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. 2021 Mar 8;11:5418. doi: 10.1038/s41598-021-84123-x

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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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Time course regarding the occurrence and impact of key factors on the calcification process analyzed in collagen scaffold. The timing of the various factors involved was investigated (A–F) using the CAM assay. A marked increase in cell infiltration and vessel density in the scaffold was observed on day 1 (A, B). On day 3, there was a distinct increase in collagen and glycosaminoglycan (GAG) content (C, D) and from day 5 on, an accumulation of calcium deposits could be sufficiently detected (E), which could also be shown by microCT analysis (F). Schematic scaffold with the localization of key factors is shown after 7 days of incubation on the CAM.