Defined Electrical Stimulation Emphasizing Excitability for the Development and Testing of Engineered Skeletal Muscle

Division of Neurobiology, Physiology and Behavior, University of California Davis, Davis, California, USA.
Tissue Engineering Part C Methods (Impact Factor: 4.64). 11/2011; 18(5):349-57. DOI: 10.1089/ten.TEC.2011.0364
Source: PubMed


Electrical stimulation is required for the maturation of skeletal muscle and as a way to nondestructively monitor muscle development. However, the wrong stimulation parameters can result in electrochemical damage that impairs muscle development/regeneration. The goal of the current study was to determine what aspect of an electrical impulse, specifically the pulse amplitude or pulse width, was detrimental to engineered muscle function and subsequently how engineered muscle responded to continuous electrical stimulation for 24 h. Acute stimulation at a pulse amplitude greater than six-times rheobase resulted in a 2.4-fold increase in the half-relaxation time (32.3±0.49 ms vs. 77.4±4.35 ms; p<0.05) and a 1.59-fold increase in fatigability (38.2%±3.61% vs. 60.6%±4.52%; p<0.05). No negative effects were observed when the pulse energy was increased by lengthening the pulse width, indicating electrochemical damage was due to electric fields at or above six-times rheobase. Continuous stimulation for 24 h at electric fields greater than 0.5 V/mm consistently resulted in ∼2.5-fold increase in force (0.30±0.04 kN/m² vs. 0.67±0.06 kN/m²; p<0.05). Forty per cent of this increase in force was dependent on the mammalian target of rapamycin (RAP) complex 1 (mTORC1), as RAP prevented this portion of the increase in force (CON=0.30±0.04 kN/m² to 0.67±0.06 kN/m² compared with RAP=0.21±0.01 kN/m² to 0.37±0.04 kN/m²; p<0.05). Since there was no increase in myosin heavy chain, the remaining increase in force over the 24 h of stimulation is likely due to cytoskeletal rearrangement. These data indicate that electrochemical damage occurs in muscle at a voltage field greater than six-times rheobase and therefore optimal muscle stimulation should be performed using lower electric fields (two- to four-times rheobase).

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Available from: Alastair Khodabukus, Mar 06, 2015
    • "However, the dimensions of these grooves were generally at the micron scale, which poorly approximates the diameter of myotubes in vivo (30–100 μm) [20] [21] [22]. Significantly, these 2D micropatterned substrates have been used only as tools for investigating mechanisms of cell function, and have not yet been developed into implantable tissue engineering constructs [23] [24]. Finally, 3D tubular scaffolds with an aligned pore structure and biologically relevant pore sizes appear to be promising for skeletal muscle tissue engineering, which generally requires a pore size of 100–150 μm; however, these dimensions are much larger than the cellular scale (∼10 μm) [18] [25]. "
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    ABSTRACT: Skeletal muscle injury can lead to severe motor deficits that adversely affect movement and quality of life. Current surgical treatments for skeletal muscle are hindered by the poor formation of organized myotube bundles at the wound site. Tissue-engineered skeletal muscle constructs to date have been unable to generate high degrees of myotube density and alignment. Generating a suitable in vitro tissue-engineered skeletal muscle construct requires the design of a scaffold that recapitulates the structural combination of nanoscale collagen fibrils and aligned microscale basal lamina tracks present in the native extracellular matrix (ECM). We hypothesized that a 3D aligned tubular porous scaffold containing aligned nanofibers inside the pores can mimic the native muscle tissue environment. We constructed a laminar section of the hypothesized scaffold with aligned chitosan-PCL nanofibers arranged co-axially with the aligned microscale chitosan scaffold bands to mimic the required myogenic environment. A 6-day study of C2C12 mouse myoblast cells cultured on this hybrid scaffold indicated that the nanofibers and scaffold bands in the scaffold played a synergetic role in directing cell orientation, interaction, migration and organization. Our results showed that aligned nanofibers mediated cell alignment and the aligned scaffold bands induced the formation of a more compact assembly of myotube cells as compared to various control substrates including chitosan films, nanofibers, and chitosan bands. The expression levels of both early and late-stage myogenic differentiation genes associated with myogenin and myosin heavy chain, respectively, were higher on the hybrid substrate than on control substrates. Our study suggests that the combination of nano and microscale topological features in the ECM can direct myogenic differentiation, and the hybrid material has the potential to improve the outcome of skeletal tissue engineering.
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    • "In the future, the in vitro skeletal muscle tissues will have to be adapted and refined, for example, by co-culturing with motor neurons to improve the comparability of the tissue-engineered constructs with in vivo muscle. Recently, Khodabukus and Baar reported that continuous 24-h EPS to 14-day culture tissue constructs at pulse amplitude of 1 V/mm and width of 4 ms, which corresponded to 100%Pt, resulted in a 2-fold increase in force27, suggesting that high %Pt may be required to increase force production when using short-duration, acute EPS. However, we demonstrated here that low %Pt was effective for long-duration, chronic EPS, as we achieved a 4.5-fold increase in force by low %Pt EPS culture (Fig. 4a) without a reduction in force production due to electrochemical damage. "
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    ABSTRACT: Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.
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    • "Passive longitudinal tension generated by anchor points Myotendinous junctions Collagen[43,45,53,67,68,71]; Fibrin[53,60,65,66]ECM ECM protein composition Collagen[43,45,53,67,68,71]; Fibrin[53]Morphology of cells Alignment and fusion of seeded cells in a single plane Collagen[45,53]; Fibrin[53,66]Active contractile phenotype (vector force) Excitability and contractility profile Fibrin[60,62,65]; Passive contractile phenotype (vector force) Cell-matrix passive contractile interaction Collagen[43,45]Myosin Heavy Chain (MYH) expression Increase in MYH-7 mRNA (slow genotype) Collagen[67]Responses to stimulation (mechanical and electrical) Increases in gene and protein expression similar to that with exercise Collagen[68,71]Fibrin[65]There can now be a high degree of confidence that the aforementioned systems do recreate a significant aspect of in vivo skeletal muscle biology. Their use to-date with respect to providing a pre-clinical test bed for exercise, genetic, pharmaceutical and nutritional therapies is limited, however the system are now at a stage where such investigations are warranted. "

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