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: Bone & Joint Research

Article Title: OX40L in endothelial cells promotes temporomandibular joint subchondral bone angiogenesis and osteoclastogenesis in mice

doi: 10.1302/2046-3758.154.BJR-2025-0249.R1

Figure Lengend Snippet: Changes in subchondral vascular proliferation and bone remodelling during different progression cycles of temporomandibular joint osteoarthritis (TMJOA). TMJOA was induced in C57BL/6J mice using a unilateral anterior crossbite (UAC) model, and subsequently assessed at two, four, six, and eight weeks after initiation. a) Haematoxylin & eosin (HE) staining in mouse condylar cartilage. The double arrow shows the thickness of the cartilage layer. Scale bar: 100 µm. b) Safranin O/fast green staining in mouse condylar cartilage. Scale bar: 100 µm. c) Tartrate-resistant acid phosphatase (TRAP) staining in mouse condylar subchondral bone. Scale bar: 100 µm. d) Micro-CT image in mouse condyle. e) CD31 immunofluorescence staining in mouse condylar subchondral bone. Scale bar: 100 µm. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Additionally, sagittal sections were processed for tartrate-resistant acid phosphatase (TRAP) staining (Servicebio, China) to quantify osteoclast numbers in the subchondral bone.

Techniques: Staining, Micro-CT, Immunofluorescence

Journal: Bone & Joint Research

Article Title: OX40L in endothelial cells promotes temporomandibular joint subchondral bone angiogenesis and osteoclastogenesis in mice

doi: 10.1302/2046-3758.154.BJR-2025-0249.R1

Figure Lengend Snippet: Abnormal activation of OX40L in endothelial cells can promote angiogenesis and osteoclast induction. Human bone microvascular endothelial cells were pretreated with KY1005 for six hours, followed by treatment with sOX40 for 48 hours. a) Detection of vascular endothelial growth factor (VEGF) gene expression using quantitative polymerase chain reaction (qPCR). b) Detection of VEGF protein expression using western blotting. c) Representative images of endothelial cell scratch experiments. Scale bar: 100 µm. d) Representative images of endothelial cell tubule formation experiments. Scale bar: 100 µm. e) Representative image of tartrate-resistant acid phosphatase (TRAP) staining after endothelial cells were co-cultured with RAW264.7 cells. Scale bar: 50 µm. f) Bone resorption pits were shown by scanning electron microscopy. Scale bar: 500 nm.

Article Snippet: Additionally, sagittal sections were processed for tartrate-resistant acid phosphatase (TRAP) staining (Servicebio, China) to quantify osteoclast numbers in the subchondral bone.

Techniques: Activation Assay, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Cell Culture, Electron Microscopy

Journal: Bone & Joint Research

Article Title: OX40L in endothelial cells promotes temporomandibular joint subchondral bone angiogenesis and osteoclastogenesis in mice

doi: 10.1302/2046-3758.154.BJR-2025-0249.R1

Figure Lengend Snippet: Blocking the OX40–OX40L pathway alleviates abnormal subchondral angiogenesis and osteoclastic activity in early temporomandibular joint osteoarthritis (TMJOA). After four weeks of unilateral anterior cross-bite (UAC) modeling, C57BL/6J mice received intraperitoneal injections of OX40L monoclonal antibody. a) CD4 immunofluorescence staining in mouse condylar subchondral bone. Scale bar: 100 µm. b) Statistical analysis of the proportion of CD4 + T cells in in mouse condylar subchondral bone. c) CD31 immunofluorescence staining in mouse condylar subchondral bone. Scale bar: 100 µm. d) Statistical analysis of blood vessel area in mouse condylar subchondral bone. e) Tartrate-resistant acid phosphatase (TRAP) staining in mouse condylar subchondral bone. Scale bar: 100 µm. f) Statistical analysis of the number of TRAP + osteoclasts in mouse condylar subchondral bone. g) Micro-CT image of mouse condyle. h) to k) Results of morphological analysis of condylar subchondral bone, including statistical analysis of bone volume to tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Data are presented as the mean (SD) (n = 3). Statistical significance was determined by independent-samples t -test.*p < 0.05, **p < 0.01 and ***p < 0.001. DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline.

Article Snippet: Additionally, sagittal sections were processed for tartrate-resistant acid phosphatase (TRAP) staining (Servicebio, China) to quantify osteoclast numbers in the subchondral bone.

Techniques: Blocking Assay, Activity Assay, Immunofluorescence, Staining, Micro-CT, Saline

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) inhibits receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. a), c), and e) Immunofluorescence staining confirmed that CXCL5 (1 or 50 ng/ml) inhibits RANKL-induced differentiation of RAW264.7 cells into cathepsin K (CTSK)-positive osteoclast-like cells. b), d), and f) CXCL5 decreased the differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells. Purple indicates TRAP expression, and cyan indicates nuclei. g) and h) Western blot analysis showed that CXCL5 (1 ng/ml) inhibited RANKL-induced differentiation of RAW264.7 cells by suppressing the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), resulting in decreased CTSK expression at 72 hours of treatment. Data are presented as the mean (standard error of the mean (SEM)) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Osteoclast formation was evaluated by immunostaining for cathepsin K (CTSK) (ab19027; Abcam, UK) and tartrate-resistant acid phosphatase (TRAP) (MK300; Takara Bio, Japan).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Comparison