Journal: International Journal of Molecular Sciences

Article Title: Roles of Dkk2 in the Linkage from Muscle to Bone during Mechanical Unloading in Mice

doi: 10.3390/ijms21072547

Figure Lengend Snippet: Effects of Dkk2 on the osteoblast phenotypes and osteoclast formation. ( A ) Total RNA was extracted from mouse osteoblasts cultured with the indicated concentrations of Dkk2 for 24 h, and real-time PCR analysis of Runx2, Osterix, ALP, osteocalcin, RANKL, OPG or GAPDH was performed ( n = 6 in each group). Data represent the mean ± SD of six experiments in each group. ** p < 0.01 and * p < 0.05 vs. control group (Dunn test). ( B ) ALP activity was measured in confluent mouse osteoblasts cultured with the indicated concentrations of Dkk2 for 24 h as described in Materials and Methods ( n = 6 in each group). Data represent mean ± SD of six experiments in each group. ** p < 0.01 vs. control group (Dunnett test). ( C ) Mouse osteoblasts were cultured with 10 mM β-glycerophosphate in the presence of the indicated concentrations of Dkk2 for 3 weeks ( n = 5 in each group). Mineralization was determined by Alizarin red staining as described in Materials and Methods. Data represents mean ± SD of five experiments in each group. ** p < 0.01 vs. control group (Dunnett test). ( D ) Total protein was extracted from mouse osteoblasts cultured with (+) or without (−) 500 ng/mL Dkk2 for 24 h ( n = 4 in each group). Then, Western blot analysis for phosphorylated β-catenin (pβ-Catenin) and β-actin was performed. The images represent experiments performed independently three times. Protein signals were quantified by densitometry and adjusted by the density of GAPDH. Data represent the mean ± SD. * p < 0.05 vs. control group (Mann-Whitney U test). ( E ) Total RNA was extracted from ST2 cells cultured with (+) or without (−) 200 ng/mL BMP-2 in the presence of the indicated concentrations of Dkk2 for 72 h, and real-time PCR analysis of Osterix, ALP, osteocalcin or GAPDH was performed ( n = 5 in each group). Data represent the mean ± SD of five experiments in each group. ** p < 0.01 (Tukey-Kramer test). ( F ) RAW264.7 cells were cultured with (+) or without (−) 75 ng/mL RANKL in the presence (+) or absence (−) of 500 ng/mL Dkk2 for 4 days ( n = 6 in each group). The cells were stained with tartrate-resistant acid phosphatase (TRAP) staining, and the number of TRAP-positive multinucleated cells (TRAP + MNCs) was counted in each well. The data represent the mean ± SD of six experiments. ** p < 0.01 (Tukey-Kramer test).

Article Snippet: Detection of osteoclasts was performed using a tartrate-resistant acid phosphatase (TRAP) staining kit (Wako Pure Chem.), and number of TRAP-positive multinucleated cells (MNCs) was counted in each well.

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Activity Assay, Staining, Western Blot, MANN-WHITNEY

Journal: Cell Transplantation

Article Title: Mechanical Unloading Promotes Osteoclastic Differentiation and Bone Resorption by Modulating the MSC Secretome to Favor Inflammation

