Articular cartilage is a highly organised connective tissue lining the ends of bones where it acts to provide load support and a low friction bearing surface for locomotion. Due to its limited intrinsic healing capacity, cartilage damage frequently results in further tissue deterioration, in turn leading to debilitating pain, disability, and the development of degenerative joint conditions. Currently available surgical interventions for symptomatic cartilage lesions commonly fail to restore the physiological structure and function of the tissue, prompting the development of alternative repair strategies. As such, functional tissue engineering aims to create new cartilage using a combination of cells, biomaterials, and physiological factors.

Hydrogel biomaterials and bioreactors are a key part of cartilage research and tissue engineering applications. Besides providing structural support, the molecular and physical clues encoded within the hydrogel matrix regulate chondrogenic differentiation and neo-cartilage formation, and allow the investigation of cellular behaviour in a controlled microenvironment. The biosynthetic activity of hydrogel-encapsulated cells can further be promoted using specialised mechanical stimulation bioreactors to accelerate or improve tissue growth and maturation. However, despite extensive research efforts, the generation of cartilage tissues with mechanical functionality remains challenging. To address these shortcomings, this thesis aims to advance current tissue engineering approaches by investigating the effects of the composition and properties of photocrosslinkable hydrogels and dynamic mechanical stimulation on chondrocyte behaviour and neo-cartilage formation.

In the first part of this thesis, bilayered hydrogel constructs permitting control over surface frictional properties were developed and characterised. When sliding shear motion was applied, shear strains in constructs with low surface friction were minor, inducing chondrogenesis and accumulation of cartilage-specific extracellular matrix molecules. Dynamic loading of high friction constructs, on the other hand, resulted in excessive shear strains which inhibited chondrogenesis and promoted the expression of proteases involved in the pathology of articular cartilage. These findings demonstrate that the frictional properties of tissue-engineered cartilage constructs may act as a potent regulator of chondrocyte mechanotransduction and neo-tissue formation

The progress in advancing our understanding of cartilage pathology and regeneration is hampered by a lack of hydrogels with cell-instructive bioactivity. Aiming to overcome these limitations, the second part of this thesis comprises a stepby-step protocol for the synthesis and application of photocrosslinkable hydrogels based on gelatin methacryloyl (GelMA). GelMA hydrogels allow for cell attachment, proteolytic degradation, and the control of physicochemical characteristics such as stiffness and porosity. Serving as a base material, GelMA can further be complemented with other chemically functionalised extracellular matrix components such as hyaluronic acid methacrylate (HAMA) to promote chondrogenic differentiation and neo-cartilage growth.

In the third experimental part, a novel shear and compression bioreactor system was designed, manufactured, and validated. Controlled by user-friendly software, the system facilitates precise displacement-controlled shear and compressive stimulation to tissue-engineered cartilage constructs in an oxygencontrolled environment. While the device primarily functions as a bioreactor that mimics the native biomechanical environment of articular cartilage, it is also fitted with a 50 Newton load cell to provide real-time force feedback and mechanical testing. In short-term experiments, both uni- and biaxial mechanical stimulation promoted cartilage marker gene expression of chondrocytes encapsulated in hydrogels composed of GelMA and HAMA. Further confirming the efficacy of the system, long-term intermittent biaxial stimulation of tissue-engineered cartilage constructs significantly enhanced the accumulation of cartilage-specific extracellular matrix molecules.

The final experimental part of this thesis focused on the development of mechanically tough double-network hydrogels for functional cartilage tissue engineering prepared from orthogonally crosslinked GelMA and alginate. Overcoming the brittleness of conventional hydrogels, these constructs displayed superior mechanical properties, recoverable energy dissipation, shape recovery, and withstood large magnitudes of compression without failure. Additionally, doublenetwork hydrogels promoted chondrogenesis and supported the accumulation of high quantities of hyaline cartilage-specific extracellular matrix which was further enhanced by intermittent biaxial shear and compressive stimulation.

In summary, the findings of this thesis demonstrate that (i) response of cartilage cells to physiological loading is highly dependent on the hydrogel properties, (ii) hydrogels can be engineered to mimic natural extracellular matrices, (iii) mechanical stimulation can be used to improve the outcomes of cartilage tissue engineering, and (iv) biomaterial-based hydrogels can be engineered to withstand and recover from harsh loading conditions whilst retaining cell-instructive bioactivity and cytocompatibility