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. 2018 Jun 12;10(2):20. doi: 10.1038/s41368-018-0021-2

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© The Author(s) 2018

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Release of TGF-β1 and BMP-2 proteins from the cortical bone and the induction of Smad signaling in mesenchymal stromal ST2 cells. a, b ELISA quantification of TGF-β1 (a) and BMP-2 (b) proteins contained in bone-conditioned medium extracted from the cortical bone chips with either Ringer’s solution (BCM-RS) or Ringer’s solution mixed with autologous serum (BCM-RS+S) at a 1:1 ratio. The two types of media were collected at 10, 20, and 40 min, as well as at 1, 3, and 6 d. Controls (ctrl) represent RS or RS+S, not containing bone particles (controls exhibited no detectable levels of the two proteins tested). Mean±standard deviations represent three independent BCM preparations, and significant differences to the respective controls (***P < 0.001, **P < 0.01, ns non-significant) are shown. c, d BCM induces enhanced phosphorylation of TGF-β1- and BMP-2-specific R-Smads in mesenchymal stromal ST2 cells. Immunoblot analyses of phospho-Smad2 (pSmad2) (c) and phospho-Smad1/5/8 (pSmad1/5/8) (d) proteins in whole-cell extracts from ST2 cells treated with BCM-RS or BCM-RS+S used in a, b. The bar charts represent densitometric quantification of the immunoblots. pSmad2 and pSmad1/5/8 levels are normalized to vinculin loading controls. Data represent mean±SD obtained from three independent experiments. Significant differences to the control cells treated with RS or RS+S (**P < 0.01, *P < 0.05) are shown