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. 2022 Sep 5;13:5211. doi: 10.1038/s41467-022-32868-y

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© The Author(s) 2022

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. 3 — a Workflow of sample isolation, mRNA libraries preparation, and single-cell RNA sequencing of regenerated tissue from critical-sized calvarial defects with or without the implantation of in situ culture system of NSs-loaded 3D printed hydrogel scaffold, at both 1 week and 2 weeks after defect surgery. n = 6 rats per group and per time point (Created with BioRender.com). b tSNE plot shows the distribution of 26016 cells that divided into 33 sub-clusters. c Heatmap shows expression of marker genes of the 33 sub-clusters. Marker genes were selected according to p value (Wilcoxon Rank Sum test, top 10). d–g tSNE plots of the expression of specific genes, which mark four different cell populations (osteo-lineage cell population: Col1a1 and Postn; endothelial cell population: Cdh5 and Vwf; perivascular cell population: Acta2 and Pdgfrb; immune-lineage cell population: CD45 and CD14). h Distribution of cells from different time and treatments that visualized by tSNE plots. 1w represents 1 week after defect surgery; 2w represents 2 weeks after defect surgery. i, j Barplots of the relative proportion of different sub-clusters in three main cell populations between two groups at 1 week and 2 weeks after surgery, respectively.