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. 2022 Sep 24;17:424. doi: 10.1186/s13018-022-03305-z

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© The Author(s) 2022

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — Detection calcium deposition. The calcium deposition assessment was done on 7, 14 and 21 days of differentiation in 3D (A) and 2D (B) groups and 3D conditions compared with matched 2D cultures in these time points (C). The highest level of calcium depositions was seen on Shilajit/Alg/ASCs + OM on 21 days after differentiation compared with other groups. D Micrographs of cells stained with alizarin red demonstrate the effects of shilajit on mineral deposition and nodule formation in 2D groups on 7, 14 and 21 days after differentiation. Qualitative observations showed that the number and size of the nodules increased in the presence of Shilajit (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)