Neuronal networks are critical for many body processes, such as brain function, movement and sensing. These neural networks are established during development and depend on precise axon guidance mediated by extrinsic molecular cues and extracellular matrix (ECM) proteins. Following spinal cord (SCI) or peripheral nerve injury (PNI), axon regrowth is impaired, leaving individuals with compromised movement or, in case of SCI, paralyzed. Our key objective is to elucidate how different ECM proteins and chemical cues influence axon outgrowth and pathfinding, thereby permitting the development of novel biochemical micro-patterned materials for enhanced nerve regeneration. To examine neuronal behaviour under various conditions, inkjet printing, light induced photopatterning and microstamping techniques were compared to create an in vitro neuronal guidance assay. Light induced photopatterning or light induced molecular adsorption (LIMAP) revealed to be the most robust and flexible tool to create patterns, as patterns were able to be generated digitally. This thesis illustrates the development and optimisation of LIMAP technology and validates its use as a tool to study axonal growth and turning behavior on surface patterns of different ECM proteins. Specifically, fibronectin (FN) and laminin (LM) crossed line patterns were produced and validated using LIMAP as a competition assay and cultured with rat dorsal root ganglion (DRG) as a model system for peripheral nervous system (PNS) regeneration, and a mouse CNS-derived catecholaminergic neuronal cell line (CAD) as a model of the central nervous system (CNS). Results indicate that defined tracks of FN and LM are able to guide axons of CAD cells and influence their directionality. Additionally, axons that started on FN preferred to stay on FN which could indicate a preference for FN. This could be due to integrin recycling or a protein concentration dependent effect. Furthermore, by establishing an algorithm for neurite tracking, data output was automated to give indication for directionality preferences. Future work will comprise live cell imaging technique to additionally assess growth dynamics and speed of growth on these defined patterns. This thesis introduced and improved a novel technique to create protein patterns by using LIMAP as a tool for axonal pathfinding assays. Future work includes testing different ECM proteins and guidance cues for their guidance capabilities on neurons from the PNS and CNS. Knowledge about guidance abilities of defined biochemical cues will allow the design of micro-patterned surgical nerve repair devices for in vivo testing in future.
Using micro-patterning to study axon outgrowth and pathfinding
Kolmogorova, A. (Author). 1 Aug 2020
Student thesis: Phd