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. 2021 Jun 3;11:11695. doi: 10.1038/s41598-021-91203-5

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Physiological functionality of engineered tissues is enhanced via neuromuscular junction formation. (a) Representative maximal induced tetanus contraction profiles of muscle (SkM -MN) and neuromuscular tissue (SkM + MN) and quantification of tetanic force. (b) Spontaneously occurring twitch force profiles in engineered tissues with (SkM + MN) and without (SkM–MN) iPSC motor neuron spheroids, and quantification of spontaneous twitch force driven via motor nerve inclusion. (c) Inhibition of the acetylcholine receptor in spontaneously contracting neuromuscular tissues via addition of 100 µM d-Tubocurarine. (d) Peak twitch force induced via electrical field stimulation. (e) Time to peak twitch force and (f) half relaxation time as physiological measures of contractile function. (g) Representative twitch profiles evidencing reduced time to peak twitch and shorter relaxation profiles in SkM + MN tissues. Δ change indicative of normalised data to SkM–MN. Beginning of representative tetanus and twitch contractions represents initiation of electrical field stimulation, time elapsed before this point is un-stimulated baseline recording. Individual functional data points indicative of n = 3 contraction profiles per tissue, totalling minimum of n = 12 and maximum of n = 18 contractions per condition and presented ± standard deviation (SD). **P ≤ 0.01 and ***P ≤ 0.001.