Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Scanning electron microscopy (SEM) of the 3D cell–collagen scaffold construct. Representative SEM images: ( A ) the 3D osteoblast–collagen scaffold construct (non-loading), ( B ) the 3D osteoblast–collagen scaffold construct (after 24 h mechanical loading), ( C ) the 3D osteoblast–collagen scaffold construct (after 48 h non-loading), and ( D ) the 3D osteoblast–collagen scaffold construct (after 48 h mechanical loading). ( E ) Representative SEM images of a 3D chondrocyte–collagen scaffold construct (non-loading), ( F ) the 3D chondrocyte–collagen scaffold construct (after 24 h mechanical loading), ( G ) the 3D chondrocyte–collagen scaffold construct (48 h non-loading), and ( H ) the 3D chondrocyte–collagen scaffold construct (after 48 h mechanical loading) (each subgroup/time point, n = 12).

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Electron Microscopy, Construct

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Effects of mechanical loading on osteoblast activity. Cohen’s d between the control and the mechanical loading group is shown in the above Gardner–Altman estimation plot. Both groups are plotted on the left axes; the mean difference is plotted on a floating axes on the right as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. The effect sizes and CIs are reported above as effect size (CI width lower bound; upper bound). ( A ) ALP: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group. ( B ) Osteocalcin: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group).

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Activity Assay, Control, Sampling, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Effects of mechanical loading on chondrocyte activity. Effects of mechanical loading on osteoblast activity. Cohen’s d between the control and mechanical loading group is shown in the above Gardner–Altman estimation plot. Both groups are plotted on the left axes; the mean difference is plotted on a floating axes on the right as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. The effect sizes and CIs are reported above as effect size (CI width lower bound; upper bound). ( A ) Proteoglycan: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group. ( B ) Type II collagen: ( a ) control group versus mechanical loading (+)-24 h incubation group, ( b ) control group versus mechanical loading (+)-48 h incubation group).

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Activity Assay, Control, Sampling, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Expressions of Glut1, SIRT1, and Runx2 in osteoblasts. ( A ) The expression of Glut1 in non-loaded osteoblasts, 24 h loaded osteoblasts, and 48 h loaded osteoblasts. ( B ) The expression relative to β-actin. ( C ) The expression of SIRT1 in non-loaded osteoblasts, 24 h loaded osteoblasts, and 48 h loaded osteoblasts. ( D ) The expression relative to β-actin. ( E ) The expression of Runx2 in non-loaded osteoblasts, 24 h loaded osteoblasts, and 48 h loaded osteoblasts. ( F ) The expression relative to the level of β-actin.

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Effects of the Glut1 inhibitor on Glut-1, SIRT1, and Runx2 in osteoblasts. ( A ) The expression of Glut1 in mechanically loaded or non-loaded osteoblasts in the presence or absence of a Glut1 inhibitor (Western blot). ( a ) The culture medium only, ( b ) the control group (DMSO solution + culture medium), ( c ) the Glut1 inhibitor (WZB117)-treated group (10.0 μM WZB117 in DMSO solution + culture medium), and ( d ) the expression relative to β-actin. ( B ) The expression of SIRT1 in mechanically loaded or non-loaded osteoblasts in the presence or absence of a Glut1 inhibitor (Western blot). ( a ) The culture medium only, ( b ) the control group (DMSO solution + culture medium), ( c ) the Glut1 inhibitor (WZB117)-treated group (10.0 μM WZB117 in DMSO solution + culture medium), and ( d ) the expression relative to β-actin. ( C ) The expression of Runx2 in mechanically loaded or non-loaded osteoblasts in the presence or absence of a Glut1 inhibitor (Western blot). ( a ) The culture medium only, ( b ) the control group (DMSO solution + culture medium), ( c ) the Glut1 inhibitor (WZB117)-treated group (10.0 μM WZB117 in DMSO solution + culture medium), and ( d ) the expression relative to β-actin. * p < 0.05.

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)

doi: 10.3390/ijms22169070

Figure Lengend Snippet: Summary of our current study. ( A ) Involvement of osteocytes in osteoblast differentiation. Mechanical loading suppresses the expression of osteocytic sclerostin following activation of Wnt/beta-catenin signaling in osteocytes and osteoblasts, resulting in osteoblast differentiation and bone formation activity. Cellular interaction of osteoblasts with osteocytes, via the sclerostin–Wnt/beta-catenin signaling pathways, plays an important part in physiologic mechanical stress-mediated bone metabolism. ( B ) Mechanical loading may directly induce osteoblast differentiation and bone formation activity, without the molecular mechanism of osteocyte-to-osteoblast interaction. The mechanical stress-induced expression of Glut1 and resultant uptake of glucose may suppress the level of the energy sensor SIRT1 in osteoblast energy metabolism. Since SIRT1 is recognized to negatively regulate Runx2 activity, the mechanical stress-induced suppression of SIRT1 results in increased activity of the osteogenic transduction factor Runx2 in osteoblasts, leading to osteoblast differentiation and bone formation.

