Journal: Bone research

Article Title: Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12.

doi: 10.1038/s41413-018-0041-8

Figure Lengend Snippet: Fig. 5 mTORC1 activation in subchondral preosteoblasts produce Cxcl12 to promote chondrocyte hypertrophic differentiation MSCs osteogenic differentiation. a Western blot and quantitative PCR analysis of Cxcl12 in primary preosteoblasts. b–d) Cxcl12 assessed by ELISA in the cultured medium of primary preosteoblasts or in the serum at 6 weeks post ACLT surgery. e Representative immunostaining and quantitative analysis of CXCR4+ cells in articular cartilage and subchondral bone in ΔTSC1, ΔRaptor mice, and their littermates (Ctrl). Scale bars, 100 μm. Quantitative analysis of CXCR4+ cells out of total cartilage chondrocytes. f, g Quantitative PCR analysis of Col2, ACAN, Col X, MMP-13 mRNA in ADTC5 cells treated with the cultured medium of primary preosteoblasts and Cxcl12-neutralizing antibody for 7 days. h, i Western blot analysis of Col2, Col X, MMP-13, and Toluidine blue staining in ADTC5 cells treated with CM of primary preosteoblasts and Cxcl12-neutralizing antibody for 7 days. j Western blot analysis of Col 1α, Col X, MMP-13, and Alizarin red staining in ADTC5 cells treated with CM of primary preosteoblasts, recombinant murine Cxcl12 or Cxcl12-neutralizing antibody for 14 days. Data are shown as mean ± s.d. and analyzed by Student’s t test and one-way ANOVA . n ≥8, **P < 0.01

Article Snippet: Mouse Cxcl12 antibody (R&D Systems, Minneapolis, MN, USA, #MAB310, 10–50 μg·mL−1) and recombinant murine Cxcl12 (PrimeGene BioTech, #20315, 100 ng·mL−1) was added to the CM as indicated.

Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunostaining, Staining, Recombinant

Journal: Bone research

Article Title: Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12.

doi: 10.1038/s41413-018-0041-8

Figure Lengend Snippet: Fig. 6 Administration of anti-Cxcl12 antibody attenuated subchondral bone remodeling and articular cartilage degeneration in ACLT mice. a, b, d, e Representative immunostaining and quantitative analysis of Cxcl12 in Osterix+ preosteoblasts in the subchondral bone and metaphysis in ΔTSC1, ΔRaptor mice, and their littermates (Ctrl). Boxed area is magnified on the corner. Scale bar, 100 μm. AC, articular cartilage; SCB, subchondral bone; GP, growth plate; BM, bone marrow. c Representative Safranin O-Fast green, H&E staining, 3D reconstructed μ-CT images and immunohistochemical staining of OCN, type X collagen, and MMP-13 in ΔTSC1 mice administered with anti-Cxcl12 antibody or normal IgG for 6 weeks. Scale bar, 100 μm (Safranin O, H&E staining, OCN, type X collagen, and MMP-13), 1 mm (μ-CT). f OARSI scores based on the histological analysis. g, h Quantitative analysis of type X collagen and MMP-13 in articular cartilage. i, j Quantitative analysis of bone mass in subchondral bone: bone volume/total volume (BV/TV) and bone mineral density (BMD). k Quantitative analysis of OCN+ osteoblasts in tibial subchondral bone. Data are shown as mean±s.d. and analyzed by Student’s t test. n ≥6, **P < 0.01

Article Snippet: Mouse Cxcl12 antibody (R&D Systems, Minneapolis, MN, USA, #MAB310, 10–50 μg·mL−1) and recombinant murine Cxcl12 (PrimeGene BioTech, #20315, 100 ng·mL−1) was added to the CM as indicated.

Techniques: Immunostaining, Staining, Immunohistochemical staining

Journal: Bone research

Article Title: Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12.

doi: 10.1038/s41413-018-0041-8

Figure Lengend Snippet: Fig. 7 Model of elevated active mTORC1 in the subchondral bone at the onset of OA. mTORC1 is activated in the subchondral bone in response to abnormal mechanical loading, which induces preosteo- blast abnormal proliferation and Cxcl12 secretion to promote subchondral bone formation and articular cartilage degeneration for OA progression

Article Snippet: Mouse Cxcl12 antibody (R&D Systems, Minneapolis, MN, USA, #MAB310, 10–50 μg·mL−1) and recombinant murine Cxcl12 (PrimeGene BioTech, #20315, 100 ng·mL−1) was added to the CM as indicated.

Techniques: