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. 2021 Mar 17;9:19. doi: 10.1038/s41413-021-00140-6

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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 — Progression of endochondral heterotopic ossification in AS patients. a Hematoxylin and eosin (H&E) staining and b Safranin O–Fast Green (SOFG) staining of normal interspinous ligaments and endochondral-ossified interspinous ligaments. In the AS group, the right panels show magnified views of the boxed area in the left panels. Scale bar: 100 μm (two panels on the right side); 25 μm (left panel). c Immunostaining and d quantitative analysis of collagen II-positive cells (brown) in the normal and chondrogenic interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). e Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) and f quantitative analysis of TRAP-positive osteoclast (red) surface (OCS) per bone surface (BS). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). g Immunostaining and h quantitative analysis of the number of CD68-positive osteoclast (brown) surface (OCS) per bone surface (BS). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). i Immunostaining and j quantitative analysis of pSmad2/3-positive cells (brown) in the normal ligaments and endochondral-ossified interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). k Immunostaining and l quantitative analysis of Osterix-positive cells (brown) in the normal and endochondral-ossified interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panel); 25 μm (bottom panel). m Immunostaining and n quantitative analysis of pSmad1/5/8-positive cells (brown) in the normal and inflammatory interspinous ligaments (sagittal view). The bottom panels show magnified views of the boxed area in the top panels. Scale bar: 100 μm (top panels); 25 μm (bottom panels). B, bone; BM, bone marrow; C, cartilage