human osteocalcin duoset elisa kit  (R&D Systems)

Journal: Bioactive Materials

Article Title: Dual-function thermoresponsive antibiotic-loaded hydrogel with antimicrobial and osteogenic properties for implant-related infection control

doi: 10.1016/j.bioactmat.2026.02.044

Figure Lengend Snippet: In vivo immunohistochemical analysis (n = 6). (A) Representative immunolabeling images of the Control, PNVCL, and PNVCL/TC 25 mg/mL groups showing osteopontin (OPN) and osteocalcin (OCN) expression (immunopositive cells indicated by black arrows). (B) Mean scores (0–3) ± standard deviation for OPN immunostaining. (C) Mean scores (0–3) ± standard deviation for OCN immunostaining. Bars indicate statistically significant differences between groups (p < 0.05; Tukey HSD test).

Article Snippet: Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide for 1 h, followed by blocking of nonspecific binding sites with 1% bovine serum albumin for 12 h. The sections were then incubated with goat anti-osteopontin and goat anti-osteocalcin primary antibodies (sc-21742 and sc-30044, respectively; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: In Vivo, Immunohistochemical staining, Immunolabeling, Control, Expressing, Standard Deviation, Immunostaining

Journal: Bioactive Materials

Article Title: MSC-mimicking nanovesicle embedded bio-adhesive hydrogel for dual immunomodulation and osteogenesis to promote maxillofacial bone regeneration

doi: 10.1016/j.bioactmat.2026.02.032

Figure Lengend Snippet: In vivo evaluation of bone regeneration after hydrogel implantation in the mandibular bone defect mouse model. a) Schematic illustration of the mandibular bone defect mouse model. b) Micro-CT 3D reconstruction images of the mandibular bone samples at 4 and 8 weeks post-surgery. Scale bar = 1 mm. c) Semi-quantitative analysis of BV/TV, bone surface, Tb.N and Tb.sp (n = 6) in mouse mandibular bone defects implanted with different hydrogels at 8 weeks post-surgery. d) H&E staining and Masson trichrome staining of tissue sections of mandibular defects at 8 weeks post-surgery. Scale bar = 100 μm. e, f) Immunofluorescent staining images and corresponding semi-quantitative analysis of the expression levels of RUNX2 and OCN in mandibular bone defect areas at 4 and 8 weeks post-surgery (n = 3). Scale bar = 50 μm. P-values are calculated using one-way ANOVA with Tukey's test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant ( a was created with bioRender. com).

Article Snippet: Briefly, tissue sections underwent antigen retrieval (37 °C, 30 min), blocked by 5% bovine serum albumin (BSA, RT, 1 h), then sequentially incubated (4 °C) with lineage-specific probes: macrophage marker F4/80 (1:200, Cat. sc-52664, Santa Cruz Biotechnology) and CD68 (1:250, Cat. 14-0681-81, ThermoFisher, USA), CD206 (1:500, Cat. 24595T, Cell Signaling Technology, USA) and Arg-1 (1:250, Cat. 82975, Proteintech, China) for a M2 marker, iNOS (1:500, Cat. ab178945, Abcam, UK) and CD86 (1:300, Cat. DF6332, Affinity, China) for a M1 marker, RUNX2 (1:150, Cat. sc390351, Santa Cruz Biotechnology, USA) and OCN (1:150, Cat. sc390877, Santa Cruz Biotechnology, USA) for osteogenesis markers, VEGF (1:50, Cat. sc57496, Santa Cruz Biotechnology, USA) and CD31 (1:50, Cat. sc20071, Santa Cruz Biotechnology, USA) for angiogenesis markers, and CD146 (1:200, Cat. Ab75769, Abcam, UK) for stem cell surface marker, overnight at 4 °C.

Techniques: In Vivo, Micro-CT, Staining, Expressing

Journal: Bioactive Materials

Article Title: MSC-mimicking nanovesicle embedded bio-adhesive hydrogel for dual immunomodulation and osteogenesis to promote maxillofacial bone regeneration

doi: 10.1016/j.bioactmat.2026.02.032

Figure Lengend Snippet: The PEG-pp@nMSC@MT hydrogel effectively promotes BMMSCs' osteogenesis in vitro . a) Schematic illustration of co-culture BMMSCs with hydrogels. b) ALP staining of BMMSCs co-cultured with different scaffolds after 7 days. Scale bar = 500 μm. c) Semi-quantitative analysis of ALP staining (n = 3). d) ARS staining of BMMSC co-cultured with different scaffolds after 21 days. Scale bar = 500 μm. e) Semi-quantitative analysis of ARS staining (n = 3). f) mRNA expression of osteogenic genes (BMP2, OCN, and RUNX2) of BMMSCs treated with different hydrogels in the MMP condition after 5 and 10 days (n = 3). g) Western blot analysis of osteogenic protein (BMP2 and RUNX2) of BMMSCs treated with different hydrogels in the MMP condition after 7 and 14 days. P-values are calculated using one-way ANOVA with Tukey's test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant ( a was created with bioRender. com).

