recombinant human rankl  (Sino Biological)

Journal: Clinical Implant Dentistry and Related Research

Article Title: Bone Morphogenetic Protein ( BMP ) 9 Outperforms BMP2 in Osteogenesis and Osseointegration: In Vitro and In Vivo

doi: 10.1111/cid.70135

Figure Lengend Snippet: Comparative effects of BMP9 and BMP2 on osteogenic differentiation and osteoclastogenesis in vitro. (A) Real‐time PCR analysis of key osteogenic genes (Col1, Runx2, ALP, and OCN) in MC3T3‐E1 cells treated with 8 nM of BMP2 or BMP9 for 3, 5, and 7 days. All gene‐expression levels were normalized to GAPDH. (B) Western blot analysis of osteogenic marker proteins in cell lysates harvested after 7 days of treatment with BMP2 or BMP9. GAPDH was used as the loading control. Densitometric quantification of band intensities (integrated density) normalized to GAPDH is shown below the blots and presented as relative protein expression. (C) Western blot showing dose‐dependent p‐Smad1/5/9 in MC3T3‐E1 cells exposed to varying concentrations of BMP2 or BMP9. Phosphorylation was quantified by densitometry and expressed as fold change vs. control after normalization using [(p‐Smad1/5/9)/(total Smad1/5/9)] and further normalized to GAPDH, as shown in the graph below the blots. Asterisks indicate statistical significance for pairwise comparisons between BMP2 and BMP9 at the same concentration (****, p < 0.0001), unless otherwise indicated. (D) ALP activity and representative images of ALP staining in MC3T3‐E1 cultures after 7 days of induction with BMP2 or BMP9. (E) Alizarin Red S staining illustrating mineralized nodule formation after extended culture with BMP2 or BMP9. (F) Representative TRAP‐stained images of RAW 264.7‐derived osteoclasts following treatment with RANKL (3 nM), BMP2 (8 nM), or BMP9 (8 nM) for 5 days. TRAP‐positive multinucleated osteoclasts are indicated by arrows. Scale bar, 20 μm. (G) Quantification of TRAP‐positive multinucleated cells per well. Data are presented as the mean ± SD ( n = 3 independent experiments), and p ‐values were calculated using one‐way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). BMP, bone morphogenetic protein; PCR, polymerase chain reaction; ALP, alkaline phosphatase; Col1, collagen type I; Runx2, runt‐related transcription factor 2; OCN, osteocalcin; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Recombinant human RANKL (11682‐HNCH; Sino Biological, Beijing, China) and a tartrate‐resistant acid phosphatase (TRAP) staining kit (MK300; Takara Bio, Shiga, Japan) were used for the osteoclast differentiation assay.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Marker, Control, Expressing, Phospho-proteomics, Concentration Assay, Activity Assay, Staining, Derivative Assay, Polymerase Chain Reaction

Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFα induced osteoclast formation at the late culture time point in the presence of RANKL and M-CSF. (A) Cultured bone marrow cells were treated with 100 ng/mL RANKL, 20 ng/mL M-CSF, and indicated doses of TNFα (1, 3, or 10 ng/mL) for 10 days. (B) Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 10 days. TNFα (10 ng/mL) was also added in the early (days 1–4), middle (days 4–9), and late (days 7–9) stages of cell culture. Cells were assessed by TRAP staining, and TRAP-positive multinuclear cells containing more than three nuclei (TRAP[+] MNCs) were counted. n = 8 per group, ∗ p < 0.05 compared with the control. Images are shown at low ( × 100) and high ( × 400) magnification to visualize the overall cell density and detailed morphology of osteoclasts, respectively. Scale bars = 0.25 mm.

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Cell Culture, Staining, Control

Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFα mRNA expression level was higher in non-adherent bone marrow cells, whereas TNFR1 and TNFR2 mRNA expression levels were higher in osteoclasts. Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 9 days. Cells were separated into osteoclasts and non-adherent bone marrow cells using the pronase procedure (A). RANKL and RANK (B), and TNFα, TNFR1, and TNFR2 (C) mRNA expression levels were quantified by quantitative RT-PCR. mRNA expression levels were normalized to that of β-actin mRNA. n = 3 per group, ∗ p < 0.05 compared with osteoclasts.

