Abstract
Materials used in tissue engineering and regenerative medicine (TERM) need to be biocompatible, sterile, and functional. TERM materials need to interface with patients’ bodies without activating immune responses that can lead to complications or further illness and injury. While there has been extraordinary progress made in TERM which has improved patient outcomes, there are still several challenges to overcome. The growing demand for effective solutions in regenerative medicine has catalysed significant interest in biomaterials derived from alternative biocompatible sources. Among these, fish skin collagen has emerged as a promising alternative to mammalian-derived collagens due to its favourable biological properties, reduced risk of zoonotic transmission, and ecological advantages. This thesis explores the comprehensive journey of fish skin collagen, beginning with its extraction and characterisation, and progressing through to its application in the engineering of vascularised soft tissue constructs. By bridging materials science, cell biology, and tissue engineering, this work aims to establish fish collagen as a viable scaffold material capable of supporting neovascularisation and functional tissue regeneration. This work contributes to the advancement of next-generation biomaterials for clinical use.
Collagen, an abundant fibrillar protein, is among the most common biomaterials used in TERM research due to its biological characteristics that support and enhance cellular growth. While mammalian collagen is the most widely used type of collagen in TERM research, fish collagen is emerging as an alternative collagen source for TERM applications. Fish collagen has high biocompatibility, is amenable to low-cost and high-yield extraction techniques, and fewer religious restrictions. Using fish collagen in TERM applications requires different approaches compared to mammalian collagen due to its temperature sensitivity, higher solubility, and a wider variety of physical and chemical characteristics depending on the source. This thesis explores the use of extracted fish skin collagen sourced from hoki (Macruronus novaezelandiae) and its suitability as a biomaterial for TERM applications.
The development of clinically applicable constructs using fish skin derived collagen requires rigorous assessment of sterilisation methods. Most established sterilisation techniques are either unsuitable for or damaging to biomaterials; alternative methods are needed to preserve desirable characteristics of sensitive biomaterials while meeting standards for sterilisation. Fish skin collagen in particular is thermolabile and more susceptible to denaturation under harsh conditions compared to mammalian collagen. Therefore, it was crucial to identify sterilisation techniques that effectively eliminated microbial contaminants while preserving the structural integrity and functional performance of fish collagen scaffolds. To examine the impacts of sterilisation on unmodified, extracted fish collagen, four sterilisation methods were investigated. Gamma irradiation, ultraviolet irradiation and 70% ethanol are established sterilisation methods for medical devices. The impacts of these treatments on fish collagen were compared with the impacts of supercritical carbon dioxide (CO2) sterilisation treatment, an emerging method for sterilising biomaterials. None of the treatments negatively impacted biocompatibility, and all fish collagen extracts significantly increased cellular metabolism compared to commercially available mammalian gelatin.
To improve the mechanical and thermal resilience of native fish skin collagen extracts, multiple crosslinking strategies were considered. This included methacryloyl conjugation, adding crosslinkable molecules onto collagen that can rapidly create a crosslinked matrix using light. Another strategy used periodate oxidation, altering sugars to crosslink with collagen in a way that mimics a type of crosslink found in natural tissues. These crosslinking methods were optimised to preserve the native structures and functionality of collagen, which were then used to create inks for 3D printing.
Larger three-dimensional (3D) constructs are not capable of full functionality without working vasculature, which is needed to provide nutrition and waste removal for living cells. To further enhance the biological functionality of the printed constructs, vascular endothelial growth factor (VEGF) was immobilised onto the scaffolds, which were then seeded with bone marrow derived mesenchymal stem cells to promote cell-mediated vascularisation.
The findings of this thesis present a comprehensive approach to overcoming critical challenges in the development of TERM materials tailored for soft tissue applications. Supercritical CO2 sterilisation treatments were the least disruptive to the structural and mechanical properties of native collagen extract. Further, it is possible to crosslink native fish skin collagen while preserving its structural integrity. These crosslinked collagen hydrogels were able to be processed into microgels and blended with calcium alginate to formulate an extrudable, self-healing biomaterial ink. This innovative 3Dprintable biomaterial ink does not need additional post-printing treatments, and is amenable to further functionalisation using growth factors to drive stem cell-mediated vascular tissue development.