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. 2017 Jul 18;7:5747. doi: 10.1038/s41598-017-06126-x

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© The Author(s) 2017

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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Animal weight and trabecular bone analysis. 11 to 12 week-old male SPF & GF mice were weighed; euthanized; femur harvested for histomorphometric analysis and tibia harvested for micro-CT analysis. (a) Animal weight (n = 12/gp). (b,c) Static histomorphometric analysis of distal femur trabecular bone area (n = 4/gp). (b) B.Ar/T.Ar = bone area. (c) Representative images (100×) of toluidine blue stained distal femur sections. (d–i) Micro-CT analysis of proximal tibia trabecular bone (n = 4/gp). (d) Representative reconstructed cross-sectional images, extending 360 µm distally from where analysis was initiated. (e) BMD = trabecular bone mineral density. (f) BV/TV = trabecular bone volume fraction. (g) Tb.N = trabecular number. (h) Tb.Th = trabecular thickness. (i) Tb.Sp = trabecular separation. (j–l) Dynamic histomorphometric analysis of bone formation indices in distal femur trabecular bone; calcein administered 5 and 2 days prior to sacrifice (n = 4/gp). (j) Representative images of calcein labeled secondary spongiosa (400×). (k) MAR = mineral apposition rate. (l) BFR = bone formation rate. Data reported as mean ± SEM. *p < 0.05 vs. SPF; **p < 0.01 vs. SPF; ***p < 0.001 vs SPF.