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. 2019 Aug 6;10:3520. doi: 10.1038/s41467-019-11455-8

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© The Author(s) 2019

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. 6 — Vascularization of mineralized cell-laden collagen and interaction with prostate cancer cells. a Timeline for culture and implantation in SCID mice. b HUVECs formed endothelial networks that were supported by αSMA-expressing hMSCs (scale bar: 50 µm) and were also positive for CD31 (scale bar: 400 µm). The remainder of hMSCs expressed RUNX2 as a marker for osteogenic differentiation (scale bar: 50 µm). c Von Kossa staining of the non-mineralized (top) vs. mineralized tissue sections (bottom) after 7 days of in-vitro culture and 7 days of implantation. The dark/brown staining indicates calcification. Higher-magnification images (right) show luminal structures (arrows) consistent with microvessel formation within the calcified construct. d H&E images depict the collagenous matrix populated with cells. Anti-human CD31 antibody staining (middle) suggests the formation of endothelial networks by the transplanted HUVECs as opposed to murine vasculature infiltration. Vessels in non-mineralized sections had constricted lumens (arrows), as opposed to wider HUVEC-lined vessel structures in the mineralized construct (arrows). Anti-αSMA staining (right) shows fewer αSMA+ cells in the non-mineralized sections than in mineralized constructs, which appear to be wrapped by pericyte-like cells. e Quantification of vessel number and diameter indicate robust vascularization and cell survival in mineralized groups, compared with their non-mineralized controls. Quantitative analysis of % area of CD31 and αSMA immunostaining suggests an increased vascularization and vessel stability in mineralized constructs. Data presented as Mean ± SD (N = 3) (****p < 0.0001 Student’s t-test). f Representative bioluminescence images captured before and once a week until 3 weeks, after injection of PC3/Luc cells as visualized by IVIS. g A significantly higher bioluminescence signal intensity was detected in the region implanted with mineralized construct compared with the non-mineralized control. Data presented as Mean ± SEM (two-tailed Student’s t-test, *p < 0.05; n = 8 for non-mineralized groups; n = 12 for mineralized groups from two-independent experiments)