doi: 10.1177/09636897241236584

Figure Lengend Snippet: Mechanical unloading leads to BM inflammation. (A) Schematic of HU models and WB controls. (B) Representative images of micro-CT. Femur of rats unloaded for indicated time points were used for micro-CT detection. (C) Quantification of three-dimensional microstructural parameters from micro-CT was shown as BMD, BV/TV, Tb.Sp, and Tb.N. (D) Representative images of ALP/TRAP staining of tibia sections of HU rats as well as WB controls. (E) Quantification of ALP/TRAP staining was performed by measuring the positive areas in tibia sections, and data were presented as a percentage of ALP-positive surface (BFS) or TRAP-positive surface (BRS) to the total areas (BS). Data are means ± SD, n = 6 per group. ( F) The protein levels of RANKL and OPG in the BM fluid of suspended rats and WB controls. (G) Representative images of CD11b IHC of the tibia sections from WB controls and rats suspended for 7 days. Quantification data are presented as a percentage of positively stained cells to total cells. * P < 0.05, ** P < 0.01, *** P < 0.001 versus WB (one-way ANOVA with Dunnett’s post hoc test), n = 5 per group. Data are means ± SEM. (H) Flow cytometry analysis showed the percentage of monocytes in the BM of WB rats and rats suspended for 7 days. Data are means ± SD, n = 5 per group, representative of three independent experiments. (I) Flow cytometry analysis showed the percentage of neutrophils in the BM of WB rats and rats suspended for 7 days. Data are means ± SD, n = 5 per group, representative of three independent experiments. (J) Flow cytometry showed the percentage of CD11b + cells in the peripheral blood of WB controls and rats suspended for 7 days. Data are means ± SD, n = 5 per group. (K) The protein level of inflammatory cytokines in the BM was tested by ELISA. Data are means ± SD, n = 5 per group. HU: hindlimb unloading; WB: weight-bearing; CT: computed tomography; BMD: bone mineral density; BV/TV: bone volume versus total volume; Tb.Sp.: trabecular space; Tb.N: trabecular number; ALP/TRAP: alkaline phosphatase/tartrate-resistant acid phosphatase; BFS: bone formation surface; BRS: bone resorption surface; BS: bone surface; RANKL: receptor activator of nuclear factor kappa B ligand; OPG: osteoprotegerin; IHC: immunohistochemistry; ANOVA: analysis of variance; SEM: standard error of mean; ELISA: enzyme-linked immunosorbent assay.

Article Snippet: TRAP staining was performed using a commercial kit (Solarbio, China) according to the manufacturer’s instructions.

Techniques: Micro-CT, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Computed Tomography, Immunohistochemistry

Journal: Cell Transplantation

Article Title: Mechanical Unloading Promotes Osteoclastic Differentiation and Bone Resorption by Modulating the MSC Secretome to Favor Inflammation

doi: 10.1177/09636897241236584

Figure Lengend Snippet: Conditional knockout of Piezo1 in MSCs leads to BM inflammation under the WB conditions. (A) Representative images of micro-CT of Piezo1 conditional KO mice (Piezo1 fl/fl ; LepR Cre ) and their littermate controls (Piezo1 fl/fl ). n = 5 per group. (B) Quantification of three-dimensional microstructural parameters from micro-CT scanned tibia. (C) Representative images of ALP and TRAP staining of tibia sections. Quantification data were presented as a percentage of ALP-positive area (BFS) or TRAP-positive area (BRS) to total bone surface (BS). n =5 per group. (D) Heatmap showed the protein levels of inflammatory cytokines in the BM fluid of Piezo1 fl/fl ; LepR Cre mice compared with Piezo1 fl/fl littermates. n = 5 per group. (E) Flow cytometry analysis of the percentage of monocytes and neutrophils. The statistical significance was assessed by a Student’s t test of three to six independent experiments. All data represented are means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. MSC: mesenchymal stem cell; BM: bone marrow; WB: weight-bearing; CT: computed tomography; KO: knockout; ALP: alkaline phosphatase; TRAP: tartrate-resistant acid phosphatase; BFS: bone formation surface; BRS: bone resorption surface; BS: bone surface.

Article Snippet: TRAP staining was performed using a commercial kit (Solarbio, China) according to the manufacturer’s instructions.

