Anisotropic 3D scaffolds for spinal cord regeneration

• Main Author: Sousa, Joana Patricia Marques
• Publication Date: 2024
• Language: eng
• Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Download full: http://hdl.handle.net/10773/42191
• topic: Anisotropy; Biomaterials; Neural stem cells; Tissue engineering; Spinal cord injury; Scaffolds; Nervous system

Spinal cord injuries pose major challenges to modern medicine due to the restricted capacity of the central nervous system to regenerate and the profound impact they have on patients' quality of life. Neural tissue engineering offers promising solutions, particularly through the development of three-dimensional (3D) scaffolds that serve as templates for tissue regeneration. Scaffolds possessing anisotropic characteristics, which mimic the inherent longitudinal alignment of nerve fibres in the spinal cord, have significant potential for restoring the structure and functionality of neural networks. This thesis examines the significance of scaffold anisotropy in the regeneration of neural tissue by studying the structural characteristics of scaffolds that possess axially aligned channels, pores, or fibres. The initial method utilised 3D printed moulds to fabricate a hydrogel-based multichannel 3D scaffold. The second method utilised directional freezing combined with cryopolymerization to create cryogels featuring longitudinal pores. Ultimately, an innovative automated microfabrication technique was employed to create magneto-responsive aligned fibres. The study examined how microarchitecture affects the behaviour of neural stem cells (NCS), specifically their viability, migration, and proliferation. Additionally, the study investigated how different scaffolds can promote neuronal differentiation and guide the growth of neurites. The various strategies were evaluated for their successes and limitations. The multichannel scaffolds offer precise structural control and easy fabrication but may pose challenges for irregular lesions. Importantly, they promoted unassisted cell adhesion, robust proliferation, and neuronal differentiation, fostering intricate neuronal networks with longitudinal neurite extension. The cryogels presented high deformability allowing injectability. Cryogels with longitudinal pores showed extensive cellular infiltration and migration, supporting neuronal differentiation and neurite extension compared to random cryogels. The injectable fibre-integrating hydrogels offer a minimally invasive approach adaptable to various injuries, opening the avenue for further exploration of the practical clinical application of such an approach. In this study, initial investigations performed on two-dimensional substrates highlighted the impact of fibre diameter and orientation on NSC differentiation, with larger fibres promoting enhanced neuronal differentiation and neurite outgrowth. The inclusion of magnetic particles did not affect cell viability or differentiation. Transitioning to 3D injectable scaffolds, aligned fibre tiles embedded in a hydrogel induced neurite outgrowth with preferential orientation along the fibre axis. The results obtained are promising and indicate the potential of anisotropic scaffolds as a therapeutic strategy for spinal cord injuries. In addition to the anisotropic nature of the scaffolds, their biochemical nature is also important. This thesis explores the innovative application of human amniotic membranes (AM) modified with methacryloyl domains (AMMA) to prepare scaffolds for NSC culture. This innovation has made it possible to produce fully human and photopolymerizable hydrogels from liquefied precursors. The diverse components within the AM extracellular matrix, including critical structural proteins like various collagen types and fibronectin, as well as growth factors, make it a promising candidate for serving as an engineered scaffold material in the field of nervous tissue regeneration. These hydrogels offer improved and tuneable mechanical properties, enabling innovative engineering designs. In this context, this thesis marks the pioneering introduction of AMMA into NSC research.