Interrogation of the neuromuscular interface is essential for both insights into, and therapies for, neuromuscular disease. However, animal systems are used extensively because of the difficulty in sourcing relevant human materials, despite the models not being totally ideal. The aim of this project is therefore to develop a 3D tissue engineered neuromuscular junction – this will give a more biologically relevant system but will also, in the long term, reduce the number of animals used in neuromuscular experimentation. In the short term, the number of experiments that can be completed per animal will be increased leading to a reduction in the number of animals used. Our previous work has succeeded in developing and characterising both early skeletal muscle and neural model tissues individually in culture. This project aims to investigate integrating these two models, as an in vitro neuro-muscular test-bed. We are proposing using two skeletal muscle model systems – a developmental, fibrin-based system and a regenerative, collagen-based system. In both of these systems, muscle cells are incorporated into a matrix that has a "pseudo" tendon at each pole of the construct. The formed constructs can be attached to force transducers to measure the amount of tension generated over time. It is important that in any neuromuscular system the neural component is well covered and there is extensive expertise available as successfully culturing such cells is a non-trivial task. The applicants in this grant include a well established motor neuron laboratory with a long track record of such cultures. Both of the skeletal muscle systems have been shown to generate tension over time and our preliminary data indicates that motor neurons can be successfully cultured on the regenerative/collagen system. We are seeking to extend these preliminary findings and co-culture both skeletal muscle systems with motor neurons to promote construct maturation and then to allow monitoring of functional output. The effect of the presence of motor neurons on such outputs will be studied. Various pharmacological agents will be added to the cultures to ensure that any neuromuscular junction is behaving physiologically. Finally, pathological motor neurons or muscle cells will be added to normal cells in constructs and several of the described parameters measured to see if the system can recreate the response of a pathological system.
Martin NR et al. (2015). Neuromuscular function formation in tissue-engineered skeletal muscle augments contractile function and improves cytoskeletal organization. Tissue Engineering Part A 21(19-20):2595-604. doi: 10.1089/ten.TEA.2015.0146
Martin NR et al. (2013). Factors affecting the structure and maturation of human tissue engineered skeletal muscle. Biomaterials 34(23):5759-65. doi: 10.1016/j.biomaterials.2013.04.002
Sharples AP et al. (2012). Modelling in vivo skeletal muscle ageing in vitro using three-dimensional bioengineered constructs. Aging Cell 11(6):986-95. doi: 10.1111/j.1474-9726.2012.00869.x
Smith A et al. (2012). Characterisation and optimisation of a simple, repeatable system for the long term in vitro culture of aligned myotubes in 3D. Journal of Cellular Biochemistry 113(3):1044-1053 doi: 10.1002/jcb.23437
Principal investigatorProfessor Mark Lewis
Co-InvestigatorDr Linda Greensmith
Dr Vivek Mudera