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However, the behavior of multilayer cells under cyclic stretch remains unclear. In this study, we cultured MC3T3-E1 osteoblast-like cells at high density to form multilayer cells and subjected them to cyclic stretch with an amplitude of 10% at 1\u00a0Hz. We found that the lower layer cells aligned in the direction of the stretch after 12\u00a0h, whereas the upper layer cells aligned perpendicular to the direction of stretch after 24\u00a0h. The 10% cyclic stretch was transmitted to the upper layer cells as approximately 5% at the onset of the stretch and increased over time, reaching 7% at 12\u00a0h when the lower layer cells completed alignment in the direction of stretch. This suggests that sufficient cyclic stretch transmitted to the upper layer led to the alignment of the upper layer cells in the perpendicular direction after 12\u00a0h. On the other hand, reducing intracellular tension with Y-27632 caused cells in both upper and lower layers to align in the direction of stretch. In contrast, increasing intracellular tension with calyculin A eliminated significant alignment in both layers. These findings indicate that cell alignment is closely related to intracellular tension and that the alignment of the lower layer cells in the direction of stretch may be due to a decrease in intracellular tension.<\/jats:p>","DOI":"10.1007\/s10237-025-01978-z","type":"journal-article","created":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T04:11:09Z","timestamp":1751429469000},"page":"1501-1511","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Orthogonal alignment of multilayered MC3T3-E1 cells induced by cyclic stretch"],"prefix":"10.1007","volume":"24","author":[{"given":"Shuichiro","family":"Suzuki","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ken","family":"Imajo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junfeng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeonghyun","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eijiro","family":"Maeda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuaki","family":"Nagayama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takeo","family":"Matsumoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,7,2]]},"reference":[{"issue":"16","key":"1978_CR2","doi-asserted-by":"publisher","first-page":"2999","DOI":"10.1007\/s00018-017-2511-3","volume":"74","author":"JL Bays","year":"2017","unstructured":"Bays JL, DeMali KA (2017) Vinculin in cell-cell and cell-matrix adhesions. 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