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR

Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFα treatment in the later stage of culture increased CX3CL1 and CXCL7 mRNA expression levels in non-adherent bone marrow cells. Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 9 days. TNFα (10 ng/mL) was also added in the late (days 7–9) stage of cell culture. Cells were separated into osteoclasts and non-adherent bone marrow cells using the pronase procedure. RANK, CXCR4, CXCR2, IGF1R, NFATc1, DC-STAMP, TRAP, CX3CR1, and iNOS mRNA expression levels in osteoclasts (A) and RANKL, SDF1, CXCL1, CXCL7, IGF2, OPG, and iNOS mRNA expression levels in non-adherent bone marrow cells were quantified using quantitative RT-PCR. mRNA expression levels were normalized to that of β-actin mRNA. n = 3 per group, ∗ p < 0.05 compared with the control (without TNFα stimulation).

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Control

Journal: Biochemistry and Biophysics Reports

Article Title: TNFα stimulates osteoclastogenesis and expression of CX3CL1 in non-adherent bone marrow cells

doi: 10.1016/j.bbrep.2025.102155

Figure Lengend Snippet: TNFR1-and TNFR2-neutralizing antibodies reduced the enhancement of osteoclast formation by TNFα treatment in the later stage of cell culture. (A) Cultured bone marrow cells were treated with 100 ng/mL RANKL and 20 ng/mL M-CSF for 9 days. TNFα (10 ng/mL) was also added in the late (days 7–9) stage of cell culture. Cells were also treated with anti-TNFR1 and/or anti-TNFR2 antibody (Ab) (each 3 μg/mL) 30 min before TNFα treatment. Cells were assessed by TRAP staining, and TRAP[+] MNCs were counted. n = 4 per group, ∗ p < 0.05 compared with the control (without TNFα and without anti-TNFR1 and/or anti-TNFR2 antibody stimulation). # p < 0.05 compared with TNFα alone. Images are shown at low ( × 100) and high ( × 400) magnification to visualize the overall cell density and detailed morphology of osteoclasts, respectively. Scale bars = 0.25 mm. (B) CX3CL1 and CXCL7 mRNA expression levels in non-adherent bone marrow cells were quantified using quantitative RT-PCR. mRNA expression levels were normalized to that of β-actin mRNA. n = 3 per group, ∗ p < 0.05 compared with the control (without TNFα stimulation).

Article Snippet: Recombinant soluble human RANKL was prepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).

Techniques: Cell Culture, Staining, Control, Expressing, Quantitative RT-PCR

Journal: JBMR Plus

Article Title: Cathepsin K inhibitors promote osteoclast-osteoblast communication and engagement of osteogenesis

doi: 10.1093/jbmrpl/ziaf079

Figure Lengend Snippet: Prevalence of ALP + OBs in resorption cavities and level of osteogenic markers after a 72 hr co-culture of OBs and OCs in the presence and absence of CatK inhibitors. (A) Representative images of TRAP-stained OCs (orange) and ALP-stained OBs (purple) under control, T06 and ODN treated conditions. The segmented outline (black dotted) shows the resorption excavation generated by OCs. (B) The quantification of ALP + resorption cavities (±OCs) during 72 hr of OC-OB co-culture indicated the preference of OBs towards pits compared to trenches when normalized with total number of pits and trenches. (C) The quantification of ALP + resorption cavities with OCs (+OCs). Statistics: Kruskal–Wallis’s test, 2 tailed ( * p < .05; *** p < .001); Dunn’s multiple comparisons test ( * p < .057; *** p < .001) compared to the untreated control. (D) ALP activity quantification in the culture supernatant showed significantly enhanced activity under CatK inhibition (Kruskal–Wallis’s test, 2 tailed (ns: not significant; * p < .05; ** p < .01; *** p < .001). Sample size ( n = 4 donors) for control, T06 (300 nM, 500 nM, 1 μM), and ODN (15 nM, 50 nM). For each donor, 3 replicate experiments on individual bone slices were analyzed for all conditions. The median obtained in each experiment are shown as bars. We analyzed between 450 and 800 OC-OB activity per condition per experiment for each of the donors). (E) Western blot analysis of ALP, RANKL, collagen (COL1), CatK and MMP 13 in control, T06 (0.3, 0.5, and 1 μM), ODN (15 and 50 nM) conditions and (F) their quantification ( n = 3 donors). Statistics: Kruskal–Wallis’s test, 2 tailed (ns: not significant; ** p < .01; *** p < .001); Dunn’s multiple comparisons test ( ** p < .02; *** p < .001) compared to untreated control.

Article Snippet: After 2 d, the cells were differentiated into mature OCs by further culturing them for 7 d in medium containing 25 ng/mL of both M-CSF and recombinant human RANKL (R&D Systems) and medium was changed twice.

Techniques: Co-Culture Assay, Staining, Control, Generated, Activity Assay, Inhibition, Western Blot