tgf β2 neutralizing antibody  (R&D Systems)

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Other drugs and compounds used in this study included: GW9662 (MCE, HY-16578; intraperitoneal injection, 1 mg/kg body weight/day, administered continuously for 4 weeks), T0070907 (Selleck, S2871; intraperitoneal injection, 2 mg/kg body weight/day, administered continuously for 4 weeks), rapamycin (MCE, HY-10219; subcutaneous injection, 3 mg/kg body weight/day, administered continuously for 4 weeks), Rosiglitazone (MCE, HY-17386; oral gavage, 3 mg/kg body weight/day, administered continuously for 2 weeks), LY294002 (Selleck, S1105; intraosseous injection, 10 μM, 5 μL per dose per week, administered for 1 or 4 weeks), DMH1 (Selleck, S7146; intraperitoneal injection, 5 mg/kg body weight/day, administered continuously for 4 weeks), Noggin (PeproTech, 250-38; intraosseous injection, 50 ng per dose, twice per week, administered for 2 or 4 weeks), LDN-193189 (Selleck, S2618; intraperitoneal injection, 3 mg/kg body weight/day, administered for 1 or 4 weeks), IGF-1 (PeproTech, 250-19; intraosseous injection, 4 μg per dose per week, administered for 2 weeks), IGF-1 neutralizing antibody (R&D Systems, AF-791; intraosseous injection, 2 μg per dose, twice per week, administered for 2 or 4 weeks), VEGF neutralizing antibody (R&D Systems, AF-493-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-AA neutralizing antibody (R&D Systems, AF-221-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-BB neutralizing antibody (R&D Systems, AF-220-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β1 neutralizing antibody (R&D Systems, MAB2401; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β2 neutralizing antibody (R&D Systems, AB-112-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Other drugs and compounds used in this study included: GW9662 (MCE, HY-16578; intraperitoneal injection, 1 mg/kg body weight/day, administered continuously for 4 weeks), T0070907 (Selleck, S2871; intraperitoneal injection, 2 mg/kg body weight/day, administered continuously for 4 weeks), rapamycin (MCE, HY-10219; subcutaneous injection, 3 mg/kg body weight/day, administered continuously for 4 weeks), Rosiglitazone (MCE, HY-17386; oral gavage, 3 mg/kg body weight/day, administered continuously for 2 weeks), LY294002 (Selleck, S1105; intraosseous injection, 10 μM, 5 μL per dose per week, administered for 1 or 4 weeks), DMH1 (Selleck, S7146; intraperitoneal injection, 5 mg/kg body weight/day, administered continuously for 4 weeks), Noggin (PeproTech, 250-38; intraosseous injection, 50 ng per dose, twice per week, administered for 2 or 4 weeks), LDN-193189 (Selleck, S2618; intraperitoneal injection, 3 mg/kg body weight/day, administered for 1 or 4 weeks), IGF-1 (PeproTech, 250-19; intraosseous injection, 4 μg per dose per week, administered for 2 weeks), IGF-1 neutralizing antibody (R&D Systems, AF-791; intraosseous injection, 2 μg per dose, twice per week, administered for 2 or 4 weeks), VEGF neutralizing antibody (R&D Systems, AF-493-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-AA neutralizing antibody (R&D Systems, AF-221-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-BB neutralizing antibody (R&D Systems, AF-220-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β1 neutralizing antibody (R&D Systems, MAB2401; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β2 neutralizing antibody (R&D Systems, AB-112-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker