Doctor of Philosophy
Intelligent Polymer Research Institute
Stevens, Leo Robert, Materials and processes for the biofabrication of peripheral nerve guides, Doctor of Philosophy thesis, Intelligent Polymer Research Institute, University of Wollongong, 2016. https://ro.uow.edu.au/theses/4955
Injuries sustained to the peripheral nervous system disrupt the body’s major signalling pathway, leading to pain or paralysis. The treatment of serious peripheral nerve injuries currently relies on the use of autografts, whereby secondary nerves are sacrificed to treat the pathology. Recently, scientists and clinicians have sought alternative treatments based on engineered tissue scaffolds that may effectively replace grafted tissues. Several peripheral nerve guides (PNGs) are already available, however patient recovery outcomes using PNGs have thus far been poor compared to autograft treatments. The limited efficacy of current-generation PNGs has been attributed to their failure to replicate the complex structure and biofunctionality of nerve tissue. In this thesis, we explore materials and fabrication processes with the aim of forming multifunctionalised PNG fibers with the potential to accelerate nerve repair.
The anionic polysaccharide gellan gum (GG) was assessed for its potential to directly encapsulate neural cells, as well as contribute to the PNG fabrication process. GG was purified to its sodium salt (NaGG), which exhibited processing characteristics that were favourable for biofabrication techniques including casting, reactive printing and wet spinning. NaGG hydrogels were visualised using scanning electron microscopy, confirming high levels of internal porosity. A variety of cell types including fibroblasts, skeletal muscle cells and neurons were encapsulated within NaGG hydrogels and remained highly viable. However, many cells also exhibited increased clustering and diminished differentiation compared to controls, with a reduction in neuronal differentiation and neurite extension being of most concern for PNG applications. Mechanical testing of NaGG hydrogels revealed them to be weaker and more rigid than neural tissues, in line with comparable polysaccharide hydrogels. An alternative material, type-I collagen, was successfully extracted from rat tails at high purity and confirmed to be highly supportive for PC12 cell differentiation. Electron micrographs of our type-I collagen hydrogels revealed a porous fibrillar network in line with previous reports. Whilst collagen’s gelation behaviour was not ideal, rheological studies identified a short processing window that was later applied for the wet spinning of multi-material fibers. Finally, a short peptide sequence containing the cell-adhesion motif arginine-glycineaspartate (RGD) was coupled to NaGG using carbodiimide chemistry to 40 % yield. RGD-GG retained the favourable processing characteristics of NaGG, whilst improving metabolic activity in encapsulated cells and supporting high rates of differentiation in encapsulated skeletal muscle cells and primary cortical neurons. However PC12 cells remained poorly differentiated in RGD-GG and it was concluded that the PNG should have a multi-material design incorporating distinct materials tailored for cell support, mechanical strength and processing characteristics.
Conductive materials were prepared and assessed as potential electrode materials for the stimulation of regenerating axons within a multi-functional PNG. We assessed a range of biopolymers as alternative dopants for the conducting polymer poly(3,4- ethylenedioxythiophene) (PEDOT). In initial screening, dextran sulfate (DS) was found to be a promising material, with PEDOT:DS having physical and electrical properties comparable to the widely applied PEDOT:PSS. PEDOT:DS was polymerised both chemically and electrochemically, with electrochemically deposited films being the most suitable for cell stimulation. Reduced liquid crystalline graphene oxide (rLCGO) fibers were formed by wet-spinning, and then assessed as a potential template for PEDOT:DS electropolymerisation. Whilst rLCGO was ultimately deemed to be a poor template for PEDOT:DS, the fibers exhibited promising electrical properties and an aligned topography that rendered them promising for neuronal stimulation and alignment. PC12 cell stimulation experiments performed using rLCGO, PEDOT:DS and gold stimulation electrodes further supported the application of rLCGO fibers as substrates for neuronal cell stimulation and guidance. PC12 cells adhered directly to rLCGO fibers whilst electrical stimulation was applied exhibited extensive neuronal outgrowth that aligned along the rLCGO’s microstructure.
Finally, we developed two distinct biofabrication processes to pattern biomaterials and conductors towards the ultimate goal of prototyping a multi-functional PNG. A reactive printing process based on co-extrusion of polysaccharide and cross-linking solutions was developed for both automated and hand-held devices. Hand-held bioprinting was found to be an inexpensive yet versatile method of fabricating complex cell-laden hydrogels, and we applied the technique to encapsulate primary mouse cortical neurons and glia within a multi-layered RGD-GG hydrogel reflective of the cerebral cortex. Despite significant benefits, the reactive bioprinting process was not suited to the formation of multi-functional PNG fibers, and an alternative approach based on wet spinning was pursued instead. Using customised, multi-outlet spinnerets, PNG fibers were generated with a wide variety of internal geometries and material compositions. Applying a ‘pultrusion’ technique, rLCGO fibers, collagen channels and live PC12 cells were all successfully positioned within prototype PNG fibers. Once encapsulated within the fibers, PC12 cells exhibited high degrees of differentiation and aligned neural outgrowths, behaviours that underlined the potential of these multi-functionalised fibers for application as PNGs.
Overall, this thesis makes a significant contribution to the fields of biomaterials science, biofabrication and tissue engineering. As well as furthering the design of nextgeneration PNGs, our materials and processing techniques may also aid in the fabrication of a wide range of engineered tissues towards the ultimate goal of providing improved clinical therapies for soft tissue pathology.