Professor Rensselaer Polytechnic Institute Troy, New York, United States
Introduction:: Current treatments often fail to fully restore biomechanical function to damaged skeletal muscle, leading some researchers to explore tissue engineering approaches for skeletal muscle replacements [1]. One of the greatest challenges to engineering functional skeletal muscle is creating constructs that can generate the active contractile forces skeletal muscles produce in the body. Our lab studies the influence of biophysical stimuli (e.g., mechanical, electrical stimulations) on skeletal muscle fiber development in vitro to: engineer constructs that better mimic native skeletal muscle, accelerate construct maturation, and inform future tissue engineering approaches [2].
Materials and Methods:: To engineer fibers, u-shaped growth channels (150µm-wide, 300µm-deep, 17.5mm-long) are molded in agar, and 4-mm holes are punched on each end, wherein collagen discs are inserted to act as anchors [3]. Then, diluted fibronectin is wicked through the growth channel, and the channel is seeded with primary heterogeneous cells (~5 million cell/mL; from minced mouse gastrocnemius muscle), which self-assemble to form a fiber. A custom single-fiber bioreactor is employed to provide biophysical stimulation [4] Previously, we studied mechanical and electrical stimulation, independently. Herein we investigate concurrent stimulation to explore their potential synergies. Our mechanical stimulation utilized a 4hr-on 4hr-off, 0.5% cyclic strain at 0.1Hz, starting 10 hours post-seeding, and the electrical stimulation utilized a 0.5V biphasic square waveform at either 0.2Hz (Concurrent 1) or 1.0Hz (Concurrent 2), starting 3 hours post-seeding, and continued for 72 hours. Fibers were evaluated for their material and contractile properties at 1, 3, and 7 days post-seeding. We evaluated active (contractile) properties using a previously established calcium-activation technique, wherein the fibers are permeabilized, sectioned, then t-clipped [5]. Each t-clipped fiber sample was placed into a well of low-calcium (relaxing) solution, and subjected to 1% stretch to determine the relaxed modulus (stress-strain curve’s linear region slope), then switched to a high-calcium (activating) solution and subjected to the same 1% stretch to calculate the “total” modulus. Relaxed stress-strain values were subtracted from the total trace to obtain the active component. In addition, passive material properties were characterized in intact fibers samples, using a constant rate elongation-to-failure [3].
Results, Conclusions, and Discussions:: Concurrent stimulation significantly improved active and passive biomechanical properties of engineered skeletal muscle fibers, compared to the same electrical and mechanical stimuli delivered independently – for both concurrent stimulation protocols explored. Concurrent electrical and mechanical stimulation caused a 10-fold increase in the strength and toughness of engineered skeletal muscle fibers (data not shown), suggesting an increase in matrix deposition and/or alignment. In general, when delivered independently, electrical stimulation had a greater influence than mechanical stimulation on the active component of instantaneous modulus (Fig. 1). However, when the electrical stimulation was supplemented with mechanical stimulation, the active instantaneous modulus was strikingly increased – by ~10-fold in Concurrent 1, and ~3-fold in Concurrent 2 (Fig. 1). These findings suggest much more robust actin-myosin cross-bridging within the engineered fiber. Excitedly, active characterization also revealed that supplementing 1.0Hz electrical stimulation with mechanical stimulation showed signs of accelerated maturation, with combined stimulation producing similar work and power at day 3 compared to 7d electrically stimulated fibers, with these values continuing to increase by day 7 (data not shown). Conclusion: Taken together, these findings show the potential synergies of electrical and mechanical stimuli, where their combined delivery produced biomechanical properties far greater than the sum of their individual influences. Utilizing this scaffold-free approach will provide a novel platform for unique insight into the role of biophysical stimuli on muscle development, and how to leverage these stimuli to accelerate maturation and tune the biomechanical performance of engineered skeletal muscle replacements.
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References (Optional): : [1] Hawke TJ et.al., (2001) J App Physio; [2] Benam KH et al., (2015), Annu. Rev. Pathol. Mech. Dis.; [3] Mubyana K et al., (2018) Tissue Eng. Part A; [4] Van Houten et. al, (2022) J Biomech Eng. [5] Swank DM (2012), Methods.
