Applying electron microscopy analysis to assess neural tissue engineering outcomes

Lindop, Georgina Elizabeth

Abstract

Spinal cord injury (SCI) has a major socio-economic impact with treatments providing little to no functional recovery due to the SCI environment hindering regeneration through chemical and physical barriers. Novel approaches to alter the injury environment are needed for repair. Biomaterial implants can potentially alter the physical and chemical properties of injury environments to promote regeneration and functional recovery. Evaluation of biomaterial influence over neural cells, in sites of pathology, are reliant on fluorescence microscopy techniques. Whilst useful for providing an overview, biomaterials are difficult to observe and their influence on ultrastructural cellular properties is missed when using fluorescence microscopy. Electron microscopy (EM) is an important tool within research due to its high resolution. However, EM processes require optimisation for novel tissue engineering applications.

In this thesis, we tested two new biomaterial-based neural tissue engineering strategies (magnetoelectric hydrogels and PODS, protein-based crystals, slow-release growth factor crystals) and established EM processes for ultrastructural analysis alongside standard imaging procedures. We demonstrated that a magnetoelectric hydrogel can support the neural transplant population of neural stem cells (NSCs). Further, NSCs could differentiate into their daughter cells – astrocytes, neurons, and oligodendrocytes. Applying an oscillating magnetic field to the constructs during NSC differentiation appeared to enhance neurite length without affecting overall cell proportions. Using Scanning EM (SEM), we could identify differentiated cell types and showed features of membranes such as pits, filopodia and circular ruffles could be observed and quantified. Subsequently, we investigated PODS delivery to a novel NSC derived injury model. We demonstrated observations made in phase and fluorescence microscopy are supported in EM, including identification of neurite protrusion, proliferation and invading immune cells. Further, we showed PODS–cell interactions are observable under SEM and membrane features were identifiable. Lastly, we showed for the first time that Transmission EM methods could be applied to a 2D multicellular and pathological in vitro model for testing biomaterials, with a key point of witnessing the internalisation of PODs into the cell. Here, we identified key cellular features such as mitochondria, endoplasmic reticulum, and microtubules, important when considering axonal function and growth.

Taken together, we believe the work demonstrates EM can be incorporated into the workflow for developing neuro-regenerative biomaterials and provide additional insights compared to standard imaging. Methodology needs to be developed to improve throughput. For hydrogels this could be adopting methods such as freeze drying to reduce the shrinkage of samples. For the multicellular injury model adopting and adapting serial block face imaging (SBF) volume EM offers advantages of a larger overview of ultrastructure and the lesion region at high magnification. Alongside, TEM embedding and sectioning could be improved for ultrastructural analysis.

Citation

Lindop, G. E. (2024). Applying electron microscopy analysis to assess neural tissue engineering outcomes. (Thesis). Keele University. Retrieved from https://keele-repository.worktribe.com/output/884963

• Thesis Type: Thesis
• Deposit Date: Aug 16, 2024
• Publicly Available Date: Aug 16, 2024
• Public URL: https://keele-repository.worktribe.com/output/884963
• Award Date: 2024-08