Development of Biomimetic Hydrogels as Cell-Laden Devices for Muscle Regeneration

The aim of this thesis was to develop a novel cross-linking strategy to prepare defined collagen-based hydrogel networks to investigate stiffness-induced cell differentiation for skeletal muscle tissue engineering. Volumetric muscle loss (VML) occurs with traumatic injury or aggressive tumour ablation and results in a diminished natural capacity for repair. Whilst autologous muscle transfer offers the gold standard treatment option, the level of success is limited by surgical expertise, availability of healthy tissue, donor site morbidity and a loss of muscle strength and function due to scar tissue formation. As a result, a clear need exists for therapeutic strategies that can enhance the innate ability of skeletal muscle to regenerate following VML.
Thiol-ene photo-click collagen hybrid hydrogels were systematically developed and prepared via step-growth reaction using thiol-functionalised type-I collagen and 8-arm poly(ethylene glycol) norbornene terminated (PEG8NB). Collagen was thiol-functionalised by a ring opening reaction with 2-iminothiolane (2IT), whereby up to 80% functionalisation and 90% triple helical preservation were recorded in addition to improved solubility of the material. Type, i.e. Irgacure 2959 (I2959) or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and concentration of photoinitiator were varied to ensure minimal photoinitiator- induced cytotoxicity in line with the in vitro study. Gelation kinetics proved to be largely affected by the specific photoinitiator and the concentration, with LAP- containing thiol-ene mixtures leading to 8 times faster gelation times compared to I2959-containing mixtures. Photo-click hydrogels with tunable storage moduli (G’: 0.54- 6.4 kPa), elastic modulus (Ec: 1.2- 12.5 kPa), gelation time (ԏ: 73- 331 s) and swelling ratio (SR: 1500- 3000 wt.%) were prepared. Three of these hydrogels (Ec: 7, 10, 13 kPa) were taken forward for in vitro tests with a myoblast cell line due to their similarity to the elasticity of natural muscle. These three hydrogels were shown to support cell attachment, spreading, proliferation and maturation/ differentiation of myoblasts into myotubes. A subcutaneous model was used to analyse the immune response of these new materials at 1, 4 and 7 days after implantation using Mucograft® as the control. Mucograft® was shown to present a lower immune response and reduced inflammation, whereas, the immune response from the hydrogel, promoted angiogenesis, which can be more beneficial for muscle regeneration.