Journal: Bioactive Materials

Article Title: Geometry-driven immunomodulation in 3D-printed bioceramics: Negative curvature promotes macrophage M2 polarization via Ras-MAPK/HIF-1α signaling for vascularized osteogenesis

doi: 10.1016/j.bioactmat.2026.01.001

Figure Lengend Snippet: (A–C) Cell adhesion, relative fluorescence intensity of CCR7/CD206 and ROS for Raw264.7 on different Gaussian curvatures. (D) CCR7 & CD206 staining of Raw264.7 cells (blue-nucleus, red-CCR7, green-CD206, scale:100 μm). (E, F) F-actin & DAPI staining and statistical scatterplot of hBMSC adhesion. (G) ALP intensity of hBMSCs on different Gaussian curvatures. (H, I) Cell adhesion and CD31 fluorescence intensity of HUVECs. (J) Chord diagram for comprehensive normalized data of Raw264.7, hBMSCs and HUVECs behavior detection with different Gaussian curvatures on HCGC chips.

Article Snippet: The reagents used in the experiment included: H-DMEM(11965118, Gibco, USA.), α-DMEM medium(12571063, Gibco, USA.), TritonX-100(ST1723, Beyotime, China), 4 % paraformaldehyde (BL539A, Biosharp, China),FBS(A5256701, Gibco, USA.),ECM medium (Science Cell, USA.),and DAPI staining solution (C1006, Beyotime, China),BCIP/NBT(C3206, Beyotime, China), reactive oxygen species kit (S0033S, Beyotime, China), BSA (B2064, ≥98 %, Sigma-Aldrich, USA.),CD31 antibody (ab28364, Abcam, USA.), secondary anti-IGg (ab175773, Alexa Fluor® 680, Abcam, USA.), Phalloidin-iFluor 488(ab176753, Abcam, USA.), CCR7(AF5293, Bioss, China), CD206 (bsm-60761R, Bioss, China), iNOS (bs-22924R, Bioss, China), RIPA (P0013, Beyotime, China), p-ERK1/2 (AF3687, Affinity, USA.) and ERK1/2 (#AF0155, Affinity, USA), luminol detection reagent (sc-2048, Santa Cruz, USA.), GAPDH (Cat#KC-5G5, Kangchen Biotechnology, China), Trizol(15596026CN, Invitrogen, USA.), DEPC(R0601, Thermo Scientific, USA.), TBST(R017R.0000, Thermo Scientific, USA.), HIF-1a(GTX127309, GeneTex, USA.), β-Tubulin(10094-1-AP, Proteintech, UK) Adezmapimod (SB 203580, MCE, USA.) medium and Paclitaxel (99.88 %, HY-B0015R, MCE), ELISA Arg-1(E-EL-M3092, ELabSci@, China), TNF-α (E-EL-M3063, ELabSci@, China), OPN(22952-1-AP, Proteintech, UK.), F4/80 (GB11027-100, Servicebio, China) Alkaline phosphatase activity kit (P0321S, Beyotime, China), Matrigel (CLS356234, Corning, USA), Microfill MV120 (Flow tech, USA), EDTA(17892, Thermo Scientific, USA.) xylene(X112051, AR,99 %, Aladdin, China), ethanol (107-21-1, AR,99 %, Aladdin, China).

Techniques: Fluorescence, Staining

Journal: Bioactive Materials

Article Title: Geometry-driven immunomodulation in 3D-printed bioceramics: Negative curvature promotes macrophage M2 polarization via Ras-MAPK/HIF-1α signaling for vascularized osteogenesis

doi: 10.1016/j.bioactmat.2026.01.001

Figure Lengend Snippet: Macrophage polarization analysis of Raw264.7 on structures with different gaussian curvature: (A, B) Chord Diagram for qPCR analysis of CCR7, IL6, iNOS-inflammatory and M1 marker genes, and Arg-1, CD206, IL10-M2 related protein genes in different Gaussian curvature groups. (C) Protein content of Arg-1 in different Gaussian curvature groups at 1 and 3 days. (D) Integral plots of the five experimental groups. IL4 group is the positive control for CD206 expression and lipopolysaccharide (LPS) group is the negative control.

