Abstract
The nervous system is a crucial component of the body and damages to this system, either by injury or disease, can result in serious or potentially lethal consequences. For example, stroke is one of the leading causes of death and disability in the world, with more than 15 million people suffering annually. Restoring the damaged nervous system is a great challenge due to its complex physiology and limited regenerative capacity.
The current project aimed at developing three-dimensional nanoscaffolds (3DNSs) from a biomaterial blend of silk fibroin (SF), polyethylene glycol (PEG), and polyvinyl alcohol (PVA) for regenerating brain tissue after stroke. PEG is a highly biocompatible polymer well suited for neural tissue engineering because of its hydrophilic and mechanical properties that can match the stiffness of the native brain, but it is not bioactive. Therefore, the current project aimed to add different concentrations of SF as a bioactive additive to a blend of PEG and PVA. PVA was added because of its high affinity for electrospinning, suitable viscosity, and its FDA approval for human application. This novel biomaterial was processed via electrospinning using the nanofibre electrospinning unit TL-BM. Four different formulations of 3DNSs were electrospun: F1 (SF=60%), F2 (SF=50%), F3 (SF=40%), F4 (SF=30%).
The 3DNSs were thoroughly characterised, their biological activity was investigated in vitro using PC12 cells, and their effects on reactive astrogliosis were assessed in vivo using a photothrombotic model of ischemic stroke in mice. Biophysical characterisation showed that concentration of SF directly affected the mechanical characteristics and internal structure of 3DNSs that presented either a gel-like structure (SF 50%) or a nanofibrous structure (SF 40%). FTIR analysis and rheological measurements confirmed the results seen in SEM nanographs, demonstrating that the higher number of crosslinks between PEG and PVA coupled with a reduced concentration of SF yielded mechanically stable nanofibres (G´(ω) < G´´(ω) and Tan(∂) > 1). In addition, DSC and TGA thermograms indicated that the concentration of SF was inversely proportional to the thermal stability of 3DNSs (DSC endothermic curves shifted from 230C for SF 50% to 270C for SF 40%). The different internal structures of 3DNSs were also evident in the swelling studies as the gel-like structures were capable of faster and higher water intake (> 125.15% for F1 vs > 64.76% for F4). Biodegradation kinetics were investigated using mouse blood serum from young stroked mice as a biodegradation buffer and they were modulated and optimised via annealing (e.g. 19.37% of mass loss after annealing vs 90.41% of mass loss without annealing after 12 days for F4).
PC12 cell viability was measured using a resazurin assay and all formulations showed improved cell viability over time (mortality rate estimated at 0.433% after 24h and 0.031% after 3 days for F4). Cell growth and proliferation showed no statistical differences in the quantity of cells growing on each 3DNS (p<0.05) and morphological analysis revealed no differences in the morphological features compared to control. However, a difference in PC12 cell aggregate size and positioning throughout the 3DNSs was identified and linked to the two different internal structures. In vivo analysis of animals treated with different formulation of 3DNSs after photothrombotic stroke indicated that the 3DNSs implant did not have any negative effects on immunological response to stroke (p<0.05). A significant decrease in levels of glial fibrillar acidic protein (GFAP) expression in the peri-infarct area (p<0.001 for F2 and p<0.05 for F4), suggested possible suppression of chronic astrogliosis.
These findings demonstrated the potential of 3DNSs for neural regeneration after stroke and encouraged the use of SF in tissue engineering and regenerative medicine. Furthermore, the results of the current project validate the use of electrospinning as a fabrication technique for neural tissue engineering, enhance our understanding of interactions between SF and polymers, investigate rheology of biological complex fluids, and elucidate the bioactivity and the biocompatibility of SF in vitro and in vivo.