Mechanical loading stimulates hypertrophy in tissue‐engineered skeletal muscle: Molecular and phenotypic responses

Figure 1. Experimental diagrams of: (a) CAD model of the MSB used for mechanical overload, i) tissue engineered skeletal muscle mould, ii) mechanical loading bar, iii) culture plate border containing three loading portals, iv) movement arm containing two rods attached to loading bar, and v) stepper motor. (b) Graphical illustration of progressive mechanical overload regime [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. Relative mRNA expression of IGF‐1, MMP‐2, MMP‐9, MAFbx, and MuRF‐1 after mechanical overload (Control [CON] [n = 4], 0 [n = 3] ,21 [n = 6] and 45 hr [n = 4]). Values are normalized to POLR2B and made relative to static engineered muscles (CON). Data are expressed as mean ± SD. Significant values are identified using * where a significance of p ≤ 0.05 was achieved. IGF‐1, insulin like growth factor‐1; MMP‐2, matrix metalloprotease‐2; MMP‐9, matrix metalloprotease‐9; MuRF‐1, muscle ring finger protein‐1; MAFbx, muscle atrophy F box; mRNA, messenger RNA; POLR2B, RNA polymerase II beta; SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3. Phosphorylation of p70S6 kinase, 4EBP‐1, and Akt at various time points after mechanical loading. Phosphorylated p70S6 kinase, 4EBP‐1, and Akt is normalized to the Gel Code blue stain reagent stain. Data are expressed as mean ± SD for n = 7 engineered muscles. Significant values are identified using * where a significance of p ≤ .05 was achieved. SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4. Fluorescent staining of the nucleic DNA (blue) and the actin cytoskeleton (red) in engineered muscles with (×40 magnification) CON, 21 and 45 hr postmechanical load (a) myotubes subject to no stretch (CON; n = 6), (b) myotubes subject to no stretch 21 hr post (n = 3), (c) myotubes subject to no stretch 45 hr post (n = 3), (d) myotubes 21 hr after load (n = 6), (e) myotube 45 hr after load (n = 6), (f) Myotube width (μm), (g) nuclei per myotube, (h) fusion index (%), and (i) total nuclei of CON(no stretch), 21 and 45 hr after mechanical loading (blue bars represent static engineered muscles and red bars represent loaded engineered muscles). Scale bar represents 50 μm. Significantly different values are identified using*. Data representative of three experimental repeats and presented as mean ± SD. SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5. Maximal contractile force from 50 µl engineered skeletal muscle cultures at each time point 21 and 45 hr after mechanical loading. All cultures were compared to CON at day 14 within individual experimental repeats to calculate relative force. Data are representative of three experimental repeats and expressed as mean ± SD, for n = 9 engineered muscles. SD, standard deviation [Color figure can be viewed at wileyonlinelibrary.com]