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. 2020 Feb 12;11(4):267–285. doi: 10.1007/s13238-020-00692-z

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© The Author(s) 2020

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/.

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Heterotopic ossification of hydrogels with gradient mechanical cuesin vivo. (A) H&E staining of the hydrogels when implanted subcutaneously for 4 weeks, the black arrows indicate the oriented growth of cells around the GSNF hydrogels; (B) Immunofluorescence analysis of osteogenic/chondrogenic-related markers of the hydrogels when implanted subcutaneously for 4 weeks. Cell nuclei are stained in blue, Runx2, OCN, and OPN are stained in green, SOX9, COL II and Acan are stained in red. Scale bar 100 μm; (C) H&E staining of the hydrogels when implanted subcutaneously for 8 weeks; (D) Immunofluorescence analysis of osteogenic/chondrogenic-related markers of the hydrogels when implanted subcutaneously for 8 weeks. Cell nuclei are stained in blue, Runx2, OCN, and OPN are stained in green, SOX9, COL II and Acan are stained in red. Scale bar 100 μm; (E and F) Quantitative analysis of fluorescence intensity of osteogenic/chondrogenic specific proteins when implantation for 4 and 8 weeks. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001