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. 2021 Oct 28;12:6229. doi: 10.1038/s41467-021-26454-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 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. 4 — a Schematic of the experimental protocol for assessing osteogenic differentiation of hMSCs cultured on TCP for 1-7 d, followed by 3 d (for RUNX2) or 7 d (for OCN and ALP) culture on 20 kPa Scram/RGD or HAVDI/RGD hydrogels. hMSCs were cultured on TCP (dark purple) in growth medium (pink) for 1, 3, or 7 d and then transferred (light yellow, day 0) to soft Scram/RGD (orange) or HAVDI/RGD (blue) hydrogels for additional 3 d or 7 d of culture in osteogenic medium (light blue) before collection and analysis (gray, day 3 or day 7). RUNX2 expression was assessed in the conditions denoted So3 (soft control, without culture on TCP), DT1 + So3, DT3 + So3, or DT7 + So3. OCN and ALP expression were assessed in the conditions denoted So7 (soft control, without culture on TCP), DT1 + So7, DT3 + So7, or DT7 + So7. b Representative confocal images of cells stained for RUNX2, a marker of early osteogenic differentiation, in hMSCs following treatment as shown in a. Scale bars, 20 µm. c Analysis of confocal images showed a significant increase in the RUNX2 n/c ratio with increasing exposure to TCP in hMSCs transferred to Scram/RGD hydrogels (from left to right n = 77, 49, 59, 73, 72, 44, 84, 92 cells examined over 7, 15, 19, 20, 12, 24, 23, 26 images respectively, p-values were obtained using one-way ANOVA followed by Tukey’s post hoc test, mean ± s.e.m.). However, effects of TCP were evident only after 7 days of exposure to TCP in hMSCs transferred to HAVDI/RGD hydrogels, and was greatly attenuated compared to Scram/RGD for 7 days of mechanical dosing on TCP. d–g Representative images of staining for late osteogenic differentiation markers OCN (d) and ALP (f) in hMSCs following the treatment shown in a. Scale bars, 20 µm. Quantification of the percentage of OCN-positive cells (e) and ALP-positive cells (g) affirmed that HAVDI ligation significantly attenuated osteogenic differentiation after mechanical priming on TCPS (n = 3 experiments per group, p values were obtained using one-way ANOVA followed by Tukey’s post hoc test, mean ± s.e.m.). Source data are provided as a Source Data file.