Techniques: Knock-Out, Micro-CT, Staining, Flow Cytometry, Computed Tomography

Journal: Cell Transplantation

Article Title: Mechanical Unloading Promotes Osteoclastic Differentiation and Bone Resorption by Modulating the MSC Secretome to Favor Inflammation

doi: 10.1177/09636897241236584

Figure Lengend Snippet: CM of 10% CMS-treated MSCs inhibited monocyte proliferation and osteoclastic differentiation. (A) Representative images of the EdU cooperation assay showed the proliferation rate of BM monocytes under the treatment of MSC-CM. (B) Quantification of EdU-positive cells presented as a percentage of total cells per field of view. (C) CCK8 assay showed the metabolic activity of monocytes under the treatment of MSC-CM. (D) Flow cytometry analysis of apoptotic cells treated by MSC-CM. (E) Representative images of TRAP staining of osteoclasts after 5 days of differentiation under the treatment of MSC-CM. (F) Representative images of SA-β-gal staining of MSCs 24 h after CMS application. Quantification data were presented as a percentage of β-gal-positive cells to total cells per field of view. The statistical significance was assessed by two-tailed Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the respective nc group. Data are means ± SD of three independent experiments. CM: conditioned medium; CMS: cyclic mechanical stretch; MSC: mesenchymal stem cell; BM: bone marrow; TRAP: tartrate-resistant acid phosphatase; SA-β-gal: senescence-associated beta-galactosidase.

Article Snippet: TRAP staining was performed using a commercial kit (Solarbio, China) according to the manufacturer’s instructions.

Techniques: CCK-8 Assay, Activity Assay, Flow Cytometry, Staining, Two Tailed Test

Journal: Cell Transplantation

Article Title: Mechanical Unloading Promotes Osteoclastic Differentiation and Bone Resorption by Modulating the MSC Secretome to Favor Inflammation

doi: 10.1177/09636897241236584

Figure Lengend Snippet: Transplantation of CMS-treated MSCs effectively rescued bone resorption and BM inflammation under mechanical-unloading conditions. (A) The expression of genes was tested by qRT-PCR at the indicated time points after CMS application for 24 h. (B) Representative images of TRAP staining of osteoclasts. The culture medium was changed after 24 h of CMS application, and MSCs were cultured in a static state for the indicated time point before the conditioned medium was collected for the treatment of osteoclast differentiation. (C) Representative images of bone histomorphology analysis of HU rats as well as WB controls. micro-CT was performed on femurs 21 days after suspension, and ALP and TRAP staining was performed on tibia sections 7 days after suspension. (D) Quantification (BMD, BV/TV, Tb.N, and Tb.Th) of three-dimensional microstructural parameters from micro-CT scanned femur. Quantification data of ALP and TRAP staining were presented as a percentage of BFS and BRS, respectively, to total bone areas (BS). n = 6 per group. (E) Flow cytometry analysis of the percentage of CD11b + cells in the BM of rats treated as indicated. n = 6 per group. (F) ELISA showed the protein levels of cytokines in the BM fluid of rats treated as indicated. n = 6 per group. The statistical significance was assessed by two-tailed Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are means ± SD. Gene expression levels were normalized to GAPDH. CMS: cyclic mechanical stretch; MSC: mesenchymal stem cell; BM: bone marrow; qRT-PCR: quantitative real-time polymerase chain reaction; TRAP: tartrate-resistant acid phosphatase; HU: hindlimb unloading; ALP: alkaline phosphatase; BMD: bone mineral density; BV/TV: bone volume versus total volume; Tb.N: trabecular number; BFS: bone formation surface; BRS: bone resorption surface; BS: bone surface; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: TRAP staining was performed using a commercial kit (Solarbio, China) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Expressing, Quantitative RT-PCR, Staining, Cell Culture, Micro-CT, Suspension, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: iSN40 inhibits the RANKL-induced osteoclastogenesis of RAW264.7 cells. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL and 0.1–3.0 μM iSN40 for 7 days. Scale bar, 100 μm. ( B ) The number of nuclei in the TRAP + osteoclasts was quantified. No TRAP + cells were observed in any of the RANKL(−) groups. ** p < 0.01 vs. RANKL(+)/control. n = 4 fields. ( C ) Representative images of the pits generated in the bone matrix resorption assay of RAW264.7 cells treated with 100 ng/mL RANKL and 0.3 μM iSN40 for 6 days. Scale bar, 100 μm. ( D ) The pit area was quantified. ** p < 0.01 vs. RANKL(−)/control; †† p < 0.01 vs. RANKL(+)/control. n = 3 fields.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control, Generated

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: iSN40 inhibits osteoclastogenesis in a TLR9-dependent manner. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODN, and 1 μM E6446 for 6 days. Scale bar, 100 μm. ( B ) The number of nuclei in TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40/E6446(−) and CpG-2006/E6446(−) groups. NS, no significant difference, ** p < 0.01 vs. each E6446(−) group. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODNs, and 1 μM E6446 for 24 h. ** p < 0.01 vs. control/E6446(−), †† p < 0.01 vs. iSN40/E6446(−), ‡‡ p < 0.01 vs. CpG-2006/E6446(−). n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: The CpG motif is essential for the anti-osteoclastogenic effect of iSN40. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL and 0.3 μM ODN for 7 days. Scale bar, 100 μm. ( B ) The number of nuclei in the TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40 group. NS, no significant difference vs. control. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODNs for 24 h ( Il1b ) or 5 days ( Nfatc1 , Ctsk , and Dcstamp ). * p < 0.05, ** p < 0.01 vs. RANKL(−)/control; † p < 0.05, †† p < 0.01 vs. RANKL(+)/control; ‡ p < 0.05, ‡‡ p < 0.01 vs. RANKL(+)/iSN40. n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: Anti-osteoclastogenic effects of the iSN40 variants. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL and 0.3 μM ODN for 7 days. Scale bar, 100 μm. ( B ) The number of nuclei in the TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40 group. * p < 0.05, ** p < 0.01 vs. control. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL and 0.3 μM ODN for 24 h. * p < 0.05, ** p < 0.01 vs. control. n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: Anti-osteoclastogenic effect of iSN40 in the coculture of RAW264.7 and MC3T3-E1 cells. ( A ) Representative images of the TRAP staining of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL for 4 days. RW, undifferentiated RAW264.7 cells (dense nuclei in black); OC, differentiated TRAP + multinucleated osteoclasts; OB, fibroblast-like MC3T3-E1 osteoblasts. Scale bar, 25 μm. ( B ) qPCR results of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL, 50 μg/mL ascorbic acid (AA), and 0.3–10 μM iSN40 for 4 days. ** p < 0.01 vs. RANKL(+)/AA(+)/iSN40(−). n = 3. ( C ) Representative images of the TRAP staining of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL and 0.3 μM ODN for 4 days (top panels). Representative images of the pits generated in the bone matrix resorption assay of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL and 0.3 μM ODN for 6 days (bottom panels). Scale bars, 100 μm. ( D ) The number of nuclei in TRAP + osteoclasts in panel C was quantified. ** p < 0.01 vs. RANKL(−)/control, †† p < 0.01 vs. RANKL(+)/control. n = 3 fields. ( E ) Scale bar, 100 μm. ( E ) The pit area in panel C was quantified. ** p < 0.01 vs. RANKL(−)/control, †† p < 0.01 vs. RANKL(+)/control. n = 3 fields. ( F ) qPCR results of the ratio of RANKL ( Tnfsf11 ) to OPG ( Tnfrsf11b ) of the same samples in panel B. * p < 0.05 vs. RANKL(−)/AA(−)/iSN40(−), †† p < 0.01 vs. RANKL(+)/AA(−)/iSN40(−). n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Generated, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: Time course of anti-osteoclastogenic effect of iSN40. ( A ) Representative images of TRAP staining of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL for 4 days and 0.3 μM iSN40 for defined periods. d, differentiation days with iSN40 treatment. Scale bar, 100 μm. ( B ) The number of nuclei in TRAP + osteoclasts was quantified. ** p < 0.01 vs. control/RANKL(−); †† p < 0.01 vs. control/RANKL(+); ‡‡ p < 0.01 vs. d0–3, d0–4, d1–3, and d1–4; §§ p < 0.01 vs. d2–3 and d2–4. n = 3 fields.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control