Tendon, ligament, and skeletal muscle are highly-organized tissues that largely rely on a hierarchical collagenous matrix to withstand high tensile loads experienced in activities of daily life. This critical biomechanical role predisposes these tissues to injury, and current treatments fail to recapitulate the biomechanical function of native tissue. This has prompted researchers to pursue engineering functional tissue replacements, or dysfunction/disease/development models, by emulating in vivo stimuli within in vitro tissue engineering platforms; specifically mechanical stimulation, as well as active contraction in skeletal muscle. Mechanical loading is critical for matrix production and organization in the development, maturation, and maintenance of native tendon, ligament, and skeletal muscle, as well as their interfaces. Tissue engineers seek to harness these mechanobiological benefits using bioreactors to apply both static and dynamic mechanical stimulation to tissue constructs, and induce active contraction in engineered skeletal muscle. The vast majority of engineering approaches in these tissues are scaffold-based, providing interim structure and support to engineered constructs, and sufficient integrity to withstand mechanical loading. Alternatively, some recent studies have employed developmentally-inspired scaffold-free techniques, relying on cellular self-assembly and matrix production to form tissue constructs. Whether utilizing a scaffold or not, incorporation of mechanobiological stimuli has been shown to improve the composition, structure, and biomechanical function of engineered tendon, ligament, and skeletal muscle. Together, these findings highlight the importance of mechanobiology and suggest how it can be leveraged to engineer these tissues and their interfaces, and to create functional multi-tissue constructs.

A muscle undergoing cyclical contractions requires fast and efficient muscle activation and relaxation to generate high power with relatively low energetic cost. To enhance activation and increase force levels during shortening, some muscle types have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. SA's complementary phenomenon is shortening deactivation (SD), a delayed decrease in force following muscle shortening. SD increases muscle relaxation, which decreases resistance to subsequent muscle lengthening. While it might be just as important to cyclical power output, SD has received less investigation than SA. To enable mechanistic investigations into SD and quantitatively compare it to SA, we developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. When normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease, Lethocerus IFM dropped by 97%, and Drosophila jump muscle decreased by 37%. The same order was found for normalized SA tension: Drosophila IFM increased by 233%, Lethocerus IFM by 76%, and Drosophila jump muscle by only 11%. SD occurred slightly earlier than SA, relative to the respective length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. Our results suggest SA and SD evolved to enable highly efficient IFM cyclical power generation and may be caused by the same mechanism. However, jump muscle SA and SD mechanisms are likely different, and may have evolved for a role other than to increase the power output of cyclical contractions.

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Bioreactors are commonly used to apply biophysically-relevant stimulations to tissue-engineered constructs in order to explore how these stimuli influence tissue development, healing, and homeostasis, and they offer great flexibility because key features of the stimuli (e.g., duty cycle, frequency, amplitude, duration) can be controlled to elicit a desired cellular response. However, most bioreactors that apply mechanical and electrical stimulations do so to a scaffold after the construct has developed, preventing study of the influence these stimuli have on early construct development. To enable such exploration, there is a need for a bioreactor that allows the direct application of mechanical and electrical stimulation to constructs as they develop. Herein, we develop and calibrate a bioreactor, based on our previously established modified Flexcell system, to deliver precise mechanical and electrical stimulation, either independently or in combination, to developing scaffold-free tissue constructs. Linear calibration curves were established, then used to apply precise dynamic mechanical and electrical stimulations, over a range of physiologically relevant strains and voltages respectively. Following calibration, applied mechanical and electrical stimulations were not statistically different from their desired target values, and were consistent whether delivered independently or in combination. Concurrent delivery of mechanical and electrical stimulation resulted in a negligible change in mechanical (< 2%) and electrical (<1%) values, compared to their independently-delivered values. With this calibrated bioreactor, we can apply precise, controlled, reproducible mechanical and electrical stimulations, alone or in combination, to scaffold-free, tissue engineered constructs as they develop.

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A muscle undergoing a cyclical lengthening and shortening contraction pattern requires fast and efficient muscle activation and relaxation. To boost activation rate and increase force levels during shortening, some muscles have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. Equally important is a fast relaxation rate and low force during muscle lengthening. If a muscle incompletely relaxes, its antagonist muscle expends more energy lengthening it, causing decreased power generation and efficiency. Thus, increasing relaxation rate can increase work and power. However, increasing relaxation rate is costly because it typically occurs via ATPases pumping calcium out of the myoplasm. Some flying insect species have greatly reduced this cost in their indirect flight muscle (IFM) by evolving a property known as shortening deactivation (SD), which decreases force levels during a contraction cycle without having to change calcium concentration. SD is a delayed decrease in force following rapid shortening of an active muscle. In IFM, the timing of this force decrease allows the antagonist muscle (DVM) to be lengthened with much less resistance from the agonist muscle (DLM). While there is general appreciation for SD in IFM, there have been few investigations into whether SD is present in other muscle types and if so, how much it benefits these muscles. Even less is known about SD’s mechanism as there have been no investigations into the SD mechanism for any muscle type. To enable mechanistic investigations into SD and quantitatively compare it to SA, we developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. All three muscle types exhibited a 4‐phase tension transient in response to a 1% length decrease, with phase 3 defined as SD. Lethocerus IFM presented the greatest SD tension drop, 20 mN/mm2, which was ~1.5‐fold greater than Drosophila jump muscle and ~10‐fold greater than DrosophilaIFM. However, isometric tension in Drosophilajump muscle, 34.3 mN/mm2, was higher than Lethocerus, 18.4 mN/mm2, and DrosophilaIFM, 1.6 mN/mm2. Thus, when normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease. Similarly, SD tension in Lethocerus IFM dropped by 97%, while Drosophilajump muscle decreased by 37%. The same order occurred for normalized phase 3 SA tension: Drosophila IFM displayed the largest gain, 233%, Lethocerus IFM increased by 76%, and Drosophila jump muscle showed only a 11% increase. SD phase 3 occurred a bit earlier than SA phase 3, relative to the length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. We suggest that SA and SD evolved together to enable fast and efficient IFM cyclical power generation and may be caused by the same molecular mechanism. However, the SA and SD molecular mechanisms in jump muscle are likely different and might have evolved for an alternate role besides increasing the power output of cyclical muscle contracti...