Article Snippet: The reagents used in the experiment included: H-DMEM(11965118, Gibco, USA.), α-DMEM medium(12571063, Gibco, USA.), TritonX-100(ST1723, Beyotime, China), 4 % paraformaldehyde (BL539A, Biosharp, China),FBS(A5256701, Gibco, USA.),ECM medium (Science Cell, USA.),and DAPI staining solution (C1006, Beyotime, China),BCIP/NBT(C3206, Beyotime, China), reactive oxygen species kit (S0033S, Beyotime, China), BSA (B2064, ≥98 %, Sigma-Aldrich, USA.),CD31 antibody (ab28364, Abcam, USA.), secondary anti-IGg (ab175773, Alexa Fluor® 680, Abcam, USA.), Phalloidin-iFluor 488(ab176753, Abcam, USA.), CCR7(AF5293, Bioss, China), CD206 (bsm-60761R, Bioss, China), iNOS (bs-22924R, Bioss, China), RIPA (P0013, Beyotime, China), p-ERK1/2 (AF3687, Affinity, USA.) and ERK1/2 (#AF0155, Affinity, USA), luminol detection reagent (sc-2048, Santa Cruz, USA.), GAPDH (Cat#KC-5G5, Kangchen Biotechnology, China), Trizol(15596026CN, Invitrogen, USA.), DEPC(R0601, Thermo Scientific, USA.), TBST(R017R.0000, Thermo Scientific, USA.), HIF-1a(GTX127309, GeneTex, USA.), β-Tubulin(10094-1-AP, Proteintech, UK) Adezmapimod (SB 203580, MCE, USA.) medium and Paclitaxel (99.88 %, HY-B0015R, MCE), ELISA Arg-1(E-EL-M3092, ELabSci@, China), TNF-α (E-EL-M3063, ELabSci@, China), OPN(22952-1-AP, Proteintech, UK.), F4/80 (GB11027-100, Servicebio, China) Alkaline phosphatase activity kit (P0321S, Beyotime, China), Matrigel (CLS356234, Corning, USA), Microfill MV120 (Flow tech, USA), EDTA(17892, Thermo Scientific, USA.) xylene(X112051, AR,99 %, Aladdin, China), ethanol (107-21-1, AR,99 %, Aladdin, China).

Techniques: Marker, Positive Control, Expressing, Negative Control

Journal: Bioactive Materials

Article Title: Geometry-driven immunomodulation in 3D-printed bioceramics: Negative curvature promotes macrophage M2 polarization via Ras-MAPK/HIF-1α signaling for vascularized osteogenesis

doi: 10.1016/j.bioactmat.2026.01.001

Figure Lengend Snippet: (A) Immunofluorescence images of β-Tubulin /CD206 and β-Tubulin /HIF-1α of macrophages in different Gaussian curvature groups before and after treatment with Adezmapimod . (B, C) Protein quantification and Western blotting image of ERK1/2, p-ERK1/2, β-tubulin, HIF-1α and CD206 proteins in all groups before and after treatment with Adezmapimod and Paclitaxel. (D) Mechanism summary of Gaussian curvature-driven macrophage polarization.

