Calcifying nanoparticles induce apoptosis and calcification in bone marrow mesenchymal stem cells via the transforming growth factor-β/Smad pathway

Figure 1 Ultrastructural morphology of calcifying nanoparticles observed by scanning electron microscope and transmission electron microscope. A: Scanning electron microscope images of calcifying nanoparticles (CNPs); B-D: Transmission electron microscope images of CNPs at different magnifications; E: Distribution of CNP diameters.

Figure 2 The trends of bone marrow mesenchymal stem cell proliferation and apoptosis on exposure to different concentrations of nanoscale hydroxyapatites and different concentrations of calcifying nanoparticles groups compared with controls. A: The trends of bone marrow mesenchymal stem cell proliferation on exposure to different concentrations of nanoscale hydroxyapatites and different concentrations of calcifying nanoparticles groups compared with controls; B: The trends of bone marrow mesenchymal stem cell apoptosis on exposure to different concentrations of nanoscale hydroxyapatites and different concentrations of calcifying nanoparticles groups compared with controls. L-nHAPs: Low concentration nanoscale hydroxyapatites; M-nHAPs: Medium concentration nanoscale hydroxyapatites; H-nHAPs: High concentration nanoscale hydroxyapatites; L-CNPs: Low concentration calcifying nanoparticles; M-CNPs: Medium concentration calcifying nanoparticles; H-CNPs: High concentration calcifying nanoparticles.

Figure 3 Flow cytometric analysis of apoptosis in bone marrow mesenchymal stem cells on exposure to different concentrations of nanoscale hydroxyapatites and different concentrations of calcifying nanoparticles compared with controls. L-nHAPs: Low concentration nanoscale hydroxyapatites; M-nHAPs: Medium concentration nanoscale hydroxyapatites; H-nHAPs: High concentration nanoscale hydroxyapatites; L-CNPs: Low concentration calcifying nanoparticles; M-CNPs: Medium concentration calcifying nanoparticles; H-CNPs: High concentration calcifying nanoparticles.

Figure 4 Calcium deposition in bone marrow mesenchymal stem cells after alizarin red staining in the indicated treatment groups (× 100). L-nHAPs: Low concentration nanoscale hydroxyapatites; M-nHAPs: Medium concentration nanoscale hydroxyapatites; H-nHAPs: High concentration nanoscale hydroxyapatites; L-CNPs: Low concentration calcifying nanoparticles; M-CNPs: Medium concentration calcifying nanoparticles; H-CNPs: High concentration calcifying nanoparticles.

Figure 5 Polymerase chain reaction gene detection of apoptosis and calcification gene expression in the indicated intervention groups. A: Osteopontin expression in the indicated intervention groups; B: Bone morphogenetic protein-2 expression in the indicated intervention groups; C: Bcl-2 expression in the indicated intervention groups; D: Caspase3 expression in the indicated intervention groups; E: BAX expression in the indicated intervention groups; F: Smad3 expression in the indicated intervention groups; G: Transforming growth factor-β expression in the indicated intervention groups. ^aP < 0.05, ^bP < 0.01. OPN: Osteopontin; BMP-2: Bone morphogenetic protein-2; TGF-β: Transforming growth factor-β; M-nHAPs: Medium concentration nanoscale hydroxyapatites; M-CNPs: Medium concentration calcifying nanoparticles.

Figure 6 Western blotting analysis of calcification- and apoptosis-related proteins in bone marrow mesenchymal stem cells. OPN: Osteopontin; BMP-2: Bone morphogenetic protein-2; TGF-β: Transforming growth factor-β; M-nHAPs: Medium concentration nanoscale hydroxyapatites; M-CNPs: Medium concentration calcifying nanoparticles.

Figure 7 Western blotting quantification of protein levels. A-H: Western blotting quantification of osteopontin (A), bone morphogenetic protein-2 (B), Bcl-2 (C), caspase3 (D), BAX (E), Smad3 (F), p-Smad3 (G), transforming growth factor-β (H) levels. ^aP < 0.05, ^bP < 0.01, NS: P > 0.05. OPN: Osteopontin; BMP-2: Bone morphogenetic protein-2; TGF-β: Transforming growth factor-β; M-nHAPs: Medium concentration nanoscale hydroxyapatites; M-CNPs: Medium concentration calcifying nanoparticles.