Journal: PLoS ONE

Article Title: Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

doi: 10.1371/journal.pone.0116462

Figure Lengend Snippet: RT-PCR primers used in this study.

Article Snippet: Bone formation was confirmed by immunohistochemistry using a primary antibody against osteocalcin (1:5000, OCG3, Takara Bio).

Techniques:

Journal: PLoS ONE

Article Title: Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

doi: 10.1371/journal.pone.0116462

Figure Lengend Snippet: A: Schematic representation of the cell culture protocol. Gross images of ALP staining (ALP) and Von Kossa staining (VK) of BMSCs (B) and MuSCs (D). Quantitative analysis of the mRNA expression of ALP and osteocalcin in BMSCs (C) and MuSCs (E). The fold change in gene expression was normalized to that of BM-Dex-AG or Mu-Dex-AG. Bars show the mean and SEM. Statistical significance was confirmed between the BM and BM-Dex groups (ALP: p = 0.015, OCN: p = 0.023) and between the Mu and Mu-DEX groups (ALP: p = 0.019, OCN: p = 0.015). Effects of combinations of differentiation reagents were significant for ALP in BM-Dex (p = 0.032) and Mu-DEX (p = 0.019) and for OCN in Mu-DEX (p = 0.024).

Article Snippet: Bone formation was confirmed by immunohistochemistry using a primary antibody against osteocalcin (1:5000, OCG3, Takara Bio).

Techniques: Cell Culture, Staining, Expressing

Journal: PLoS ONE

Article Title: Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

doi: 10.1371/journal.pone.0116462

Figure Lengend Snippet: A: Bone formation capability of muscle-derived cells. Representative histological sections of a scaffold loaded with BMSCs or MuSCs cultured with both dexamethasone and BMP-2. Scale bar: 1 mm (left panels), 200 μm (middle panels) and 50 μm (right panels). Black arrows indicate new bone formation in the scaffold. Black arrow heads indicate osteocytes and green arrow heads indicate bone lining cells. B: Newly formed bone, T: β-TCP, P: Porous area. B: Recruitment of cells residing in muscle tissue to participate in BMP-2-induced ectopic bone formation. Cells labeled prior to local BMP-2 administration were detected in the newly formed woven bone area. Representative histological sections were stained with H&E and evaluated for i-QD fluorescence. The black arrows in the H&E image show the locations of fluorescently labeled cells indicated with white arrows in the fluorescent image. Scale bar: 200 μm. B: Newly formed bone, T: β-TCP, P: Porous area. C: Augmentation of ectopic bone formation by dexamethasone. C-1, 2: Representative histological sections of an excised scaffold that had been loaded with BMP-2 alone and a scaffold that had been loaded with dexamethasone and BMP-2. The sections were stained with H&E and immunostained for osteocalcin. Black arrows indicate new bone formation in the scaffold. Red arrows indicate osteocalcin positive staining area. Scale bar: 1 mm (top panels of C-1), 200 μm (bottom panels of C-1) and 50 μm (C-2). B: Newly formed bone, T: β-TCP, P: Porous area. C-3: Quantification of bone formation at 3 weeks after transplantation. The Y axis indicates the bone formation ratio calculated as total bone area/total scaffold area. Each bar represents the mean with the standard deviation (SD). *denotes P < 0.05.

Article Snippet: Bone formation was confirmed by immunohistochemistry using a primary antibody against osteocalcin (1:5000, OCG3, Takara Bio).

Techniques: Derivative Assay, Cell Culture, Labeling, Staining, Fluorescence, Transplantation Assay, Standard Deviation

Journal: PeerJ

Article Title: Long noncoding RNA Gm31629 promotes bone regeneration by maintaining bone marrow mesenchymal stem cells activity

doi: 10.7717/peerj.13475

Figure Lengend Snippet: (A) Time plan for surgical ablation of trabecular bone in distal femoral of mice. (B) Representative micro-CT images. The white square was selected to measure trabecular bone volume in bone regeneration region. (C) Quantification of trabecular bone volume in bone regeneration region. ( n = 6). (D) HE staining of distal femora. Scale bar: 200 µm. (E) Immunohistochemical staining of osteocalcin positive cells. Black arrows represent osteocalcin positive cells. Scale bar: 50 µm. (F) Quantitative analysis of osteocalcin positive cells. ( n = 6). (G) TRAP staining images. Scale bar: 50 µm. (H) Quantitative analysis of TRAP positive cells. ( n = 6). Data are expressed as mean ± sd and statistical differences were analyzed by Student’s t test. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: For immunocytochemistry, after antigen retrieval the samples were incubated with primary antibodies against osteocalcin (Takara M173) at 4 °C overnight and Horseradish peroxidase-streptavidin detection system (Dako Agilent, Santa Clara, CA) was used to detect immuno-activity.

