Journal: Stem Cells International

Article Title: Scaffold-Free Fabrication of Osteoinductive Cellular Constructs Using Mouse Gingiva-Derived Induced Pluripotent Stem Cells

doi: 10.1155/2016/6240794

Figure Lengend Snippet: Primers used for SYBR Green quantitative RT-PCR.

Article Snippet: For immunofluorescent staining, deparaffinized sections were incubated in 0.05% Triton-X and 5% skim milk (BD, NJ) in phosphate-buffered saline at room temperature for 60 min. After washing, the sections were incubated with anti-hypoxia-induced factor-1 α (HIF-1 α ) monoclonal antibody (H1alpha 67: 1/25, Santa Cruz Biotechnology, CA), anti-type II collagen monoclonal antibody (5B2.5: 1/100, Abcam, Cambridge, UK), anti-osteocalcin polyclonal antibody (FL-95: 1/100, Santa Cruz Biotechnology), or control IgG [normal mouse IgG (sc-2025) or rabbit IgG (sc-2027): 1/100, Santa Cruz Biotechnology] at 4°C overnight and then incubated for 60 min at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (1/500, Molecular Probes, Thermo Fisher Scientific, MA) or Alexa Fluor 555-conjugated goat anti-rabbit IgG (1/500, Thermo Fisher Scientific), followed by Hoechst 33258 (1/500, Thermo Fisher Scientific) nuclear staining.

Techniques: SYBR Green Assay

Journal: Stem Cells International

Article Title: Scaffold-Free Fabrication of Osteoinductive Cellular Constructs Using Mouse Gingiva-Derived Induced Pluripotent Stem Cells

doi: 10.1155/2016/6240794

Figure Lengend Snippet: Expression of osteogenic marker genes in the GF-iPSC constructs under osteogenic induction. Expression of Runx2 (a), osterix (b), collagen 1a1 (c), and osteocalcin (d) on day 10, 20, and 30 was determined by quantitative real-time RT-PCR. Gene expression of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used as an internal control. The data represent the mean values ± SD ( n = 3). Significant differences ( ∗ P < 0.01: ANOVA with Dunnett's correction for multiple comparisons) were evaluated with respect to day 0 (before osteogenic induction) values.

Article Snippet: For immunofluorescent staining, deparaffinized sections were incubated in 0.05% Triton-X and 5% skim milk (BD, NJ) in phosphate-buffered saline at room temperature for 60 min. After washing, the sections were incubated with anti-hypoxia-induced factor-1 α (HIF-1 α ) monoclonal antibody (H1alpha 67: 1/25, Santa Cruz Biotechnology, CA), anti-type II collagen monoclonal antibody (5B2.5: 1/100, Abcam, Cambridge, UK), anti-osteocalcin polyclonal antibody (FL-95: 1/100, Santa Cruz Biotechnology), or control IgG [normal mouse IgG (sc-2025) or rabbit IgG (sc-2027): 1/100, Santa Cruz Biotechnology] at 4°C overnight and then incubated for 60 min at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (1/500, Molecular Probes, Thermo Fisher Scientific, MA) or Alexa Fluor 555-conjugated goat anti-rabbit IgG (1/500, Thermo Fisher Scientific), followed by Hoechst 33258 (1/500, Thermo Fisher Scientific) nuclear staining.

Techniques: Expressing, Marker, Construct, Quantitative RT-PCR, Gene Expression, Control

Journal: Stem Cells International

Article Title: Scaffold-Free Fabrication of Osteoinductive Cellular Constructs Using Mouse Gingiva-Derived Induced Pluripotent Stem Cells

doi: 10.1155/2016/6240794

Figure Lengend Snippet: (a) H&E staining of the osteogenically induced GF-iPSC construct, which consisted of an inner region of unstructured cell mass (inside the white dotted area) and outer osseous-like tissue region (asterisks). Scale bar: 0.5 mm. (b) Magnification of the dotted square in panel (a). Aligned cells were present outside (arrows) and inside (arrow heads) the osseous-like region (asterisk). White circle indicates the inner region of the GF-iPSC construct. Scale bar: 100 μ m. (c) Alcian blue staining also indicates the presence of aligned cells outside (arrows) and inside (arrow heads) the osseous-like region (asterisk). White circle indicates the inner region of the GF-iPSC construct. (d)–(h) Staining for HIF-1 α ((e) green fluorescence), type II collagen ((f) green fluorescence), osteocalcin ((h) red fluorescence), and nuclei ((d)–(h) blue fluorescence). Staining with nonspecific control IgGs as primary antibodies was used as a negative control (d and g). Aligned cells (arrows), osseous region (asterisks), and inner region (circles) of the cellular construct are indicated. Scale bars: 100 μ m.

