d bmp2  (Cell Signaling Technology Inc)

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: Preparation of the D-Bmp2@M system and its therapeutic mechanism for osteoporosis fractures. Bmp2 fused with DSS6 were expressed in HEK293T and then encapsulated in porous PLGA microspheres to construct the D-Bmp2@M system. Upon injection into the osteoporotic fracture site, the system gradually releases D-Bmp2 as the microspheres degrade over approximately 30 days. The released D-Bmp2 actively binds to bone tissue due to the affinity of DSS6 for bone. This localized enrichment promotes osteogenic activity at the fracture site, promoting fracture healing while reducing the risk of ectopic bone formation. The sustained-release and targeted delivery systems provides a superior therapeutic strategy for fracture treatment.

Article Snippet: After blocking with 3% BSA, membranes were incubated with the following primary antibodies: anti-Flag (1:5000, Sigma‒Aldrich, Germany) to detect Bmp2 and D-Bmp2, anti-phospho-Smad1/5/9 (1:1000, Cell Signaling Technology, USA) to assess pathway activation, anti-Runx2 (1:1000, Beyotime, China), and anti-Sp7 (1:1000, MCE, China) to examine osteogenic marker expression.

Techniques: Construct, Injection, Activity Assay

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system (IVIS) (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After blocking with 3% BSA, membranes were incubated with the following primary antibodies: anti-Flag (1:5000, Sigma‒Aldrich, Germany) to detect Bmp2 and D-Bmp2, anti-phospho-Smad1/5/9 (1:1000, Cell Signaling Technology, USA) to assess pathway activation, anti-Runx2 (1:1000, Beyotime, China), and anti-Sp7 (1:1000, MCE, China) to examine osteogenic marker expression.

Techniques: Biomarker Discovery, Plasmid Preparation, Construct, Immunofluorescence, Staining, Transfection, SDS Page, Purification, Western Blot, Expressing, Activity Assay, Reporter Assay, Luciferase, Fluorescence, In Vivo Imaging, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: Preparation and characterization of self-healing sustained-release microspheres loaded with D-Bmp2. a. Representative SEM images of microspheres before (top) and after (bottom) healing; left scale bar: 10 μm; middle and right scale bar: 2.5 μm. b. Statistical analysis of the microsphere diameter before and after healing determined via SEM. c. Representative confocal microscopy images of protein-loaded microspheres: PLGA microspheres (red) and Cy5-labeled D-Bmp2 (blue). Scale bar: 2 μm. d. Morphology of lyophilized D-Bmp2@M powder. e. SDS-PAGE of lyophilized D-Bmp2@M powder at different storage times. f. Representative firefly luciferase images from bioactivity assays of lyophilized D-Bmp2@M powder at different times. g. Activity change curve of lyophilized D-Bmp2@M powder at different time points (n = 3 per group). h, i. In vitro fluorescence intensity changes of Cy7-labeled D-Bmp2 from microspheres: (h) Representative fluorescence images of Cy7-D-Bmp2 maintained in microspheres (0–30 days) (top) and representative SEM images of microsphere degradation at different time points. Scale bar: 2.5 μm (bottom); (i) Relative fluorescence intensity change of Cy7-D-Bmp2 maintained in microspheres (n = 3 per group). j. Representative firefly luciferase images from Bmp2 reporter assays. k. Protein activity normalization: ratio of luminescence intensity (data from ) to protein concentration (data from ) (n = 3 per group). The data are presented as the means ± SDs. One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant).

Article Snippet: After blocking with 3% BSA, membranes were incubated with the following primary antibodies: anti-Flag (1:5000, Sigma‒Aldrich, Germany) to detect Bmp2 and D-Bmp2, anti-phospho-Smad1/5/9 (1:1000, Cell Signaling Technology, USA) to assess pathway activation, anti-Runx2 (1:1000, Beyotime, China), and anti-Sp7 (1:1000, MCE, China) to examine osteogenic marker expression.

