating. The graphene distribution, surface morphology and chemistry were characterized in this chapter. Sheet resistance, a critical parameter of the graphene-PEM functionalized nanofibres was also studied, where fibre alignement, LbL deposition conditions and post-annelaing treatments were found important to the electrical properties of the graphene-PEM scaffolds. In the last chapter, the PEM, graphene-PEM functionalized microfibres (PLL terminating heparin terminating) of both random and aligned (graphene-PEM functionalized) fibre orientations were implanted into the rat brain to evaluate the scaffold-tissue interactions in vivo. Cellular infiltration of the scaffolds by endogeneous astrocytes and neurons was assessed for all implanted scaffolds, and the conductive graphene-PLL terminating scaffolds was found to be highly effective allowing cellular migration, which provided novel results and valuable information on using electroactive scaffolds for CNS regeneration processes.