Abstract
With the rapid progress of 3D-printing, this emerging technology is anticipated to revolutionise the biomedical industry in the near future. However, the low shape-fidelity of natural-based inks and the low biological performance of synthetic-based inks remarkably limits the number of potential inks for 3D-printing of bioengineered constructs. Therefore, it is increasingly important to develop cost-effective and efficient inks with high degrees of printability and biocompatibility to fabricate bioartificial scaffolds equivalent to human organs to ultimately solve the organ transplant problem. This dissertation reports on the efforts to fill this gap by developing new functional hydrogel inks to 3D print bone tissue-engineered scaffolds. Two hydrogel inks based on either chitooligosaccharide (COS) or hydroxyapatite (HA) were developed for extrusion-based 3D-printing. Bone tissue was selected as the target tissue because in the event of excessive damage to the bone tissue, the self-healing process alone is not sufficient to restore the bone integrity in the lifetime of a patient.
Chitosan, being biocompatible, biodegradable and antimicrobial, has been immensely utilised in the field of biomedical sciences. Consequently, chitosan-based hydrogels hold great promise for developing 3D-printing inks. However, low water solubility at neutral pH and poor mechanical integrity are the main issues associated with using chitosan in regenerative medicine. To overcome the limitations of existing chitosan, COS was prepared by partial acidic hydrolysis of unstable glycosidic bonds of chitosan using a microwave-assisted process in an eco-friendly environment. Afterwards, the synthesis of a new family of acrylated-COS derivatives was investigated by employing click-based approaches as a means to prepare a number of efficient dual-curing hydrogel inks. In these dual-curing inks, step-growth Michael addition polymerisation was combined with chain-wise acrylate homo-photopolymerisation. The initial Michael addition permitted the green synthesis of acrylated-COS derivatives under ambient and solvent-free conditions. Later, as a second curing stage, the unreacted acrylate groups participated in UV-induced photopolymerisation, becoming part of the final polymer network, thereby increasing the crosslinking density and shape-fidelity of the 3D-printed scaffolds.
Simultaneously, a new set of HA-based inks were developed. These inks, which were also prepared using the same carrier ink, had HA as the osteoconductive and mechanical reinforcement agent in their structure. The printing hydrogel inks in both groups were formulated to match the rheological properties requirements of the extrusion-based 3D-printing machine. The chemical compositions were optimised to permit rapid crosslinking of the hydrogel components and provide high shape fidelity of the printed scaffolds. The physiochemical properties of scaffolds including swelling behaviour, degradation rate, and compression strength were measured. It was shown that the material properties of these scaffolds were dependent on the weight fractions of COS and HA. The optimised COS-based (H0C2) and HA-based (H2C0 and H5C0) scaffolds were selected for further investigation into their potential to provide a three- dimensional environment suitable for the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) towards osteoblast-like cells. 3D-printed scaffolds promoted osteogenic differentiation of hBMSCs where scaffolds containing 2 wt% COS (H0C2) showed higher cell viability and alkaline phosphatase (ALP) activity while scaffolds containing 2 wt% and 5 wt% HA (H2C0 and H5C0) exhibited higher calcium deposition as measured by Alizarin Red S (ARS) staining. All 3D-printed scaffolds demonstrated in vitro bioactivity in simulated body fluid (SBF), suggesting the osseointegration of these scaffolds in vivo. Altogether, optimised 3D-printed COS-based and HA-based scaffolds have promising applications for bone tissue regeneration.