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. 2019 Jun 27;9:9309. doi: 10.1038/s41598-019-45797-6

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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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Spheroid differentiation, mineralization and matrix organization after TiO₂ NPs exposure (5 and 100 µg/ml of TiO₂ NPs during 72 hours): cross sections of paraffin-embedded spheroids stained with: (A) alkaline phosphatase for differentiation, (B) Alizarin Red S to visualize calcium deposition, (C) and Masson’s trichrome and (D) aniline blue staining for collagen. Images shown are representative of three independent experiments with triplicates. The arrows indicate the presence of TiO₂ NPs. (E,F) TEM image of an ultrathin section of an osteoblast spheroid where inset delineated by a dotted line in E is magnified in F and demonstrate the presence of collagen fibrils. (G) Quantification of collagen deposition was performed by Blue pixel analysis and quantification in aniline blue sections using Image J software. Data show mean ± SD of three independent experiments in triplicates for each condition. *P < 0.05 vs control.