Abstract
Stroke is a devastating disease that affects millions of people every year worldwide. For those who survive after stroke, many live with lasting impairments in sensorimotor and cognitive functions. Unfortunately, to date, no regenerative therapies are available that can restore the lost functions after stroke. Therefore, it is necessary to find therapeutic interventions to promote recovery of the damaged brain following stroke.
Biopolymers are macromolecules that are either derived from living tissue or chemically synthesised to interact with biological systems for therapeutic or diagnostic purposes. In recent years, delivery of biopolymer hydrogels directly into the stroke cavity has been shown to remodel the hostile stroke microenvironment and enable persistent release of drugs and neurotrophic factors to the peri-infarct tissue surrounding the fully formed stroke. However, no biopolymer hydrogel has yet been approved for clinical trials in stroke patients. Successful clinical translation of biopolymer hydrogel-based treatments requires robust tuning of physicochemical properties and biodegradation kinetics to meet the specific requirements of the human stroke microenvironment. The overall aim of this thesis was to develop and characterise a novel thermoresponsive biopolymer hydrogel for brain tissue regeneration following stroke. Specifically, we aimed to develop and characterise a novel hydrogel by blending multiple polymers and evaluate the hydrogels with or without neurotrophic factor combinations to modulate reactive astrogliosis, inflammation, neurogenesis and functional recovery following stroke.
We aimed to investigate the physicochemical properties including temperature-induced gelation, shear thinning, morphology, and biodegradation of chitosan (CS) / β-glycerophosphate (β-GP) hydrogels, consisting of 0.5 – 2% CS and 2 – 3% β-GP, in order to assess their suitability for injectable neural tissue engineering application. Furthermore, we tested the in vitro biocompatibility of CS/β-GP hydrogels using PC12 cells, an immortalised cell line with neuronal cell-like properties. The resulting CS/β-GP hydrogels showed a varying range of gelation temperatures and gelation times depending on their CS / β-GP blend ratio. Among those tested, 0.5% CS / 3% β-GP and 0.75% CS / 3% β-GP formulations underwent rapid gelation at physiological temperature (37°C) and pH (7.4). In addition, both formulations showed shear thinning properties, porous microstructure, biodegradation, and in vitro biocompatibility, which are crucial for injectable tissue engineering application. These two CS/β-GP hydrogels were then blended with silk fibroin (SF), polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) in order to prepare hybrid hydrogel formulations. The physicochemical properties of the hybrid hydrogels, including gelation behaviour, morphology, biodegradation rate, and in vitro cytotoxicity were assessed. Based on the results of physicochemical characterisations and in vitro cytotoxicity, four hybrid hydrogel formulations, F1, F2, F3 and F6, were tested in vivo to determine their effect on post-stroke reactive astrogliosis, inflammation and neurogenesis in a mouse model of photothrombotic stroke. Interestingly, treatment with F1, F2, and F6 hydrogels decreased reactive astrogliosis in peri-infarct tissue and increased neurogenesis in the ipsilateral subventricular zone. In addition, both F2 and F6 hydrogels decreases inflammation in peri-infarct tissue.
Considering the results of in vitro and in vivo assessments, F6 hydrogel was then chosen to combine with two neurotrophic factors, brain-derived neurotrophic factor (BDNF) and liposome-encapsulated vascular endothelial growth factor (VEGF) to assess their therapeutic potential on behavioural function, reactive astrogliosis, inflammation, neurogenesis, and angiogenesis following photothrombotic stroke to the motor cortex in mice. Intracerebral administration of F6 + BDNF and F6 + VEGF either alone or in combination (F6 + BDNF + VEGF) improved motor function on both the grid-walking, and cylinder tasks. However, the combined delivery of BDNF and VEGF from F6 hydrogel improved motor function more rapidly and significantly than F6 + BDNF or F6 + VEGF alone. In addition, treatment with F6 hydrogel with or without neurotrophic factors decreased reactive astrogliosis (both 2- and 8-weeks post-stroke) and inflammation (2 weeks post-stroke) in peri-infarct tissue. Furthermore, administration of F6 hydrogel with or without neurotrophic factors induced neuronal proliferation in the subventricular zone and formation of new blood vessels in the peri-infarct tissue at both 2- and 8-weeks following stroke. Overall, this thesis has developed and optimised a hybrid hydrogel system by blending biopolymers CS, SF, PVA and PVP. In addition, this thesis has provided evidence that the developed hybrid hydrogels have potential to be used in concert with pharmacological interventions to promote post-stroke functional recovery.