Article Snippet: The reagents used in the experiment included: H-DMEM(11965118, Gibco, USA.), α-DMEM medium(12571063, Gibco, USA.), TritonX-100(ST1723, Beyotime, China), 4 % paraformaldehyde (BL539A, Biosharp, China),FBS(A5256701, Gibco, USA.),ECM medium (Science Cell, USA.),and DAPI staining solution (C1006, Beyotime, China),BCIP/NBT(C3206, Beyotime, China), reactive oxygen species kit (S0033S, Beyotime, China), BSA (B2064, ≥98 %, Sigma-Aldrich, USA.),CD31 antibody (ab28364, Abcam, USA.), secondary anti-IGg (ab175773, Alexa Fluor® 680, Abcam, USA.), Phalloidin-iFluor 488(ab176753, Abcam, USA.), CCR7(AF5293, Bioss, China), CD206 (bsm-60761R, Bioss, China), iNOS (bs-22924R, Bioss, China), RIPA (P0013, Beyotime, China), p-ERK1/2 (AF3687, Affinity, USA.) and ERK1/2 (#AF0155, Affinity, USA), luminol detection reagent (sc-2048, Santa Cruz, USA.), GAPDH (Cat#KC-5G5, Kangchen Biotechnology, China), Trizol(15596026CN, Invitrogen, USA.), DEPC(R0601, Thermo Scientific, USA.), TBST(R017R.0000, Thermo Scientific, USA.), HIF-1a(GTX127309, GeneTex, USA.), β-Tubulin(10094-1-AP, Proteintech, UK) Adezmapimod (SB 203580, MCE, USA.) medium and Paclitaxel (99.88 %, HY-B0015R, MCE), ELISA Arg-1(E-EL-M3092, ELabSci@, China), TNF-α (E-EL-M3063, ELabSci@, China), OPN(22952-1-AP, Proteintech, UK.), F4/80 (GB11027-100, Servicebio, China) Alkaline phosphatase activity kit (P0321S, Beyotime, China), Matrigel (CLS356234, Corning, USA), Microfill MV120 (Flow tech, USA), EDTA(17892, Thermo Scientific, USA.) xylene(X112051, AR,99 %, Aladdin, China), ethanol (107-21-1, AR,99 %, Aladdin, China).

Techniques: Immunofluorescence, Western Blot

Journal: Bioactive Materials

Article Title: Geometry-driven immunomodulation in 3D-printed bioceramics: Negative curvature promotes macrophage M2 polarization via Ras-MAPK/HIF-1α signaling for vascularized osteogenesis

doi: 10.1016/j.bioactmat.2026.01.001

Figure Lengend Snippet: Immune-related analysis of macrophages in scaffolds with various Gaussian curvature after muscle bag implantation: (A, B) Immunohistochemical sections and statistical analysis of IL10 positive cells. (C, D) Immunofluorescence staining images and statistical analysis of CD68, CD206 and CCR7 positive cells. (E) The M2-phenotypical genes and inflammatory genes expression of macrophage (n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Article Snippet: The reagents used in the experiment included: H-DMEM(11965118, Gibco, USA.), α-DMEM medium(12571063, Gibco, USA.), TritonX-100(ST1723, Beyotime, China), 4 % paraformaldehyde (BL539A, Biosharp, China),FBS(A5256701, Gibco, USA.),ECM medium (Science Cell, USA.),and DAPI staining solution (C1006, Beyotime, China),BCIP/NBT(C3206, Beyotime, China), reactive oxygen species kit (S0033S, Beyotime, China), BSA (B2064, ≥98 %, Sigma-Aldrich, USA.),CD31 antibody (ab28364, Abcam, USA.), secondary anti-IGg (ab175773, Alexa Fluor® 680, Abcam, USA.), Phalloidin-iFluor 488(ab176753, Abcam, USA.), CCR7(AF5293, Bioss, China), CD206 (bsm-60761R, Bioss, China), iNOS (bs-22924R, Bioss, China), RIPA (P0013, Beyotime, China), p-ERK1/2 (AF3687, Affinity, USA.) and ERK1/2 (#AF0155, Affinity, USA), luminol detection reagent (sc-2048, Santa Cruz, USA.), GAPDH (Cat#KC-5G5, Kangchen Biotechnology, China), Trizol(15596026CN, Invitrogen, USA.), DEPC(R0601, Thermo Scientific, USA.), TBST(R017R.0000, Thermo Scientific, USA.), HIF-1a(GTX127309, GeneTex, USA.), β-Tubulin(10094-1-AP, Proteintech, UK) Adezmapimod (SB 203580, MCE, USA.) medium and Paclitaxel (99.88 %, HY-B0015R, MCE), ELISA Arg-1(E-EL-M3092, ELabSci@, China), TNF-α (E-EL-M3063, ELabSci@, China), OPN(22952-1-AP, Proteintech, UK.), F4/80 (GB11027-100, Servicebio, China) Alkaline phosphatase activity kit (P0321S, Beyotime, China), Matrigel (CLS356234, Corning, USA), Microfill MV120 (Flow tech, USA), EDTA(17892, Thermo Scientific, USA.) xylene(X112051, AR,99 %, Aladdin, China), ethanol (107-21-1, AR,99 %, Aladdin, China).

