The Development of a Novel 3D Bioprinting Strategy to Drive Vascularisation, and its Application for Bone Tissue Engineering

Abstract

One of the major challenges facing the field of tissue engineering today is vascularisation. Without a system in place to circulate oxygen, nutrients and metabolites, the size of many tissue engineered constructs is restricted, as embedded cells must rely on the diffusion of these factors for their survival. This has limited the clinical translation of engineered tissues and organs. The inclusion of a vascular network within tissue engineered constructs could overcome this constraint, thereby enabling the scaling up of such tissues to previously unattainable, clinically relevant sizes. One branch of tissue engineering which has a considerable need for more effective vascularisation strategies is the field of bone tissue engineering. Bone tissue engineering endeavours to produce bone substitutes and provide a viable alternative to autologous bone grafts. To achieve this, new approaches to produce large, viable bone tissues must be identified. The overall aim of this thesis was to develop a 3D bioprinting strategy to vascularise tissue engineered constructs. This thesis sought to achieve this through preforming microvessels in vitro within a 3D printed construct prior to its implantation in vivo, a process known as 'prevascularisation'. The first phase of this thesis focused on identifying a suitable bioink formulation which could support endothelial sprouting and be 3D bioprinted. After determining printability, the optimum cell combinations and culture conditions to produce stable microvessels in vitro and in vivo were established. The next phase of this thesis applied this prevascularisation strategy to multiple bone tissue engineering strategies to investigate whether prevascularisation could enhance in vivo vascularisation of these constructs and whether this could enhance bone formation. Three bone tissue engineering strategies were examined. Firstly, a cartilage template was 3D bioprinted. This template underwent endochondral ossification in vivo. Prevascularising such a template prior to implantation significantly enhanced vascularisation of the engineered tissue but did not significantly enhance bone formation. Secondly, hypertrophic μTissues were fabricated in a relatively high-throughput manner in vitro. These μTissues underwent endochondral ossification in vivo, producing mineralised bone tissue. Prevascularising these μTissue constructs accelerated this process of endochondral ossification. Lastly, the prevascularising bioink was used to prevascularised 3D printed PCL scaffolds coated with nano hydroxyapatite. This led to enhanced vascularisation and accelerated bone regeneration in large bone defects created in the femurs of rats. To conclude, this thesis demonstrates a novel 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into multiple bone tissue engineering strategies to improve vascularisation and enhance bone formation.