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Investigation of the Effects of IGF-1 Slow Release on Human Neuromuscular Junctions Formation and Functions

Karim, Ikbal (2020) Investigation of the Effects of IGF-1 Slow Release on Human Neuromuscular Junctions Formation and Functions. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Insulin-like growth factor 1 (IGF-1) plays central roles in neuromuscular junction (NMJ) development, skeletal muscle and motor neurons growth. The NMJ is the interface between the nerve and muscle fibre, and thus they may receive (patho) physiological input from both the muscle and the motor neuron. Several conditions including ageing, disuse, neurodegenerative diseases, diabetes, and cancer are associated with NMJ dysfunctions which lead to profound muscle wasting and impaired NMJ function. Recent attention has focused on the use of anabolic growth factors such as IGF-1 in preventing muscle atrophy associated with NMJ dysfunction, however, difficulties in isolating and extracting functional NMJs, low bioavailability and potential side effects such as hypoglycaemia appear to limit the usefulness of IGF-1 for clinical treatment of muscle wasting conditions linked with NMJs degeneration. Therefore, there is a need for a simplified and reproducible system of engineered in vitro NMJs to facilitate the investigation of sustained slow release of IGF-1 on NMJs function. The aim of this thesis was to fabricate mesoporous silica nanoparticles (MSNPs) loaded with IGF-1 to manipulate the function of NMJ using physiologically relevant human in vitro NMJ model. MSNPs were generated using the Stöber method and fully characterised using different techniques (dynamic light scattering, scanning electron microscopy, powder X-ray diffraction and nitrogen adsorption) and then loaded with IGF-1. Human in vitro co-culture model of NMJ was engineered using human immortalised myoblast and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs). In the co-culture, myoblasts differentiated into aligned myotubes and NPCs differentiated spontaneously into cholinergic motor neurons sprouted axons that branched to form multiple NMJ innervation sites along myotubes, which showed extensive spontaneous contractile activity. The NMJ model was characterised using preand postsynaptic markers and functional assessment was verified using agonist and antagonist to NMJ. Finally, the NMJ platform was treated with IGF-1 loaded MSNPs for 7 days. The data showed that the slow IGF-1 release enhanced the formation and the functions of NMJs significantly compared with exogenous IGF-1. Furthermore, the slow release of IGF-1 enhanced the bi-directional communications between motor neurons and muscle cells through the elevation of endogenously secreted essential neural growth factors providing regenerative niche for NMJ formation and function. The outcome of this project may provide a breakthrough approach to enable research in designing new therapeutic treatments using IGF-1 for muscle wasting linked with NMJ deterioration.

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