Techniques: Micro-CT, Staining, Immunohistochemical staining

Journal: PeerJ

Article Title: Long noncoding RNA Gm31629 promotes bone regeneration by maintaining bone marrow mesenchymal stem cells activity

doi: 10.7717/peerj.13475

Figure Lengend Snippet: (A) Time plan for surgical ablation of trabecular bone in distal femoral of mice. (B) Representative micro-CT images. The white square was selected to measure trabecular bone volume in bone regeneration region. (C) Quantification of trabecular bone volume in bone regeneration region. ( n = 6). (D) HE staining of distal femora. Scale bar: 200 µm. (E) Immunohistochemical staining of osteocalcin positive cells. Black arrows represent osteocalcin positive cells. Scale bar: 50 µm. (F) Quantitative analysis of osteocalcin positive cells. ( n = 6). (G) TRAP staining images. Scale bar: 50 µm. (H) Quantitative analysis of TRAP positive cells. ( n = 6). Data are expressed as mean ± sd and statistical differences were analyzed by Student’s t test. ∗ P < 0.05; ∗∗ P < 0.01; N.S, no significance.

Article Snippet: For immunocytochemistry, after antigen retrieval the samples were incubated with primary antibodies against osteocalcin (Takara M173) at 4 °C overnight and Horseradish peroxidase-streptavidin detection system (Dako Agilent, Santa Clara, CA) was used to detect immuno-activity.

Techniques: Micro-CT, Staining, Immunohistochemical staining

Journal: PeerJ

Article Title: Long noncoding RNA Gm31629 promotes bone regeneration by maintaining bone marrow mesenchymal stem cells activity

doi: 10.7717/peerj.13475

Figure Lengend Snippet: (A) Time point at which mice were treated with TF2A or vehicle, and performed with surgical ablation of trabecular bone in distal femoral . (B) The body weight of 12-month-old mice before and after treated with TF2A or vehicle. ( n = 6). (C) Representative micro-CT images. The white square was selected to measure trabecular bone volume in bone regeneration region. (D) Quantification of trabecular bone volume in bone regeneration region. ( n = 6). (E) HE staining of distal femora of middle-aged mice. Scale bar: 200 µm. (F) Immunohistochemical staining of osteocalcin positive cells. Black arrows represent osteocalcin positive cells. Scale bar: 50 µm. (G) Quantitative analysis of osteocalcin positive cells. ( n = 6). (H) TRAP staining images. Scale bar: 50 µm. (I) Quantitative analysis of TRAP positive cells. ( n = 6). Data are expressed as mean ± sd and statistical differences were analyzed by Student’s t test. ∗ P < 0.05; N.S, no significance.

Article Snippet: For immunocytochemistry, after antigen retrieval the samples were incubated with primary antibodies against osteocalcin (Takara M173) at 4 °C overnight and Horseradish peroxidase-streptavidin detection system (Dako Agilent, Santa Clara, CA) was used to detect immuno-activity.

Techniques: Micro-CT, Staining, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Platelet-derived growth factor-BB regenerates functional periodontal ligament in the tooth replantation

doi: 10.1038/s41598-022-06865-6

Figure Lengend Snippet: PDGF-BB decreases osteocalcin-positive cells and β-catenin accumulation in nucleus in vivo. ( A ) Representative images of sections immunostained for osteocalcin (OCN). Sections were immunostained with primary anti-OCN antibody and peroxidase-conjugated secondary antibody, and the immune complex was visualized with DAB substrate. The sections were then counterstained with haematoxilin. Pictures were taken of periodontal ligament (PDL) of the mesial root of the replanted teeth in the AC (a) and PDGF-BB (b) groups 3 days after tooth replantation. ( B ) Representative images of sections immunostained for β-catenin. Sections were incubated with primary anti-β-catenin antibody and secondary Alexa 488-conjugated antibody, and then counterstained with DAPI. DAPI (Ba and Bd), ß-catenin (Bb and Be), and overlay (Bc and Bf) images of the periodontal tissues. Pictures were taken of PDL of the mesial root of the replanted teeth day 3 in the AC (a-c) and PDGF-BB (d-f) groups. White arrows indicate nuclear b-catenin positive cells. B bone, Cm cementum, D dentine.

Article Snippet: Briefly, deparaffinized sections from the AC and PDGF-BB groups were incubated with a primary antibody against bovine osteocalcin (OC4-30, Takara, Ohtsu, Japan) and incubated with peroxidase-conjugated secondary antibody.

Techniques: In Vivo, Incubation