Untreated articular cartilage focal defects tend to progressively worsen over time due to poor intrinsic healing properties. Such conditions frequently lead to the development of osteoarthritis, a common joint disorder associated with pain and impaired quality of life which affects 1 in 5 Australians over the age of 45. With a growing ageing population and increasing obesity levels, the rate of osteoarthritis diagnoses poses a significant global socioeconomic challenge. The difficulty in clinically managing osteoarthritis has prompted significant research into early intervention strategies to mitigate progressive tissue degradation as a result of focal defects. Tissue engineering has emerged as a promising therapeutic approach for the regeneration of articular cartilage. By combining biomaterials, cells and cell signaling stimuli, it may be possible to facilitate the regeneration of functional native tissue.

Hydrogels are a class of biomaterials that provide an aqueous, three-dimensional environment that promotes chondrocyte matrix synthesis and the formation of nascent articular cartilage. Gelatin methacryloyl (GelMA)-based hydrogels have been extensively utilised in articular cartilage tissue engineering because they are both cell-instructive and highly biocompatible for chondrocyte encapsulation. The presence of photocrosslinkable functional groups also allows for the fabrication of hydrogels with finely tuned mechanical and shape properties. The mechanical properties of GelMA hydrogels can be further enhanced through reinforcement with highly organised microfibre scaffolds produced with melt electrowriting (MEW). Additionally, matrix biosynthesis of GelMA-encapsulated chondrocytes can be promoted by the application of mechanical stimulation bioreactors that reproduce the dynamic mechanical environment of articular cartilage. Although promising, the use of fibre-reinforced hydrogels for articular cartilage tissue-engineering faces several challenges for preclinical investigations and ultimately, the regeneration of patient articular cartilage defects. Therefore, the aim of this thesis was to investigate how the fibre architecture of reinforcing scaffolds behaves under loading as well as how hydrogel fabrication and mechanical stimulation influence chondrocyte function in fibre reinforced GelMA hydrogel constructs.

In the first experimental portion of this thesis, the bulk and depth-dependent mechanical properties of GelMA/hyaluronic acid methacrylate (HAMA) hydrogels reinforced with orthogonally patterned MEW medical-grade polycaprolactone (mPCL) scaffolds with either monophasic fibre spacing throughout the depth, or scaffolds organized into a gradient pattern to mimic the zonal structure of articular cartilage were investigated. Hydrogels reinforced with gradient scaffolds exhibited depth-dependent strain characteristics that resembled the native tissue. Analysis of fibre behavior revealed that mPCL fibres did not straighten significantly under compressive loading, whereas lateral expansion of the hydrogel phase appeared to play a more prominent role in strengthening the construct.

Biomaterial characterisation is important for determining the suitability of tissue-engineered constructs for clinical translation. In the second part of this thesis, chondrocyte-laden gradient mPCL-reinforced GelMA-HAMA hydrogels were prepared with gelatin derived from either porcine and bovine animal sources as well as photocrosslinked with either ultraviolet or visible light-based photocrosslinking systems to determine how chondrocyte redifferentiation of encapsulated human articular chondrocytes might be affected after in vitro monolayer expansion. It was found that GelMA-HAMA hydrogels which were synthesised using bovine-derived type B gelatin and photocrosslinked with the UV-based photoinitiator Irgacure 2959 (IC2959) produced constructs with superior mechanical properties and glycosaminoglycan (GAG) accumulation. Furthermore, chondrogenic gene expression was upregulated in IC2959 photocrosslinked constructs, whereas the use of the visible light-based photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) increased catabolic gene expression.

In the final experimental portion of this thesis, chondrocyte-laden gradient mPCL fibre-reinforced GelMA-HAMA hydrogels were mechanically loaded in an in vitro biaxial mechanical stimulation bioreactor to determine whether progressively increasing loading intensity would influence matrix accumulation and organisation. Constructs were mechanically stimulated with a regimen of either increasing compressive strain or compressive frequency during dynamic cell culture. Chondrocyte morphology was strongly influenced by the loading protocols, with lower compressive frequency correlating with larger cell size and less rounded cell shape. Mechanical stimulation did not significantly affect construct mechanical properties and in some cases decreased GAG accumulation. However, the application of dynamic loading with increasing compressive strain appeared to enhance proteoglycan synthesis towards the surface of the construct, indicating a potential avenue for developing tissue-engineered constructs with depth-dependent properties. Ultimately, progressively increasing the intensity of mechanical loading was less effective at promoting a chondrogenic phenotype when compared with constructs stimulated with a higher compressive strain across the duration of the dynamic cell culture period.

In summary, the results of this thesis show that MEW fibre scaffolds can be designed to both strengthen hydrogels as well as recapitulate the depth-dependent strain properties of articular cartilage. Furthermore, the fabrication and dynamic cell culture of GelMA-based hydrogels can be optimised to promote chondrocyte redifferentiation, improve bulk physical and biochemical properties as well as promote depth-dependent matrix synthesis and accumulation.