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. 2021 Jul 16;11:14570. doi: 10.1038/s41598-021-93951-w

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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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(a–d) mRNA relative expression analysis of COL1A1, SPARC and Sox2 genes and WB assay of COL1A1 and SPARC proteins. (a–c) mRNA relative expression analysis (mean + SEM) of COL1A1, SPARC and Sox2. (a,b) mRNA relative expression analysis of the osteogenic differentiation genes COL1A1 (i.e. pro-alpha1 chains of type I collagen) and SPARC (i.e. secreted protein acidic and rich in cysteine) in non-pored (NP) and pored (P) scaffolds, 7 and 14 days after seeding. Expression levels of both genes are increased in the d14 scaffolds in comparison to the d7 ones. (c) Conversely, a progressive decrease of Sox2 mRNA level in non-pored (NP) and pored (P) scaffolds was detected at d7 and d14, confirming the differentiation of MSCs during this time window. Real-time PCR analysis was performed in triplicate (n = 6 pig per experimental group; statistics by two-way ANOVA and Bonferroni post hoc). Data are expressed as mean ± SEM. *p < 0.05; ***p < 0.001. (d) Immunoblot analysis and bar graphs showing the levels of COL1A1 and SPARC in non-pored (NP) and pored (P) scaffolds seeded with hBM-MSCs for 14 days. Cropped images are from samples run on the same gel. Pored scaffolds showed a significant increase in both COL1A1 and SPARC protein levels compared to the non-pored samples (n = 3; statistics using unpaired Student’s t test). GAPDH was used as loading control. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01. Positive and negative controls, as well as molecular size markers and full-length blots are presented in Supplementary Figure 2.