title: Neuromuscular Junction Formation in Tissue-Engineered Skeletal Muscle Augments Contractile Function and Improves Cytoskeletal Organization.
abstract: Neuromuscular and neurodegenerative diseases are conditions that affect both motor neurons and the underlying skeletal muscle tissue. At present, the majority of neuromuscular research utilizes animal models and there is a growing need to develop novel methodologies that can be used to help understand and develop treatments for these diseases. Skeletal muscle tissue-engineered constructs exhibit many of the characteristics of the native tissue such as accurate fascicular structure and generation of active contractions. However, to date, there has been little consideration toward the integration of engineered skeletal muscle with motor neurons with the aim of neuromuscular junction (NMJ) formation, which would provide a model to investigate neuromuscular diseases and basic biology. In the present work we isolated primary embryonic motor neurons and neonatal myoblasts from Sprague-Dawley rats, and cocultured the two cell types in three-dimensional tissue-engineered fibrin hydrogels with the aim of NMJ formation. Immunohistochemistry revealed myotube formation in a fascicular arrangement and neurite outgrowth from motor neuron cell bodies toward the aligned myotubes. Furthermore, colocalization of pre- and postsynaptic proteins and chemical inhibition of spontaneous myotube twitch indicated the presence of NMJs in the innervated constructs. When electrical field stimulation was employed to evoke isometric contractions, maximal twitch and tetanic force were higher in the constructs cocultured with motor neurons, which may, in part, be explained by improved myotube cytoskeletal organization in these constructs. The fabrication of such constructs may be useful tools for investigating neuromuscular pharmaceuticals and improving the understanding of neuromuscular pathologies.
authors.affiliation: 1 Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, National Centre for Sport and Exercise Medicine (NCSEM), School of Sport, Exercise and Health Sciences, Loughborough University , Loughborough, United Kingdom .
authors.forename: Neil R W
authors.initials: NR
authors.lastname: Martin
authors.affiliation: 1 Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, National Centre for Sport and Exercise Medicine (NCSEM), School of Sport, Exercise and Health Sciences, Loughborough University , Loughborough, United Kingdom .|2 Department of Pharmacology and Therapeutics, University of Melbourne , Parkville, Victoria, Australia .
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Passey
authors.affiliation: 1 Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, National Centre for Sport and Exercise Medicine (NCSEM), School of Sport, Exercise and Health Sciences, Loughborough University , Loughborough, United Kingdom .
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Player
authors.affiliation: 3 Institute of Orthopedics and Musculoskeletal Sciences, University College London , London, United Kingdom .
authors.forename: Vivek
authors.initials: V
authors.lastname: Mudera
authors.affiliation: 4 Division of Neurobiology, Physiology and Behavior, University of California Davis , Davis, California.
authors.forename: Keith
authors.initials: K
authors.lastname: Baar
authors.affiliation: 5 The Sobell Department of Motor Neuroscience and Movement Disorders, University College London , London, United Kingdom .
authors.forename: Linda
authors.initials: L
authors.lastname: Greensmith
authors.affiliation: 1 Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, National Centre for Sport and Exercise Medicine (NCSEM), School of Sport, Exercise and Health Sciences, Loughborough University , Loughborough, United Kingdom .
authors.forename: Mark P
authors.initials: MP
authors.lastname: Lewis
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004558:Electric Stimulation; D005337:Fibrin / Q000378:metabolism; D007150:Immunohistochemistry; D009046:Motor Neurons / Q000166:cytology; D009119:Muscle Contraction / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D018482:Muscle, Skeletal / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D005337:Fibrin