The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle

Abstract

One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogelbased neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca 2+ handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force-length and force-frequency relationships achieving tetanus at 40 Hz. The use of 2 mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4±0.3 mN as compared to 0.9±0.4 mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8±0.5 mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio=11.4±4.1 vs. 6.5±1.9 μg/μg in control) and a prolonged Ca 2+ transient half-width (Ca 50 =232.8±33.3 vs. 101.7±19.8 ms). The use of growth-factor-reduced Matrigel instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle.

Local Tissue Geometry Determines Contractile Force Generation of Engineered Muscle Networks

“…Parameters of a single twitch including amplitude (A t ), the time-to-peak twitch (TPT, from the onset of electrical stimulus to the time of peak twitch) and half relaxation time (RT 1/2 , from the time of peak twitch to 50% recovery) were derived from the force traces, as previously described. 24 The network was then stimulated every 5 min by a 1 s long pulse train with increasing frequency (5,10,20,40, and 60 Hz) until tetanus was reached. Parameters of tetanus including peak amplitude (A T ) and the tetanus-to-twitch ratio (TtR = A T /A t ) were calculated from the force traces.…”

Tissue Contraction Force Microscopy for Optimization of Engineered Cardiac Tissue

“…Finally, constrained gel compaction is the primary effector of cell and fiber alignment in threedimensional engineered tissues, and comparisons between aligned and isotropic ECTs show that alignment leads to increased twitch force. 16 All these factors associated with differential gel thickness can and will play into the contractile force measured for gel-based ECTs by any method [16][17][18][19][20][21][22] and need to be considered when interpreting the data.…”

“…18 However, free-floating assays do not measure cell forces directly, instead rely on theory to relate the tissue compaction to cell forces. 19,20 In addition, while macrosized tissues 16 are easy to prepare and conducive to force measurement without a highly specialized equipment, they are low throughput, and screening many factors quickly becomes cumbersome and taxing on resources.…”

Biomimicry, Biofabrication, and Biohybrid Systems: The Emergence and Evolution of Biological Design

“…The bioactuator was shown to generate passive tension forces comparable to other demonstrations of tissue engineered skeletal muscle. [118,119] The passive tension force generated could be increased by imposing a static mechanical stretch stimulus during differentiation, and by the addition of biochemical factors, such as human insulin-like growth factor 1 (IGF-1) to the differentiation medium. This was an important confirmation that the adaptive behavior inherent to natural biological systems could be mimicked in biohybrid systems cultured in vitro.…”