Functional evaluation of nerve–skeletal muscle constructs engineered in vitro

Department of Biomedical Engineering, Division of Geriatric Medicine, Muscle Mechanics Laboratory, Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109-2007, USA.
In Vitro Cellular & Developmental Biology - Animal (Impact Factor: 1.15). 03/2006; 42(3-4):75-82. DOI: 10.1290/0509064.1
Source: PubMed


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. Nerve- muscle 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.

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Available from: Robert Dennis, Aug 10, 2014
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    • "Unlike fibroblast and muscle cell monolayers, a nerve monolayer does not contract and remodel into a 3-D construct. Previous work from our lab indicated that neural cells would proliferate and form a network of cells across an established monolayer of muscle (Larkin et al. 2006b). The objective of this research was to use similar technologies to grow a nerve monolayer on an existing fibroblast monolayer so that when the fibroblast monolayer contracts due to passive tension between the fibroblast cells, a 3-D construct with an external fibroblast sheath and an internal core of interconnected nerve cells forms. "
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    • "These nerve–muscle constructs show spontaneous contractions and can also be stimulated (Wagner et al., 2003). Larkin et al. showed in vitro that stimulated nerve–muscle constructs had better contractility characteristics than muscle-only constructs: muscle-only constructs produced a twitch force of 40 µN and a tetanic force of 95 µN, while stimulated nerve–muscle constructs produced a twitch force of 100 µN and a tetanic force of 200 µN (Larkin et al., 2006). Dhawan et al. (2007) showed that in vivo neurotization of skeletal muscle constructs led to better contractibility ex vivo than nonneurotizated skeletal muscle constructs. "
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