Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A–D) qPCR analysis of PLR genes Mmp13, Mmp14 and Ctsk and Serpine1 upon TGFβ (5ng/mL) treatment in MLO-Y4 (A, B) and OCY454 (C, D) cells. (n=3 replicates/group). (E, F) Intracellular pH (pHi) of MLO-Y4 cells after 3 days of TGFβ (5ng/ml), TβRI inhibitor SB-431542 (10 μM), or recombinant sclerostin (rhSCL, 10 ng/ml). The representative image (E) shows the shift in the emission peak from 580 nm to 640 nm after TGFβ treatment of MLO-Y4 cells. Scale bar, 100 μm). TGFβ-induced acidification is blocked by SB-431542 (F) (n=4 replicates/group). Error bars indicate mean ± SD of 3 independent experiments, *p<0.05 different from control mRNA, a-p<0.05 different from control pHi, b-p<0.05 different from TGFβ pHi, and c-p<0.05 different from rhSCL pHi. Statistics calculated from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: Recombinant, Control

Journal: Cell reports

Article Title: Osteocyte intrinsic TGFβ signaling regulates bone quality through perilacunar/canalicular remodeling

doi: 10.1016/j.celrep.2017.10.115

Figure Lengend Snippet: (A, B) TβRII-stained osteocytes (A) (arrow, scale bar, 50 μm) in the femoral cortical bone from WT and TβRIIocy−/− mice (8-week old males) were quantified as percentage of positively stained osteocytes normalized to total bone area (B) (n=5 mice/group) (C) qPCR analysis of TβRII and Serpine1 in WT and TβRIIocy−/− femoral bones. (n=8–10 mice/group). (D, E) Silver nitrate stained images of WT and TβRIIocy−/− femoral cortical bone shows the osteocyte lacuno-canalicular network (D) and canalicular length (E) (scale bar, 20 μm, n=5 mice/group). (F, G) qPCR analysis of PLR genes, Mmp2, Mmp13, Mmp14, Ctsk, and Acp5 (F) and OCY-specific genes, Sost, Dmp1 and Phex (G) in the WT and TβRIIocy−/− bones (n=8–10 mice/group) (H, I) IHC of MMP13, MMP14, CTSK and H&E staining of WT and TβRIIocy−/− femoral cortical bone. Arrows in the image indicate positively stained osteocytes (H) that were quantified and normalized to total bone area (I), (n=4 mice/group).(J–M) SRμT shows volume (J), degree of anisotropy (K), orientation (L) and mineralization (N) of osteocyte lacunae of WT and TβRIIocy−/− bone (n=3–4 mice/group). Error bars indicate mean ± SEM with *p<0.05 compared to WT from Student’s t test.

Article Snippet: For treatment, cells were cultured in α-MEM containing 0.5–1% fetal bovine serum, supplemented with 5 ng/ml TGFβ1 (Humanzyme, HZ-1011), 10 μM SB431542 (Sigma, S4317) or 10 ng/ml recombinant human sclerostin (rhSCL, R&D Systems) for the indicated times.

Techniques: Staining

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Surface plasmon resonance analysis of sclerostin/GAG interaction, reported as resonance units (RU). ( A ) Relative binding levels of labeled and unlabeled sulfated or native hyaluronan and corresponding sensorgrams ( B ). GAGs were injected at 50 and 500 µM related to the molecular weight of their disaccharide units (D.U.).

Article Snippet: The concentration of recombinant human sclerostin (R&D Systems, Minneapolis, MN, USA) used during these scavenging experiments was 8 ng/mL.

Techniques: SPR Assay, Binding Assay, Labeling, Injection, Molecular Weight

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Sclerostin binding to variously decorated material films. ( A – C ) Differently modified surfaces were incubated with 8 ng/mL sclerostin. Binding was quantified by the amount of unbound sclerostin in the supernatant after 24 h ( A , B ). ( C ) Cumulative sclerostin release from a saturated and preincubated (40 ng/mL) sHA3 surface. Sclerostin was presented in either 0.1% BSA and 0.2% Tween 80 in PBS ( A ) or DMEM with 10% serum ( B , C ). Film surfaces: sHA3: covalent ED900 and sHA3-coating; ED900: ED900 modified film preincubated with soluble sHA3, Ac. ED900: Acetylated ED900 acetylated to neutralize positive charge; T134LA6: nonmodified film. All values represent the mean ± SD of n ≥ 4. *** p < 0.001 denotes significant difference vs. the respective surface modification.

Article Snippet: The concentration of recombinant human sclerostin (R&D Systems, Minneapolis, MN, USA) used during these scavenging experiments was 8 ng/mL.

Techniques: Binding Assay, Modification, Incubation

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Sclerostin and DKK1 are the preferred binding partner of sHA3 functionalized films. Differently modified films were incubated with the same molar amounts of Sclerostin, Wnt3a, or both proteins combined. Unbound ( A ) Sclerostin, ( B ) Wnt3a, and ( C ) DKK1 was quantified after 24 h from the supernatant via ELISA. Recovery normalized to controls without any films (blank well plates). Film surfaces: ED900: ED900 incubated with soluble sHA3; sHA3: ED900 covalently coated with sHA3. All values represent the mean ± SD of n ≥ 4. *** p < 0.001 denotes significant difference vs. the respective surface modification.

Article Snippet: The concentration of recombinant human sclerostin (R&D Systems, Minneapolis, MN, USA) used during these scavenging experiments was 8 ng/mL.