Article Snippet: Briefly, tissue sections underwent antigen retrieval (37 °C, 30 min), blocked by 5% bovine serum albumin (BSA, RT, 1 h), then sequentially incubated (4 °C) with lineage-specific probes: macrophage marker F4/80 (1:200, Cat. sc-52664, Santa Cruz Biotechnology) and CD68 (1:250, Cat. 14-0681-81, ThermoFisher, USA), CD206 (1:500, Cat. 24595T, Cell Signaling Technology, USA) and Arg-1 (1:250, Cat. 82975, Proteintech, China) for a M2 marker, iNOS (1:500, Cat. ab178945, Abcam, UK) and CD86 (1:300, Cat. DF6332, Affinity, China) for a M1 marker, RUNX2 (1:150, Cat. sc390351, Santa Cruz Biotechnology, USA) and OCN (1:150, Cat. sc390877, Santa Cruz Biotechnology, USA) for osteogenesis markers, VEGF (1:50, Cat. sc57496, Santa Cruz Biotechnology, USA) and CD31 (1:50, Cat. sc20071, Santa Cruz Biotechnology, USA) for angiogenesis markers, and CD146 (1:200, Cat. Ab75769, Abcam, UK) for stem cell surface marker, overnight at 4 °C.

Techniques: In Vitro, Co-Culture Assay, Staining, Cell Culture, Expressing, Western Blot

Journal: Bioactive Materials

Article Title: A continuous adhesion-enhanced osteogenic pathway in artificial scaffold drives cellular infiltration and condensed mineralization for rapid bone regeneration

doi: 10.1016/j.bioactmat.2026.02.026

Figure Lengend Snippet: In vitro study of osteogenic capacity and mechanisms of the CPH/rGO-3/0.6 scaffold (a) Fluorescent staining of hMSCs grown on the surface of Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for 7, 14 and 21 days and intensity statistics of osteocalcin (OCN) on 21 days (Cell nuclei of hMSCs were visualized using DAPI (blue); Cytoskeleton was stained with Phalloidin-FITC (green); OCN proteins were stained with Alexa Fluor 594 (red)) (n = 16, 12, 15 for Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 groups respectively. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (b) Fluorescent staining of MSCs grown on the surface of CPH/rGO-3/0.6 scaffold for 28 days. (c) Osteogenesis related genes expression of MSCs including alkaline phosphatase ( ALP ), type I collagen (COL-I), runt-related transcription factor 2 ( Runx2 ), SP7 transcription factor ( SP7 ), Bone sialoprotein ( BSP ), dentin matrix acidic phosphoprotein 1( DMP1 ), OCN and osteopontin ( OPN ) after 7, 14 and 21 days' incubation on CPH/rGO-3/0, CPH/rGO-3/0.6 scaffolds and Blank (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (d) OD value obtained from the ALP reagent of sample Blank, CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds after osteogenic induction of hMSC for 4, 8 and 12 days (n = 3 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001). (e) Volcano map and (f) GO enrichment analysis of differentially expressed genes in hMSCs cultured on rGO/CS vs rGO and on CPH/rGO-3/0.6 vs CPH/rGO-3/0. (g) Hotmap of differentially expressed genes between rGO/CS and rGO samples, CPH/rGO-3/0.6 and CPH/rGO-3/0 scaffolds. (h) Western blot images of KCNN3 , Integrin β1 , ANK3 , FAK , MAPK , OCN , and BSP following 14 days of osteogenic induction co-culture of hMSCs with rGO, rGO/CS, Blank. (i) Schematic diagram of osteogenic gene pathways mediated by CPH/rGO-3/0.6.

Article Snippet: The staining of OCN was performed with polyclonal antibody to osteocalcin (Proteintech, USA) and goat anti-rabbit IgG H&L (Abcam, USA) after the blocking of bovine serum albumin (BSA, BioFroxx, Germany).

Techniques: In Vitro, Staining, Expressing, Incubation, Cell Culture, Western Blot, Co-Culture 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: 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: Serum concentrations of the osteogenic marker osteocalcin (NOVUS, NBP2-68151) were also measured.