Article Snippet: For immunofluorescent staining, deparaffinized sections were incubated in 0.05% Triton-X and 5% skim milk (BD, NJ) in phosphate-buffered saline at room temperature for 60 min. After washing, the sections were incubated with anti-hypoxia-induced factor-1 α (HIF-1 α ) monoclonal antibody (H1alpha 67: 1/25, Santa Cruz Biotechnology, CA), anti-type II collagen monoclonal antibody (5B2.5: 1/100, Abcam, Cambridge, UK), anti-osteocalcin polyclonal antibody (FL-95: 1/100, Santa Cruz Biotechnology), or control IgG [normal mouse IgG (sc-2025) or rabbit IgG (sc-2027): 1/100, Santa Cruz Biotechnology] at 4°C overnight and then incubated for 60 min at room temperature with Alexa Fluor 488-conjugated goat anti-mouse IgG (1/500, Molecular Probes, Thermo Fisher Scientific, MA) or Alexa Fluor 555-conjugated goat anti-rabbit IgG (1/500, Thermo Fisher Scientific), followed by Hoechst 33258 (1/500, Thermo Fisher Scientific) nuclear staining.

Techniques: Staining, Construct, Fluorescence, Control, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Naringenin is a Potential Anabolic Treatment for Bone Loss by Modulating Osteogenesis, Osteoclastogenesis, and Macrophage Polarization

doi: 10.3389/fphar.2022.872188

Figure Lengend Snippet: Naringenin alleviates LPS-induced bone loss. (A) Representative μCT analysis of trabecular bone of the distal femur. (B–D) Calculations of bone volume/total volume (BV/TV), trabecular bone mineral density (BMD), and trabecular number (Tb.N). (E) Representative H&E staining of distal femoral sections. (F) Quantification of trabecular area (Tb.Ar) from H&E staining. (G) Osteocalcin (OCN) stained sections of the distal femur. Original magnification ×40. (H) Osteoblast (OCN positive) number per bone surface (Ob.N/BS). (I) Serum level of osteoblast marker OCN. (J) Representative calcein staining of trabecula of distal femoral sections. (K) Quantification analysis of calcein staining. Mineral apposition rate (MAR) and bone formation rate per unit of bone surface (BFR/BS). (L) Tartrate-resistant acid phosphatase (TRAP) stained sections of the distal femur. Scale bar: 20 μm. (M) Osteoclast (nuclei>3; TRAP positive) number per bone surface (Ob.N/BS). (N) Serum level of osteoclast marker CTX-I. n = 5 replicates per group in all panels. Data are expressed as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Naringenin (BioChemParter, China; BCP31780; purity 98%); dimethyl sulfoxide (DMSO, Yeasen, China; 60313ES60); lipopolysaccharide (LPS, Beyotime, China; S1732); Dulbecco’s modified eagle medium (DMEM, Cytiva, China; SH30021.01); Minimal essential medium alpha modification (α-MEM, Cytiva, China; SH30265.01); fetal bovine serum (FBS, gibco, United States; 10099141); penicillin/streptomycin solution (Yuanye, China; R20016);; ACK lysing buffer (Thermo Fisher scientific, United States; A1049201); β-glycerophosphate (sigma, United States; G9422); L-ascorbic acid (sigma, United States; A4403); dexamethasone (MKBio, China; MX3252); BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China; C3206); Alizarin Red S (sigma, United States; A5533); M-CSF (Novoprotein, China; CB34); RANKL (Novoprotein, China; CR06); Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma, United States; 387A); TRITC Phalloidin (Solarbio, China; CA1610); CCK8 kit (Dojindo, Japan; CK04); RNA extracting kit (Yishan, China; RN001); PrimeScriptTM RT Master Mix (Takara, Japan; RR036); TB Green ® Premix Ex TaqTM (Takara, Japan; RR420); anti-RUNX2 rabbit pAb (Servicebio, China; GB13264); anti-osteocalcin (Bglap) rabbit mAb (Abclonal, China; A20800); anti-CTSK rabbit pAb (Abclonal, China; A1782); Anti-DCSTAMP Antibody, clone 1A2 (EMD Milipore, Sigma, United States; MABF39-I); anti-osteocalcin polyclonal antibody (Proteintech, United States; 16157-1-AP); PE anti-mouse CD197 (CCR7) antibody (BioLegend, United States; 120105); APC anti-mouse CD206 (MMR) antibody (BioLegend, United States; 141707); osteocalcin ELISA Kit (J&L Biological, China; JL20366); and CTX-I ELISA Kit (J&L Biological, China; JL20123).