Techniques: Binding Assay, Modification, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Effect of film modification on secreted protein content in the supernatant and mineralization during osteogenic differentiation of SaOS-2 cells. Cells were seeded on differently modified surfaces at a density of 10,000/cm 2 and differentiated for 10 days. ( A ) Sclerostin, ( B ) Wnt3a, and ( C ) DKK1 were then quantified from the cell supernatant via ELISA after 1, 3, 6, 8, and 10 days. Calcium content ( D ) was quantified at day 10. Film surfaces: ED900: surface coating with ED900 preincubated with soluble sHA3; sHA3: covalent ED900 and sHA3-coating; sHA3 pre: sHA3 surface preincubated with 40 ng/mL sclerostin; w / o cells: sHA3 surface without seeded cells. All values represent the mean ± SD of n ≥ 4. *** p < 0.001 denotes significant difference vs. the respective surface modifications.

Article Snippet: The concentration of recombinant human sclerostin (R&D Systems, Minneapolis, MN, USA) used during these scavenging experiments was 8 ng/mL.

Techniques: Modification, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: A Versatile Macromer-Based Glycosaminoglycan (sHA3) Decorated Biomaterial for Pro-Osteogenic Scavenging of Wnt Antagonists

doi: 10.3390/pharmaceutics12111037

Figure Lengend Snippet: Effect of film modification on secreted protein content in the supernatant and mineralization during osteogenic differentiation of hMSC. Cells were seeded on differently modified surfaces at a density of 15,000/cm 2 and differentiated for 12 days. ( A ) Sclerostin was then quantified from the cell supernatant via ELISA after 1, 3, 7, 10, and 12 days. OsteoImage TM staining was assessed at day 12 and the fluorescence signal quantitatively ( B ) and qualitatively( C , D ) analyzed. Film surfaces: ED900: surface coating with ED900 preincubated with soluble sHA3; sHA3: covalent ED900 and sHA3-coating; w / o cells: sHA3 surface without seeded cells. All values represent the mean ± SD of n ≥ 4. * p < 0.05; *** p < 0.001 denotes significant difference vs. the respective surface modifications.

Article Snippet: The concentration of recombinant human sclerostin (R&D Systems, Minneapolis, MN, USA) used during these scavenging experiments was 8 ng/mL.

Techniques: Modification, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence

Journal: Arthritis and rheumatism

Article Title: Stimulation of superficial zone protein accumulation by hedgehog and Wnt signaling in surface zone bovine articular chondrocytes.

doi: 10.1002/art.37768

Figure Lengend Snippet: Figure 5. Increase in SZP accumulation following inhibition of the Wnt/-catenin pathway. A and B, Surface zone articular chondrocytes were cultured in monolayer for 4 days in serum-free chemically defined medium with antagonists of the low-density lipoprotein receptor– related protein 5 (LRP-5)/LRP-6 coreceptors (sclerostin [SOST] or dickkopf-1 [Dkk-1]) in various concentrations (A), and surface zone cartilage explants were cultured for 4 days with 3 g/ml SOST (B). SZP accumulation in the medium was quantified by enzyme-linked immu- nosorbent assay. Bars show the mean SD SZP levels in 6 samples (A) or 16 samples (B) per group. C, SZP accumulation was also assessed by immunohistochemical staining of the cartilage explants, following treatment with 3 g/ml SOST. Untreated samples served as control. P 0.01; P 0.05 versus untreated control. Bars 100 m. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/ journal/10.1002/(ISSN)1529-0131.

Article Snippet: Human recombinant proteins SHH, IHH, Wnt-11, sclerostin (SOST), dickkopf-1 (Dkk-1), and TGF 1 were purchased from R&D Systems.

Techniques: Inhibition, Cell Culture, Immunohistochemical staining, Staining, Control

Journal: Arthritis and rheumatism

Article Title: Stimulation of superficial zone protein accumulation by hedgehog and Wnt signaling in surface zone bovine articular chondrocytes.

doi: 10.1002/art.37768

Figure Lengend Snippet: Figure 6. Influence of SHH, Wnt-3a, and sclerostin (SOST) on proliferation of bovine articular chondrocytes, and synergistic effects of transforming growth factor 1 (TGF1) and hedgehog proteins or antagonists of the Wnt/-catenin pathway on SZP accumulation in monolayer or explant cultures. A, Cell proliferation assays were performed in 4-day monolayer cultures by counting the number of cells on a hemocytometer with trypan blue exclusion. Bars show the mean SD in 6 samples per group (SHH-, Wnt-3a–, or SOST-treated cultures compared with untreated control cultures). B and C, Synergistic effects of TGF1 (10 ng/ml) and hedgehog proteins (SHH or IHH) or antagonists of the Wnt/-catenin pathway (SOST or dickkopf-1 [Dkk- 1]) were assessed in monolayer cultures of primary cells (B) or in explant cultures (C). Surface zone articular chondrocytes were cul- tured with or without TGF1 and treated with or without SHH, IHH, SOST, or Dkk-1 (A), while surface zone cartilage explants were cultured with or without TGF1 and treated with or without SHH or SOST (B). SZP accumulation in the medium after the 4-day treatment was quantified by enzyme-linked immunosorbent assay. Values are the mean SD in 6 samples (B) or 16 samples (C) per group. See Figure 1 for other definitions.

Article Snippet: Human recombinant proteins SHH, IHH, Wnt-11, sclerostin (SOST), dickkopf-1 (Dkk-1), and TGF 1 were purchased from R&D Systems.

Techniques: Control, Cell Culture, Enzyme-linked Immunosorbent Assay