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. 2021 Jun 24;11:13204. doi: 10.1038/s41598-021-92607-z

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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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(A) Schematic of the experimental time line for spheroids incubated in the osteogenesis induction medium for 7 days. (B) Morphology of the spheroid after a 7-day incubation period. The scale bar represents 100 μm. (C) Mean values of projected area of the spheroids after 2 and 7 days. F-test was performed to examine the significance of area changes in spheroids (n = 60 from 5 independent experiments; *p < 0.05, **p < 0.005). (D) Relative mRNA expressions of stem cell markers (Nanog, Oct4, and Sox2), osteoblast markers (Runx2, Alpl, and Col1a1), and osteocyte markers (Opn, Phex, and Sost) in the spheroids at 2-day and 7-day incubation were measured by real-time PCR. All the mRNA expressions were normalized to Gapdh expressions while the results were expressed as relative amounts against the expression of monolayer sample (n = 8). The bars represent the mean ± standard error. P-value was calculated from Student’s t-test; ^#p < 0.05, ^##p < 0.005. (E) Immunostaining images of spheroid after 2-day incubation; cell nuclei (DAPI in cyan), actin filaments (F-actin in red), and sclerostin (SOST in green). The scale bar represents 100 μm.