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. 2018 Jan 19;8:1256. doi: 10.1038/s41598-018-19677-4

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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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CPP in fresh plasma. Total CPP levels were measured in plasma samples from three control subjects (C1, C2, and C3, indicated as blue bars) and three dialysis patients (D1, D2, and D3, indicated as red bars). (a) Total CPP in fresh plasma. (b) Total CPP in the plasma samples frozen and thawed once. The aliquots of the fresh plasma samples used in (a) were snap-frozen in liquid nitrogen and stored at −80 °C until thawed for the CPP assay. (c) Pull-down of CPP from the fresh plasma and the freeze-thawed plasma samples using alendronate beads as in Fig. 1c. The arrow indicates fetuin-A. The full-length gel was shown in Supplementary information. (d) The transition of amorphous calcium-phosphate to crystalline calcium-phosphate induced by freeze-thaw. Nanoparticles of amorphous calcium-phosphate were suspended in DMEM with different pH (6.8, 7.4, and 8.0). The binding capacity of OsteoSense to the particles were quantified before and after one cycle of freeze-thaw.