Techniques: Immunohistochemical staining, Immunofluorescence, Staining, Expressing

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: hfNCSC-sEVs are taken up by PCs in vitro and enhance their proliferation and migration. (A) Primary cultures of hfNCSCs were established from male Sprague–Dawley rats. (B) Immunofluorescence staining of the neural crest cell marker p75 (red) and the stem cell marker nestin (green) in hfNCSCs, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. (C) Western blot analysis demonstrated the presence of surface markers (cluster of differentiation [CD]9, CD81, and tumor susceptibility gene 101 protein [TSG101]) and the absence of an endoplasmic reticulum marker (calnexin) in hfNCSC-sEVs. (D) Nanoparticle tracking analysis was used to quantify the concentration and size distribution of hfNCSC-sEVs. (E) Transmission electron microscopy was used to visualize the characteristic morphology of hfNCSC-sEVs. (F) Immunofluorescence staining indicated that the third-generation PCs cultured in vitro were positive for claudin-1, zonula occludens 1 (ZO1), and glucose transporter 1 (GLUT1) but negative for S100, with DAPI staining marking the nuclei. (G) The internalization of PKH26-labeled hfNCSC-sEVs (red) by ZO1-positive PCs (green) was visualized using immunofluorescence staining, with DAPI staining to mark the nuclei. (H) The Cell Counting Kit-8 assay was used to evaluate the cell viability of PCs across concentrations of 0, 2 × 10 8 , 5 × 10 8 , and 10 × 10 8 particles/mL hfNCSC-sEVs at 3, 5, and 7 days of in vitro culture ( n = 5 per group). (I) The Transwell assay was used to quantify the number of migrating PCs at 6, 12, and 18 hours post-treatment with the aforementioned concentrations of hfNCSC-sEVs, in in vitro culture ( n = 6 per group). (J) Western blot and (K) statistical analyses revealed the relative protein expression levels of proliferating cell nuclear antigen (PCNA) and vimentin in PCs from the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for H and I; Student’s t -test for K). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; GLUT1: glucose transporter 1; hfNCSCs: hair follicle neural crest stem cells; ns: not significant; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: In Vitro, Migration, Immunofluorescence, Staining, Marker, Western Blot, Concentration Assay, Transmission Assay, Electron Microscopy, Cell Culture, Labeling, Cell Counting, Transwell Assay, Expressing, Saline, Comparison, CCK-8 Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs augments cell proliferation and migration by enhancing HAS2 expression in PCs. (A, B) Western blot (A) and statistical analyses (B) revealed the relative protein expression levels of HAS2, proliferating cell nuclear antigen (PCNA), and vimentin in PCs across the –/–, –/si- Has2 , hfNCSC-sEVs/–, and hfNCSC-sEVs/si- Has2 groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (C, D) The wound healing assay (C) and statistical analysis (D) demonstrated the migration rates of PCs in the aforementioned groups ( n = 3 per group). (E) The Cell Counting Kit-8 assay was used to assess cell viability in PCs across the same groups on day 5 of in vitro culture ( n = 5 per group). (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of HAS2, PCNA, and vimentin in PCs treated with phosphate-buffered saline (PBS), hfNCSC-sEVs, or hfNCSC-sEVs + miR-21-5p inhibitor on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (H–J) Immunofluorescence staining visualized the expression of HAS2 (red) and 5-ethynyl-2′-deoxyuridine (EdU; green) in PCs (H), and statistical analysis revealed the integrated optical density (IOD) of zonula occludens 1 (ZO1; I) and the cell proliferation rates (J) in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 of in vitro culture ( n = 3 per group). (K, L) Western blot (K) and statistical analyses (L) showed the relative protein expression levels of HAS2, PCNA, and vimentin in regenerated tissue from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 post-operation (normalized to β-tubulin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, E, G, I, J, and L). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; EdU: 5-ethynyl-2′-deoxyuridine; HAS2: hyaluronan synthase 2; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: Migration, Expressing, Western Blot, In Vitro, Wound Healing Assay, Cell Counting, Saline, Immunofluorescence, Staining, Comparison, CCK-8 Assay