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. 2021 May 7;11:9751. doi: 10.1038/s41598-021-89164-w

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Pediatric end-stage CAVS valves have a greater number of collagen fibers with localized regions of collagen density. (A) Collagen fiber visualization of select pCAVS and normal valves. From left to right: Second Harmonic Generation (SHG); SHG multichannel; Polarized picrosirius red (PSR); PSR Regions on Increased Fiber Density (RIFD) Masque; Movat’s Pentachrome. Movat’s Pentachrome: blue = GAGs, yellow = collagen, and purple = elastin. Scale Bar = 200 μm. (B) Boxplot quantification of picrosirius red color deconvolution analysis, showing percent thick stain over total PSR stain. (C) Patient-specific data of PSR color deconvolution analysis, showing percent thick stain over total PSR stain. pCAVS and AVI patients show larger heterogeneity across age groups compared to normal. (D) Boxplot quantification of collagen RIFD areas normalized to total valve area. *p-value 0.024, MWU test. pCAVS and AVI patients have significantly more regions of high collagen fiber density compared to normal patients. (E) Boxplot showing quantification of total number of collagen fibers as measured by CT-FIRE analysis. *p-value 0.014, MWU test. pCAVS valves have significantly more collagen fibers than normal valves. (F,G) Hinge, Belly, and Free Margin (respectively) 10 × zooms of PSR-polarized light signal overlayed with CT-FIRE mask of collagen fibers for normal (F; SKU DB27) and pCAVS (G; SKU DB16) (Supplmentary Figure S3). Normal valves show stratified collagen fibers throughout the leaflet, while pCAVS valves show densely deposited fibers increasing in destratification from hinge to free margin. (H) Polar histogram showing frequency distribution of collagen fiber angles from 0–180°, across all normal valve samples (left) and pCAVS (right). pCAVS valves have a large distribution of fiber angels indicating destratification, while normal valve collagen fiber angles stratify to 0° and 180° relative to the endothelium. Normal n = 4; pCAVS n = 11; AVI n = 5. CT-FIRE V2.0 Beta was used (https://eliceirilab.org/software/ctfire/).