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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 2595-2604 |
| Number of pages | 10 |
| Journal | Tissue Engineering - Part A |
| Volume | 21 |
| Issue number | 19-20 |
| DOIs | |
| State | Published - Oct 1 2015 |
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ASJC Scopus subject areas
- Bioengineering
- Biochemistry
- Biomedical Engineering
- Biomaterials
Cite this
Neuromuscular junction formation in tissue-engineered skeletal muscle augments contractile function and improves cytoskeletal organization. / Martin, Neil R W; Passey, Samantha L.; Player, Darren J.; Mudera, Vivek; Baar, Keith; Greensmith, Linda; Lewis, Mark P.
In: Tissue Engineering - Part A, Vol. 21, No. 19-20, 01.10.2015, p. 2595-2604.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Neuromuscular junction formation in tissue-engineered skeletal muscle augments contractile function and improves cytoskeletal organization
AU - Martin, Neil R W
AU - Passey, Samantha L.
AU - Player, Darren J.
AU - Mudera, Vivek
AU - Baar, Keith
AU - Greensmith, Linda
AU - Lewis, Mark P.
PY - 2015/10/1
Y1 - 2015/10/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84944062972&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84944062972&partnerID=8YFLogxK
U2 - 10.1089/ten.tea.2015.0146
DO - 10.1089/ten.tea.2015.0146
M3 - Article
C2 - 26166548
AN - SCOPUS:84944062972
VL - 21
SP - 2595
EP - 2604
JO - Tissue Engineering - Part A
JF - Tissue Engineering - Part A
SN - 1937-3341
IS - 19-20
ER -