Development and utilisation of biofunctionalised scaffolds to support neural stem cells in vitro, in the intact brain and during neural regeneration
thesisposted on 2017-02-27, 03:19 authored by Wang, Ting-Yi
Currently there is no effective treatment for injuries to the central nervous system. Cell replacement therapy has been investigated for a number of neural injuries/neurodegenerative disease and in some instances led to clinical trials. However, the effectiveness of the treatment, most notably observed in the context of replacing dopamine neurons in Parkinson’s disease patients, has been highly variable. In part, this has been attributed to: (i) poor cell survival and (ii) poor integration of the grafted cells into the host tissue. It is likely that the non-conducive environment of the adult brain is partially responsible for these short comings and that providing an improved niche environment, enriched with chemical and physical support for newly implanted cells, could have a significant impact on transplantation outcomes. In this regard, this thesis examines the potential of bio-engineered scaffolds to support neural cells in vitro and subsequently provide a stimulating micro-environment to satisfy both physical and biological needs for grafted cells in vivo. Electrospun scaffolds possess many features reminiscent of the brains extracellular matrix and were therefore examined their ability to support of neural cells in vitro and in vivo (chapter 4, 5 and 6). The scaffolds were additionally chemically modified (with neurotrophic factors) to maximise their bio-functionality. Whilst previous studies have demonstrated the benefits of covalently tethered proteins onto scaffold to prolong exposure, little attention has been paid to the stability and functionality of these proteins. Chapter 4 demonstrates long-term stability of glial-cell derived neurotrophic factor (GDNF), its maintained ability to activate intracellular signalling pathways and, its ability to influence cellular responses (survival, differentiation and neurite growth). Prior to examining the ability of electrospun scaffolds to support neural transplants, methodologies were established to determine how best to introduce these scaffolds, so as to support the graft (chapter 5). The results showed that cells implanted adjacent to the electrospun scaffold were superior to efforts of implanting scaffolds pre-seeded with neural cells or efforts to implant cells into the cavity of the scaffold in vivo. The data illustrated that Poly(ε-caprolactone) (PCL) scaffolds supported graft survival and neurite penetration inside implanted scaffolds. Subsequently, a more extensive evaluation of the ability of electrospun scaffolds, incorporating tethered GDNF, to support neural cells was performed (Chapter 6). In vitro, PCL with immobilised GDNF (iGDNF) significantly enhanced cell viability and neural stem cell/progenitor proliferation compared to conventional 2-dimensional cultureware. Upon implantation into the intact brain of rats, PCL scaffolds including iGDNF enhanced the survival, proliferation, migration, and neurite growth of transplanted cortical cells, whilst suppressing inflammatory reactive astroglia in the comparison with unmodified PCL scaffolds and cell transplantation alone. The results illustrate the potential of biofunctionalised scaffolds to support neural grafts, findings that could have a significant impact on promoting regeneration in the injured brain. Finally this thesis examines the potential of scaffolds to support transplanted cells in an animal model of neural injury (a Parkinson’s disease model). Whilst chapter 6 explore the potential of electrospun scaffolds for supporting grafts, the final results chapter (Chapter 7) concentrates on developing a more advanced bio-engineered scaffold that is less invasive for implantation. A composite scaffold that could be easily injected into the brain was fabricated by combining a hydrogel with bio-functionalised electrospun short fibres. The composite scaffolds modified with GDNF were shown to significantly promote cell viability as well as the differentiation and neuritogenesis of dopaminergic neurones (the cell population requiring replacement in Parkinson’s disease) in the comparison with other scaffolds. After 28days in vivo, the composite scaffolds were demonstrated to maintain the survival and integration of transplanted dopamine neurone. The tethering of GDNF onto the short fibres in the composite scaffolds was shown to also suppress microglia activation when compared to the scaffolds without GDNF. Collectively, this thesis makes a significant contribution to understanding the potential of biofunctionalised scaffolds to support neural stem cells in vitro and in vivo, and may have important implications in the future for the development of cell based therapies for the treatment of neural injuries.