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. 2022 May 11;7:155. doi: 10.1038/s41392-022-00957-5

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© The Author(s) 2022

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. 1 — Klotho deficiency in Osx⁺-cells causes impaired bone remodeling. a, c Three-dimensional reconstruction from micro-computed tomography scans of the alveolar bone of OsxCre;KL^fl/fl mice and their control littermates at 3 (a) and 11 weeks old (c). b, d Quantitative analyses of bone volume/tissue volume (BV/TV, %), trabecular thickness (Tb.Th, μm), and trabecular separation (Tb.Sp, μm) in 3- (b, n = 5 in OsxCre group and n = 8 in OsxCre;KL^fl/fl group) and 11-week-old mice (d, n = 3). e, f Hematoxylin and eosin (H&E) staining of alveolar bone at root bifurcation of mandibular 1st molar in 3- (e) and 11-week-old mice (f). Higher magnifications of boxed areas show increased alveolar bone mass in OsxCre;KL^fl/fl mice. n = 5. g, h Calcein double labeling of alveolar bone from control and OsxCre;KL^fl/fl mice. Representative image (g) and quantitative analyses of bone formation rate/bone surface (BFR/BS, μm³/μm²/day), mineral apposition rate (MAR, μm/day) and mineralizing surface/bone surface (MS/BS, %) (h). n = 8. i Double fluorescence staining of RUNX2 and OSX:GFP. Higher magnifications of boxed areas show RUNX2 and OSX:GFP-positive cell distribution at 1st molar root bifurcation. j, k Quantification of RUNX2 or OSX:GFP-positive cells/total osteoblasts in 3- (j) and 11-week-old mice (k). n = 6 in OsxCre group and n = 4 in OsxCre;KL^fl/fl group. l TRAP staining. Higher magnifications of boxed areas show the number of TRAP-positive cells decreased in OsxCre;KL^fl/fl mice. m Quantification of TRAP-positive cells/bone surface in 3- (left) and 11-week-old mice (right). n = 4 in OsxCre group and n = 5 in OsxCre;KL^fl/fl group. n qRT-PCR analysis of Acp5, Mmp9, Rankl, Opg, and Rankl/Opg transcription in 11-week-old mice. n = 4 in OsxCre group and n = 6 in OsxCre;KL^fl/fl group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data are shown as the mean ± SEM. Scale bar, 1 mm (a, c), 200 μm (e), 50 μm (g, i), and 100 μm (l)