Functional evaluation of nerve-skeletal muscle constructs engineered in vitro

Summary 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.     

Novel method for fabrication of skeletal muscle construct from the C2C12 myoblast cell line using serum‐free medium AIM‐V

We have fabricated muscle tissue from murine myoblast cell line C2C12 by modifying the previously reported method. Fabrication of skeletal muscle tissue has been performed in many ways including the use of a biodegradable scaffold, a collagen gel‐embedded culture, or cell sheet tissue engineering, but the extent of tension generation remains low. Recently, a new skeletal muscle tissue engineering technique involving self‐dissociation of a cell sheet from a laminin‐coated polydimethylsiloxane surface was reported which mostly involved a primary cell culture or co‐culture of C2C12 and 10T1/2 cells. In this study, we succeeded in fabricating muscle tissue using C2C12 cells alone by enhancing cell–cell attachment by the use of serum‐free medium AIM‐V. C2C12 cells were seeded on to a laminin‐coated PDMS surface in a 35 mm culture dish with two silk sutures of 5 mm in length each pinned at two places 18 mm apart. Then, cells were allowed to differentiate in AIM‐V, and the cells started to dissociate in a sheet‐like manner after 5–8 days of differentiation. The cells remained attached to the silk sutures, and tissue having a cylindrical morphology was fabricated. After the cylindrical morphology had been obtained, the medium was changed to DMEM supplemented with 2% horse serum, followed by culture for an additional 5–8 days for maturation. Tissue fabricated using this method was excitable with electric pulse stimulation and the generated active tension was approximately 1.4× greater than that reported previously for a co‐culture of C2C12 and 10T1/2 cells. Immuno‐fluorescence study revealed the presence of a sarcomere structure within the fabricated tissue, and Western blotting confirmed the expression of muscle specific‐proteins. The increased active tension generation compared to that with the previously reported method is probably attributable to the increased proportion of myogenic cells in the tissue. Myooid fabricated from mono‐culture of C2C12 will be useful in the muscle study, especially in the area where gene modification is needed. Biotechnol. Bioeng. 2009;103: 1034–1041. © 2009 Wiley Periodicals, Inc.     

Glucose transporter content and glucose uptake in skeletal muscle constructs engineered in vitro

Engineered muscle may eventually be used as a treatment option for patients suffering from loss of muscle function. The metabolic and contractile function of engineered muscle has not been well described; therefore, the purpose of this experiment was to study glucose transporter content and glucose uptake in engineered skeletal muscle constructs called myooids. Glucose uptake by way of 2-deoxyglucose and GLUT-1 and GLUT-4 transporter protein content was measured in basal and insulin-stimulated myooids that were engineered from soleus muscles of female Sprague-Dawley rats. There was a significant increase in the basal 2-deoxyglucose uptake of myooids compared with adult control (fivefold), contraction-stimulated (3.4-fold), and insulin-stimulated (threefold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). In addition, there was a significant increase in the insulin-stimulated 2-deoxyglucose uptake of myooids compared with adult control soleus muscles in basal conditions (6.5-fold) and adult contraction-stimulated (4.5-fold) and insulin- stimulated (3.9-fold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). There was a significant 30% increase in insulin-stimulated compared with basal 2-deoxyglucose uptake in the myooids. The myooid GLUT-1 protein content was 820% of the adult control soleus muscle, whereas the GLUT-4 protein content was 130% of the control soleus muscle. Myooid GLUT-1 protein content was 6.3-fold greater than GLUT-4 protein content, suggesting that the glucose transport of the engineered myooids is similar in several respects to that observed in both fetal and denervated skeletal muscle tissue.     

Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro

Summary Our purpose was to engineer three-dimensional skeletal muscle tissue constructs from primary cultures of adult rat myogenic precursor cells, and to measure their excitability and isometric contractile properties. The constructs, termed myooids, were muscle-like in appearance, excitability, and contractile function. The myooids were 12 mm long and ranged in diameter from 0.1 to 1 mm. The myooids were engineered with synthetic tendons at each end to permit the measurement of isometric contractile properties. Within each myooid the myotubes and fibroblasts were supported by an extracellular matrix generated by the cells themselves, and did not require a preexisting scaffold to define the size, shape, and general mechanical properties of the resulting structure. Once formed, the myooids contracted spontaneously at approximately 1 Hz, with peak-to-peak force amplitudes ranging from 3 to 30 μN. When stimulated electrically the myooids contracted to produce force. The myooids (n=14) had the following mean values: diameter of 0.49 mm, rheobase of 1.0 V/mm, chronaxie of 0.45 ms, twitch force of 215 μN, maximum isometric force of 440 μN, resting baseline force of 181 μN, and specific force of 2.9kN/m². The mean specific force was approximately 1% of the specific force generated by control adult rat muscle. Based on the functional data, the myotubes in the myooids appear to remain arrested in an early developmental state due to the absence of signals to promote expression of adult myosin isoforms.