Techniques: Staining, Marker

Journal: Stem Cells International

Article Title: The Histone Demethylase KDM3B Promotes Osteo-/Odontogenic Differentiation, Cell Proliferation, and Migration Potential of Stem Cells from the Apical Papilla

doi: 10.1155/2020/8881021

Figure Lengend Snippet: KDM3B enhanced the osteo-/odontogenic differentiation potential of SCAPs. (a) The knockdown efficiency of KDM3B in SCAPs was tested by western blot. (b) KDM3B knockdown significantly depressed the ALP activity in SCAPs. (c) The Alizarin red staining and (d) the quantitative calcium analysis showed that KDM3B knockdown reduced the mineralization capacity of SCAPs compared with the control group. (e, f) Real-time RT-PCR analysis confirmed that KDM3B knockdown reduced the expression of (e) RUNX2 and (f) OSX in SCAPs. (g) Western blot analysis showed the expression of RUNX2 and OSX in the KDM3B knockdown group and the control group. Histone H3 served as an internal control. (h) Western blot analysis revealed that the expression of DSPP and OCN was decreased after KDM3B was knocked down. Histone H3 served as an internal control. (i) The KDM3B overexpression was tested by western blot. (j) KDM3B overexpression significantly enhanced the ALP activity in SCAPs. (k, l) The results of (k) the Alizarin red staining and (l) the quantitative calcium analysis revealed that KDM3B overexpression enhanced the mineralization capacity of SCAPs compared with the control group. (m, n) Real-time RT-PCR analysis revealed that KDM3B overexpression increased the expression of (m) RUNX2 and (n) OSX in SCAPs. (o) Western blot analysis showed the expression of RUNX2 and OSX in the KDM3B overexpression group and the control group. Histone H3 served as an internal control. (p) Western blot analysis revealed that the expression of OCN and DSPP was enhanced after KDM3B was overexpressed. Histone H3 served as an internal control. Statistical significance was determined using Student's t -test. All error bars represent SD ( n = 3). P < 0.05. ∗∗ P ≤ 0.01.

Article Snippet: The primary antibodies used in the study are as follows: KDM3B antibody (cat no. 19915-1-AP, Rabbit Polyclonal, USA), RUNX2 antibody (cat no. ab76956, Abcam), OSX antibody (cat no. ab209484, Abcam), OCN antibody (cat no. bs-4917R, Bioss, China), DSPP antibody (cat no. bs10316R, Bioss, China), and histone H3 antibody (cat no. SC-56616, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Activity Assay, Staining, Quantitative RT-PCR, Expressing, Over Expression

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bioinspired Protein/Peptide Loaded 3D Printed PLGA Scaffold Promotes Bone Regeneration

doi: 10.3389/fbioe.2022.832727

Figure Lengend Snippet: The immumohistochemical staining for detecting the expression of Runx2, OCN and COL-1 at 4 (A) and 12 (B) weeks (400 ×).

Article Snippet: Additionally, the expression of osteogenic factors including runt-related transcription factor 2 (Runx2) (Ab-AF5186,Affinity), osteocalcin (OCN) (OM266707, OmnimAbs)and Collagen I antibody (bs-10423R,Bioss) were analyzed through immunohistochemistry.

Techniques: Staining, Expressing