Techniques: Confocal Microscopy, Labeling, SDS Page, Luciferase, Activity Assay, In Vitro, Fluorescence, Protein Concentration

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: In vitro validation of D-Bmp2@M osteogenic efficacy and inhibition of ectopic ossification. a. Schematic diagram of the osteoblast-bone Transwell model. Bmp2/D-Bmp2@M microspheres or free Bmp2/D-Bmp2 were loaded in the upper chambers, MC3T3-E1 cells were cultured on two coverslips (one of which was precoated with HA) in the lower compartments, and the medium was refreshed every day for 7 or 14 days. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed at days 7 and 14, respectively. b. Osteogenic differentiation staining: ALP (early-stage, day 7) and ARS (late-stage, day 14) staining. Scale bar: 200 μm. c. ALP activity was quantitatively analyzed using an ALP kit (n = 3 per group). d. Relative quantitative analysis of ARS staining was performed at an OD of 562 nm (n = 3 per group). e. qPCR analysis of Bmp2 signaling-related mRNA in MC3T3-E1 cells (n = 3 per group). f. Schematic diagram of the muscle-bone Transwell model. Bovine bone slices were co-incubated with Bmp2/D-Bmp2@M or free Bmp2/D-Bmp2 in the upper chambers, and C2C12 cells were cultured in the lower chambers and the medium was refreshed every day for 7 days. D-Bmp2 and Bmp2 retention on bone slices and ALP staining of C2C12 cells were analyzed on day 7. g. Representative fluorescence imaging of bone slices incubated with AF647-conjugated anti-Flag antibodies (above) (yellow arrows: bone slice) and C2C12 ALP staining images (below), scale bar: 200 μm. h. AF647-conjugated anti-Flag antibody fluorescence intensity quantification in bone slices (n = 3 per group). i. Quantification of ALP activity in C2C12 cells (n = 3 per group). j. qPCR analysis of Bmp2 signaling-related mRNA in C2C12 cells (n = 3 per group). The data are presented as the means ± SDs. One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After blocking with 3% BSA, membranes were incubated with the following primary antibodies: anti-Flag (1:5000, Sigma‒Aldrich, Germany) to detect Bmp2 and D-Bmp2, anti-phospho-Smad1/5/9 (1:1000, Cell Signaling Technology, USA) to assess pathway activation, anti-Runx2 (1:1000, Beyotime, China), and anti-Sp7 (1:1000, MCE, China) to examine osteogenic marker expression.

Techniques: In Vitro, Biomarker Discovery, Inhibition, Cell Culture, Staining, Activity Assay, Incubation, Fluorescence, Imaging

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: In vivo testing of release kinetics and bone accumulation of D-Bmp2@M. a. Representative fluorescence images showing the changes in Cy7 fluorescence after local injection. b. Quantitative analysis of the changes in relative fluorescence intensity (n = 6 per group). c. Representative ex vivo fluorescence images of bone tissues at 1 day post-injection of free Cy7-D-Bmp2 or Cy7-Bmp2, along with quantitative analysis of the bone fluorescence intensity (n = 6 per group). d. Representative IFHC images at 1 day post-injection showing Bmp2 or D-Bmp2 localization and the bone to muscle fluorescence intensity ratio (n = 6 per group). IFHC: anti-Flag antibody (yellow), DAPI (blue); the white dotted line represents the boundary between bones and muscles (M: muscle, B: bone); scale bar: 20 μm. e. Representative ex vivo fluorescence images of bone tissues at 6 days post-injection of PLGA microspheres loaded with Cy7-D-Bmp2 or Cy7-Bmp2, along with quantitative analysis of the bone fluorescence intensity (n = 6 per group). f. Representative IFHC images at 6 days post-injection showing Bmp2 or D-Bmp2 localization and the bone-to-muscle fluorescence intensity ratio (the fluorescence intensity of the 10-μm bone boundary to muscle tissue) (n = 6 per group). IFHC: anti-Flag antibody (yellow), DAPI (blue); the white arrows highlight PLGA microspheres; the white dotted line represents the boundary between bones and muscles (M: muscle, B: bone); scale bar: 20 μm. The data are presented as the means ± standard deviations (SDs). Unpaired Student's t -test was used for two-group comparisons. Significance levels: ∗∗∗∗ p < 0.0001.