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: Serum concentrations of the osteogenic marker osteocalcin (NOVUS, NBP2-68151) were also measured.

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

Journal: Bioactive Materials

Article Title: Ribbon-shaped microgels as bioinks for 3D bioprinting of anisotropic tissue structures

doi: 10.1016/j.bioactmat.2025.12.040

Figure Lengend Snippet: μRB bioinks modulate MSC morphology, osteogenesis and bone formation in a stiffness-dependent manner. (A) Live/dead staining of MSCs after extrusion (Day 0) or after 28 days of culture in osteogenic medium. Green: live cells, Red: dead cells. (B) Metabolic activity of MSCs measured by PrestoBlue assay at day 0 and day 14 after bioprinting (n = 6 per group). (C) DNA content per scaffold measured by PicoGreen assay at day 28 (n = 3 per group). (D) Alizarin red S (ARS) staining for mineralized bone matrix, (E) Aniline Blue staining for total collagen, and (F) immunostaining of Osteocalcin (OCN), a mature bone marker, at day 14 and day 21 of osteogenesis (n = 4 per group). (G–I) Quantification of ARS and Aniline Blue percent positive area and OCN mean fluorescence intensity (MFI). Scale bar = 100 μm. Values are presented as mean ± S.D. and p-values were determined by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.005, ∗∗∗∗p ≤ 0.001.

Article Snippet: Primary antibody for osteocalcin (1:200, 23418-1-AP, Proteintech), GFP (1:100, 50430-2-AP, Proteintech) was diluted in 1 % BSA with 0.1 % Triton X-100 and incubated overnight at 4 °C.

Techniques: Staining, Activity Assay, Prestoblue Assay, Picogreen Assay, Immunostaining, Marker, Fluorescence

Journal: Bioactive Materials

Article Title: Bacteria-responsive DNAgel system for targeted delivery of photothermally enhanced MXene/MoS 2 in the treatment of pyogenic osteomyelitis

doi: 10.1016/j.bioactmat.2025.10.023

Figure Lengend Snippet: MXMoS 2 DNAgel effectively accelerates bone regeneration and suppresses infection in pyogenic osteomyelitis. (A) Schematic diagram of the treatment procedure of suppurative osteomyelitis mice. (B) Bacterial colonies in mice wounds before and after NIR treatment. (C) Quantification of bacterial colony counts. (D) 3D micro-CT reconstruction of maxilla at 4 weeks after different treatment. Representative images of H&E and Masson's trichrome staining of the maxilla at 4 weeks treatment. Immunofluorescence staining for IL-6 (red), TRAP (red) and OCN (red) in maxilla. Nuclei were counterstained with DAPI (blue). (E) BMD and (F) BV/TV analyses of newly formed bone calculated based on micro-CT. (G – I) Quantitative analysis of the ROI. Data are presented as mean ± SD (n = 5 mice per group). ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: Primary antibodies for immunocytochemistry and immunofluorescence staining—including ARG1 and iNOS, TRAP, β-Catenin, OCN and WNT4—were obtained from Proteintech (China).

Techniques: Infection, Micro-CT, Staining, Immunofluorescence

Journal: Materials Today Bio

Article Title: Immunomodulatory and dentinogenic potential of surface-engineered hesperetin-functionalized composite scaffolds for pulp-dentin regeneration

doi: 10.1016/j.mtbio.2026.102930

Figure Lengend Snippet: Immunofluorescent assessment of macrophage polarization and osteogenic markers after subcutaneous implantation. (a) iNOS (M1) and CD163 (M2) around PCL/nHA and H-PCL/nHA + HT at 1- and 4-weeks post-implantation. (b) Osteopontin (OPN) and osteocalcin (OCN) at 8 weeks post-implantation. All panels were labeled with an Alexa Fluor 488-conjugated secondary antibody (green) and imaged at 200 × ; scale bar, 50 μm. (c) Areas of positive labeling were quantified in ImageJ and reported as mean ± SD. Two-way ANOVA with Tukey's post hoc test; n = 8; α = 0.05.

Article Snippet: For immunofluorescence, sections were incubated overnight at 4 °C with primary antibodies diluted 1:100: anti-iNOS (Abcam, ab283655) together with anti-CD163 (Abcam, ab182422) to examine scaffold-driven macrophage polarization at 1 and 4 weeks, and anti-osteocalcin (Proteintech, #23418-1-AP) and anti-osteopontin (Abcam, ab216402) to evaluate mineralization potential at 8 weeks.

Techniques: Labeling