Functional evaluation of nerve-skeletal muscle constructs engineered in vitro
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Previously, we have engineered three-dimensional (3-D) skeletal muscle constructs that generate force and display a myosin heavy-chain (MHC) composition of fetal muscle. The purpose of this study was to evaluate the functional characteristics of 3-D skeletal muscle constructs cocultured with fetal nerve explants. We hypothesized that coculture of muscle constructs with neural cells would produce constructs with increased force and adult MHC isoforms. Following introduction of embryonic spinal cord explants to a layer of confluent muscle cells, the neural tissue integrated with the cultured muscle cells to form 3-D muscle constructs with extensions. Immunohistochemical labeling indicated that the extensions were neural tissue and that the junctions between the nerve extensions and the muscle constructs contained clusters of acetylcholine receptors. Compared to muscles cultured without nerve explants, constructs formed from nerve-muscle coculture showed spontaneous contractions with an increase in frequency and force. Upon field stimulation, both twitch (2-fold) and tetanus (1.7-fold) were greater in the nerve-muscle coculture system. Contractions could be elicited by electrically stimulating the neural extensions, although smaller forces are produced than with field stimulation. Severing the extension eliminated the response to electrical stimulation, excluding field stimulation, as a contributing factor. Nervemuscle constructs showed a tendency to have higher contents of adult and lower contents of fetal MHC isoforms, but the differences were not significant. In conclusion, we have successfully engineered a 3-D nerve-muscle construct that displays functional neuromuscular junctions and can be electrically stimulated to contract via the neural extensions projecting from the construct.
- Baker, E. L.; Dennis, R. G.; Larkin, L. M. Glucose transporter content and glucose uptake in skeletal muscle constructs engineered in vitro. In Vitro Cell. Dev. Biol. 39A (10): 434–439; 2003. CrossRef
- Bryers, P. S.; Ecob, M. S. Differentiation in dystrophic muscle cultures from mice of different ages. Muscle Nerve 7 (4): 332–336; 1984. CrossRef
- Burden, S. J. The formation of neuromuscular synapses. Genes Dev. 12 (2): 133–148; 1998.
- Close, R. Effects of cross-union of motor nerves to fast and slow skeletal muscles. Nature 206 (986): 831–832; 1965. CrossRef
- Close, R. Dynamic properties of fast and slow skeletal muscles of the rat after nerve cross-union. J. Physiol. 204 (2): 331–346; 1969.
- Dennis, R. G.; Kosnik, P. E. II. Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro. In Vitro Cell. Dev. Biol. 36A (5): 327–35; 2000. CrossRef
- Dennis, R. G.; Kosnik, P. E., II; Gilbert, M. E.; Faulkner, J. A. Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines. Am. J. Physiol. Cell Physiol. 280 (2): C288-C295; 2001.
- Dorchies, O. M.; Laporte, J.; Wagner, S.; Hindelang, C.; Warter, J. M.; Mandel, J. L.; Poindron, P. Normal innervation and differentiation of X-linked myotubular myopathy muscle cells in a nerve-muscle coculture system. Neuromuscul. Disord. 11 (8): 736–746; 2001. CrossRef
- Ecob, M. S. The application of organotypic nerve cultures to problems in neurology with special reference to their potential use in research into neuromuscular diseases. J. Neurol. Sci. 58 (1): 1–15; 1983. CrossRef
- Ecob, M. The location of neuromuscular junctions on regenerating adult mouse muscle in culture. J. Neurol. Sci. 64 (2): 175–182; 1984. CrossRef
- Ecob, M. S.; Butler-Browne, G. S.; Whalen, R. G. The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system. Differentiation 25 (1): 84–87; 1983. CrossRef
- Irintchev, A.; Rosenblatt, J. D.; Cullen, M. J.; Zweyer, M.; Wernig, A. Ectopic skeletal muscles derived from myoblasts implanted under the skin. J. Cell Sci. 111 (Pi. 22): 3287–3297; 1998.
- Kobayashi, T.; Askanas, V.; Engel, W. K. Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation. J. Neurosci. 7 (10): 3131–3141; 1987.
- Kosnik, P. E.; Faulkner, J. A.; Dennis, R. G. Functional development of engineered skeletal muscle from adult and neonatal rats. Tissue Eng. 7 (5): 573–584; 2001. CrossRef
- Sanes, J. R.; Lichtman, J. W. Development of the vertebrate neuromuscular junction. Annu. Rev. Neurosci. 22: 389–442; 1999. CrossRef
- Schiaffino, S.; Gorza, L.; Sartore, S.; Saggin, L.; Carli, M. Embryonic myosin heavy chain as a differentiation marker of developing human skeletal muscle and rhabdomyosarcoma: a monoclonal antibody study. Exp. Cell Res. 163 (1): 211–220; 1986. CrossRef
- Schiaffino, S.; Murgia, M.; Serrano, A. L.; Calabria, E.; Pallafacchina, G. How is muscle phenotype controlled by nerve activity?. Ital. J. Neurol. Sci. 20 (6): 409–412; 1999.
- Strohman R. C.; Bayne, E.; Spector, D.; Obinata, T.; Micou-Eastwood, J.; Maniotis, A. Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro. In Vitro Cell. Dev. Biol. 26A (2): 201–208; 1990. CrossRef
- Talmadge, R. J.; Roy, R. R. Electrophoretic separation of rat skeletal muscle myosin heavy-chain isoforms. J. Appl. Physiol. 75 (5): 2337–2340; 1993.
- Wagner, S.; Dorchies, O. M.; Stoeckel, H.; Warter, J. M.; Poindron, P.; Takeda, K. Functional maturation of micotinic acetylcholine receptors as an indicator of murine muscular differentiation in a new nerve-muscle co-culture system. Pflugers Arch. 447 (1): 14–22; 2003. CrossRef
- Whalen, R. G.; Bugaisky, L. B.; Butler-Browne, G. S.; Ecob, M. S.; Pinset, C. Synthesis and accumulation of myosin isozymes in tissue culture. Adv. Exp. Med. Biol. 182: 193–199; 1985.
- Whalen, R. G.; Johnstone, D.; Bryers, P. S.; Butler-Browne, G. S.; Ecob, M. S.; Jaros, E. A developmentally regulated disappearance of slow myosin in fast-type muscles of the mouse. FEBS Lett. 177 (1): 51–56; 1984. CrossRef
- Functional evaluation of nerve-skeletal muscle constructs engineered in vitro
In Vitro Cellular & Developmental Biology - Animal
Volume 42, Issue 3-4 , pp 75-82
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- neuromuscular junction
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- 1. Department of Biomedical Engineering, University of Michigan, 48109-2007, Ann Arbor, Michigan
- 2. Division of Geriatric Medicine, University of Michigan, 48109-2007, Ann Arbor, Michigan
- 3. Muscle Mechanics Laboratory, University of Michigan, 48109-2007, Ann Arbor, Michigan
- 5. Department of Mechanical Engineering, University of Michigan, 48109-2007, Ann Arbor, Michigan
- 6. Department of Biomedical Engineering, University of North Carolina at Chapel Hill, 27599-7575, Chapel Hill, North Carolina