Article Snippet: After blocking with 3% BSA, membranes were incubated with the following primary antibodies: anti-Flag (1:5000, Sigma‒Aldrich, Germany) to detect Bmp2 and D-Bmp2, anti-phospho-Smad1/5/9 (1:1000, Cell Signaling Technology, USA) to assess pathway activation, anti-Runx2 (1:1000, Beyotime, China), and anti-Sp7 (1:1000, MCE, China) to examine osteogenic marker expression.

Techniques: In Vivo, Fluorescence, Injection, Ex Vivo, Muscles

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: D-Bmp2@M accelerates fracture healing in mice and reduces ectopic osteogenesis. a. Schematic of the fracture treatment procedure: C57BL/6 mice underwent transverse femoral fracture induction followed by 28-day treatment. b. Representative X-ray images of the fracture healing process at different time points: white arrows (fracture location), red arrows (early callus), blue dotted lines (femur boundary), yellow dotted lines (callus boundary), and yellow arrows (ectopic ossification). Scale bar: 5 mm. c. Micro-CT 3D reconstruction of femurs on day 28 post-treatment; yellow arrows highlight heterotopic ossification; COR (coronal), SAG (sagittal), and TRA (transverse) Scale bar: 1 mm. d-g. Micro-CT quantitative analysis: (d) BMD, (e) BV, (f) TV, and (g) the BV/TV ratio of the fracture callus (n = 6 per group). The data are presented as the means ± SDs. One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: After blocking with 3% BSA, membranes were incubated with the following primary antibodies: anti-Flag (1:5000, Sigma‒Aldrich, Germany) to detect Bmp2 and D-Bmp2, anti-phospho-Smad1/5/9 (1:1000, Cell Signaling Technology, USA) to assess pathway activation, anti-Runx2 (1:1000, Beyotime, China), and anti-Sp7 (1:1000, MCE, China) to examine osteogenic marker expression.

Techniques: Micro-CT

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: D-Bmp2@M accelerates fracture healing in osteoporotic mice. a. Schematic of the osteoporotic fracture treatment procedure: C57BL/6 mice underwent bilateral ovariectomy (OVX) to establish an osteoporosis model, followed by transverse femoral fracture induction and 28 days of treatment. b. Representative X-ray images of the fracture healing process at different time points and Micro-CT 3D reconstruction of femurs on day 28 post-treatment: white arrows (fracture location), red arrows (early callus), blue dotted lines (femur boundary), and yellow dashed lines (callus boundary). Scale bar of x-ray: 5 mm; Scale bar of 3D reconstruction: 1 mm. c. Quantitative analysis of the fracture callus BMD and BV/TV (normal PBS Ctrl group data from PBS group in d–g) (n = 6 per group). d. Representative H&E staining images and representative Masson's trichrome staining images of fracture calluses at 28 days. Scale bar: 50 μm. e. Quantification of the callus area/total bone area ratio and quantification of the new bone area/total bone area ratio (n = 6 per group). f, g. Representative IFHC images of the callus region at 28 days and quantification of the relative fluorescence intensities: (f) The white dotted line represents the boundary between the callus and muscles (M: muscle, C: callus); Runx2 (red), ALP (green), and DAPI (blue). Scale bar: 50 μm; (g) Quantification of the relative fluorescence intensities of Runx2 and ALP (n = 6 per group). h, i. Representative IFHC images of the callus region at 28 days and quantification of the relative fluorescence intensities: (h) The white dotted line represents the boundary between the callus and muscles (M: muscle, C: callus); Sp7 (red), ALP (green), and DAPI (blue). Scale bar: 50 μm; (i) Quantification of the relative fluorescence intensities of Sp7 and ALP (n = 6 per group). The data are presented as the means ± standard deviations (SDs). Unpaired Student's t -test was used for two-group comparisons. One-way ANOVA was used for multiple comparisons. Significance levels: ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

Article Snippet: After blocking with 3% BSA, membranes were incubated with the following primary antibodies: anti-Flag (1:5000, Sigma‒Aldrich, Germany) to detect Bmp2 and D-Bmp2, anti-phospho-Smad1/5/9 (1:1000, Cell Signaling Technology, USA) to assess pathway activation, anti-Runx2 (1:1000, Beyotime, China), and anti-Sp7 (1:1000, MCE, China) to examine osteogenic marker expression.

Techniques: Micro-CT, Staining, Fluorescence, Muscles