Extended Data Fig. 7: µCT, histomorphometric analysis and characterization of cells isolated from Osxfl/fl;Ctskcre mouse femur. | Nature

Extended Data Fig. 7: µCT, histomorphometric analysis and characterization of cells isolated from Osxfl/fl;Ctskcre mouse femur.

From: Discovery of a periosteal stem cell mediating intramembranous bone formation

Extended Data Fig. 7

a, µCT images of longitudinal sections of femurs from Osxfl/fl;Ctskcre mice or littermate controls at 4 weeks of age. b, Haematoxylin and eosin staining showing growth plate morphology in Osxfl/fl;Ctskcre mice or littermate controls. Images in a and b are representative of 5 independent experiments. Scale bar, 100 µm. c, Bone length (i), midshaft along long axis (ii) and midshaft along short axis (iii). Osxfl/fl;Ctskcre mice show a significant reduction in bone length (i) compared to Osxfl/+;Ctskcre (*P = 0.039) and Osx+/+;Ctskcre (*P = 0.034). Two-tailed Student’s t-test. Data are mean ± s.d., n = 6 animals per group. d, Bone volume/total volume (BV/TV) for trabecular bone. Data are mean ± s.e.m., n = 4 animals per group, two-tailed Student’s t-test. e, Histomorphometric parameters. Cortical mineral apposition rate (MAR; µm day−1). *P = 0.031; two-tailed Student’s t-test; data are mean ± s.e.m., n = 5 animals per group at 4 weeks of age. f, TRAP staining of osteoclasts in the trabecular bone area of femurs of the indicated mice at 4 weeks of age. Scale bar, 100 µm. Representative images from 4 independent experiments. g, Quantification of osteoclast number/bone perimeter (No. Oc/B. Pm). Data are mean ± s.e.m., n = 4 animals per group, two-tailed Student’s t-test. h, µCT images showing the amount of bone formed when periosteal PSCs (left column) and endosteal MSCs (right column) isolated from femurs of Osx+/+;Ctskcre (top) and Osxfl/fl;Ctskcre mice (bottom) were transplanted into the kidney capsule. Scale bar, 1 mm. i, Von Kossa staining (black) of bone organoids formed after transplantation of periosteal PSCs (left column) and endosteal MSCs (right column) isolated from Osx+/+;Ctskcre (top) and Osxfl/fl;Ctskcre mice (bottom) and transplanted into the kidney capsule. Scale bar, 20 µm. Images in h and i are representative of 3 independent experiments. j, Alizarin red staining (red) of periosteal PSCs (left column) and endosteal MSCs (right column) isolated from the femur (i, ii) and calvarial sutures (iii, iv) of Osx+/+;Ctskcre (top panel) and Osxfl/fl;Ctskcre mice (bottom panel) after culture under osteoblast differentiation conditions. Images are representative of 3 independent experiments. k, l, FACS plots of contralateral unfractured femurs (k) and fractured femurs (l). Colour-coded boxes (red) indicate parent/daughter gates. Representative FACS plots from 3 independent experiments. m, A significant increase (*P = 0.019) is seen in non-CTSK MSCs in callus tissue 8 days post fracture. Values displayed represent the absolute number of cells isolated per fracture callus. Data are mean ± s.d., n = 4 independent experiments, 4 animals/group; two-tailed Student’s t-test. n, Graph displays significantly (*P = 0.017) higher fold PSC count than non-CTSK MSCs in the fractured callus. Data are mean ± s.d.; n = 3 independent experiment; two-tailed Student’s t-test. Values displayed are normalized relative to the pre-fracture numbers of the same corresponding population to demonstrate the fold expansion after fracture. op, FACS of cells from fractured callus after 3 (o) and 6 days (p) of fracture. Colour-coded boxes (green) indicate parent/daughter gates. Representative FACS plots from 3 independent experiments.

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