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. 2019 Nov 1;9:15846. doi: 10.1038/s41598-019-52358-4

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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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Breakdown of chondroitin-sulfate-based ECM increases the production of MMP-13, ADAMTS5, oxidative stress and nitric oxide (NO) through TLR2 and TLR4 in 3D-cultured chondrocytes. (A) H&E staining for gross morphology of chondrocytes. Primary chondrocytes cultured in chondroitin-sulfate-based 3D-hydrogel for 2 wks were treated with chondroitinase ABC in the presence of OxPAPC (TLR2 and TLR4 dual inhibitor) or LPS-RS (TLR4-MD2 inhibitor) for 1 wk. Black rectangles in each panel denote areas enlarged in insets. Scale bar represents 100 μm. (B) Real time qRT-PCR for the RNA expression of indicated target genes. MMP-13 and ADAMTS5 are the major matrix degrading protease and Fth1, Hmox1 and Txn are the markers for oxidative stress. *P < 0.05. (C) The supernatant of 3D-hydrogel culture collected from different groups were analyzed for NO concentration measured by measuring the absorbance at the 520 nm wavelength. *P < 0.05. (D) The representative images of immunofluorescence staining for 8-oxo-dG, MMP-13 and ADAMTS5 in frozen-sectioned 3D-hydrogel. The percentage of cells positive for target staining is indicated as dot graph in right column. *P < 0.05 and **P < 0.01.