Article Snippet: Osteoblasts: Human osteoblasts were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured according to the recommendations of the supplier in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich Japan KK., Tokyo, Japan) containing 10% fetal bovine serum (FBS: Fujifilm Wako Pure Chemical Inc., Tokyo, Japan), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Inc.), and 100 U/mL each of penicillin and streptomycin (Penicillin/Streptmycin solution, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 3 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing, Activation Assay, Activity Assay, Protein-Protein interactions, Transduction

Journal: The Journal of Experimental Medicine

Article Title: Immune regulation by glucocorticoids can be linked to cell type–dependent transcriptional responses

doi: 10.1084/jem.20180595

Figure Lengend Snippet: The direction and magnitude of transcriptional regulation by glucocorticoids are cell type dependent. Four primary human hematopoietic cell types and five primary human nonhematopoietic cell types were studied. For each cell type, cells from four unrelated healthy donors were independently cultured and treated with methylprednisolone (22.7 µM) or vehicle (0.08% ethanol). Total RNA was purified 2 and 6 h after in vitro treatment and RNA-seq was performed. Differential expression was assessed by comparing data from methylprednisolone-treated versus vehicle-treated cells in the four biological replicates. The statistical significance of differential expression was calculated with a Wald test, after accounting for dispersion, library size, and read count. The resulting P values for differential expression were adjusted for multiple testing by the method of . (a) The left panel displays the transcriptional response to glucocorticoids in hematopoietic cells versus nonhematopoietic cells for each of 56,870 genes. The log2 fold change compares methylprednisolone-treated versus vehicle-treated cells after 6 h of in vitro treatment. Each dot represents one gene. The x-axis variable is the mean log2 fold change in the five nonhematopoietic cells (endothelial cells, fibroblasts, myoblasts, osteoblasts, and preadipocytes), and the y-axis variable is the mean log2 fold change (FC) in the four hematopoietic cells (B cells, CD4 + T cells, monocytes, and neutrophils). The four tails of the distribution are color-coded and represent genes with evidence of transcriptional response to glucocorticoid (defined here as a mean log2 fold change ≥ 0.5 or ≤ −0.5) in one group of cells but not in the other. The right panel displays the baseline expression levels in hematopoietic versus nonhematopoietic cells for the genes with strongest evidence of a transcriptional response to glucocorticoid in one group of cells but not in the other (genes at the four tails of the distribution, as defined above). The values displayed are the mean log2 normalized read count at baseline in nonhematopoietic cells (x axis) versus hematopoietic cells (y axis). (b) Transcriptional response of TRIM22 to in vitro glucocorticoid treatment in nine primary human cell types. (c) Transcriptional response of ITGA5 to in vitro glucocorticoid treatment in nine primary human cell types. In b and c, the values displayed are the normalized read counts in vehicle-treated cells (VH; average of 2 and 6 h) and in glucocorticoid-treated cells (GC; 2 or 6 h). Each dot represents one biological replicate (one donor). Multiple-testing-adjusted P values ( q ) are from comparisons of glucocorticoid-treated versus vehicle-treated cells at each of the two time points. ns, not significant ( q > 0.05).

Article Snippet: Human primary osteoblasts from adult (Lonza; cat. no. CC-2538, lot no. 0000435102) or child (Lonza; cat. no. CC-2538, lot nos.

Techniques: Cell Culture, Purification, In Vitro, RNA Sequencing, Quantitative Proteomics, Dispersion, Expressing

Journal: Bone

Article Title: Ex Vivo Construction of Human Primary 3D-Networked Osteocytes

doi: 10.1016/j.bone.2017.09.012

Figure Lengend Snippet: [32] (a) microfluidic perfusion device with 6 culture chambers, (b) cross-sectional view of a 3D culture chamber with the red arrows indicating the overall direction of culture medium flow through the device, (c) schematic illustration of microbeads-guided assembly, and (d) histologic image of 3D-networked osteocytes with the red arrows indicating medium flow direction with respect to the tissue sample. Scale bar: 20 µm.

Article Snippet: A human 3D bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via: (1) the biomimetic assembly of primary human osteoblastic cells with 20–25 μm and BCP microbeads and (2) subsequent microfluidic perfusion culture.

Techniques:

Journal: Bone

Article Title: Ex Vivo Construction of Human Primary 3D-Networked Osteocytes

doi: 10.1016/j.bone.2017.09.012

Figure Lengend Snippet: (a) hip fragment shown as an example; (b) as-isolated cells after 4 collagenase digestion cycles; (c) proliferated osteoblastic cells after 10 days of 2D culture; (d) 3D tissue sample constructed using 20–25 µm microbeads and proliferated cells and 14 days of perfusion culture; (e) H&E histologic images showing the formation of 3D cellular network as indicated by black arrows in (f) and white arrows in (g); and (h) immunostaining for sclerostin (red). (d) –(f) from patient sample #6 and (g)–(h) from patient sample #4. Scale bar: 25 µm.

Article Snippet: A human 3D bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via: (1) the biomimetic assembly of primary human osteoblastic cells with 20–25 μm and BCP microbeads and (2) subsequent microfluidic perfusion culture.

Techniques: Isolation, Construct, Immunostaining