Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Curvature-controlled orientation of cytoskeletal stress fibers on concave-cylindrical surfaces. (a) Representative confocal microscopy images depicting F-actin (magenta) and nuclei (blue) of fibroblasts, mesenchymal stromal cells, osteoblasts, pre-osteoblasts and endothelial cells seeded on flat surfaces. (b) Brass mold used to fabricate the master GeoChip from which GeoChips for use in cell culture are manufactured via sugar candy molding . Photographs show the topographic surface of the brass mold and the candy mold (Scale bar: 2 mm). Scanning electron microscopy (SEM) verified the smoothness of the resulting curved surface (half-cylinder with Ø = 1000 μm, scale bar: 200 μm). (c) Representative confocal microscopy images of cells seeded on concave-cylindrical surfaces with Ø = 100 and 1000 μm. Yellow dashed lines indicate the half-cylinder boundaries. (d-i) Distribution of stress fiber orientation quantified from the F-actin signal of cells on substrates with increasing curvature (average with standard deviation). Cartesian plots include data for fibroblasts (blue), mesenchymal stromal cells (green), osteoblasts (purple), pre-osteoblasts (orange) and endothelial cells (red). The direction 0° - 180° represents the orientation along the cylindrical surface (minimum curvature) and the direction 90° represents the orientation perpendicular to the cylindrical surface (maximum curvature). The substrate curvature experienced in dependency of the orientation is indicated by the red dashed line and red scale. Random orientation is indicated by the black dashed line. Statistical significance via Mann-Whitney test (two sided) with Bonferroni correction, ∗p < 0.05. N ≥ 3 GeoChips/cell type for a total of N ≥ 12 half-cylinders/cell type, 1 donor/cell type. Scale bars 100 μm (unless otherwise stated).

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: Confocal Microscopy, Cell Culture, Electron Microscopy, Standard Deviation, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Incidence of cell spanning on concave-cylindrical surfaces. (a) Lateral view of fibroblasts exposed to cylinders with increasing diameter (decreasing curvature), with spanning cells marked by yellow arrows. (b) Probability of spanning cells in relation to the half-cylinder diameter. (c-e, top to bottom) Representative 3D reconstructed images of fibroblasts, pre-osteoblasts and endothelial cells on concave-cylindrical surfaces with Ø = 100, 200 and 300 μm. Cells were reconstructed in Imaris using the F-actin (magenta, cell surface reconstruction) and nuclei (blue) signal as obtained by confocal microscopy. Half-cylinder contour is indicated by the yellow dashed line. Spanning cells are indicated by yellow arrows in subfigures c-e for clarity. Polar plots on the right depict the percentage of spanning cells and the corresponding angle of cell orientation for fibroblasts (blue), pre-osteoblasts (orange) and endothelial cells (red). The direction 0° - 180° represents the orientation along the cylindrical surface (minimum curvature) and the direction −90° - 90° represents the orientation perpendicular to the cylindrical surface (maximum curvature). (f) Confocal microscopy images of representative cell morphologies for fibroblasts, pre-osteoblasts and endothelial cells depicting F-actin (magenta), nuclei (blue) and focal adhesions via vinculin staining (green). Focal adhesions are indicated by green arrows (example shown on fibroblasts). (g) Cell length quantified as the major axis of an ellipse fitted around the cell. (h) Cell roundness with a value of 1 representing a perfect circle and value of 0 representing a straight line. (i) FSD calculated as the distance between FA clusters (see methods part for detailed description). (j) FA size distribution per cell plotted as the percentage of FAs that fall into the indicated size classes. (k) Representative force vector maps and (l) total cell force quantified via TFM. Statistical significance via Mann-Whitney test (two sided) with Bonferroni correction, ∗p < 0.05. N ≥ 3 GeoChips/cell type for a total of N ≥ 12 half-cylinders/cell type. N ≥ 60 cells/cell type for FA and morphological analysis. 1 donor/cell type. Scale bar 50 μm.

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: Confocal Microscopy, Staining, Plasmid Preparation, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Cell spanning initiates channel closure and subsequent tissue remodeling. (a) Fabrication of full-cylindrical channels with Ø = 250 μm in PDMS substrates by direct molding from a micro-machined brass mold. (b) Degree of channel closure representing the distribution of cells within the channels at the selected points in time during live confocal imaging. A value of 0 indicates that cells are exclusively found at the wall of the channel and a value of 1 indicates cells have completely closed the channel and are homogeneously distributed. (c) Relative degree of alignment of the cell-network within the channels quantified as the maximum value of the orientation distribution for the individual cell types and time points normalized to the highest detected value of all conditions (see also Supplementary Data S2). Higher values indicate a higher degree of alignment along the channel axis. (d-f) Lateral and front view of the PDMS cylindrical channels obtained by live confocal imaging of fibroblasts (blue), pre-osteoblasts (orange) and endothelial cells (red) using CellTracker™ Green ( t = 4, 12, 24 and 48 h after seeding). Open arrows indicate cells spanning perpendicular to the channel axis. Full arrows indicate cells oriented along the direction of the channel axis after channel closure. Channel contour is highlighted by the yellow dashed lines. The surface of the forming tissue is marked by red dashed lines. White dashed lines indicate the z-volume that is shown in the corresponding lateral views. Statistical significance via Mann-Whitney test with Bonferroni correction, ∗p < 0.05. N = 3 cylindrical channels/cell type. 1 donor/cell type. Scale bars 100 μm.

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: Imaging, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Channel closure mechanism can be controlled by substrate curvature using scaffolds with well-defined geometries. (a, left) Schematic representation of the in vitro culture setup with collagen scaffold presenting channels of controlled diameter with Ø ≈ 600 μm, Ø ≈ 350 μm and Ø ≈ 150 μm. Monolayer seeding on one side of the biomaterial facilitates migration of cells from one end of the biomaterial. (a, right) SEM image of the microarchitecture (Scale bar 20 μm) and channels within the biomaterial (Scale bars 100 μm). SEM images correspond to the outermost surface of the scaffold. (b) Comparison of template diameter against resulting channel diameter after cross-linking and sterilization of the biomaterial. (c) Representative images of fibroblasts, pre-osteoblasts and endothelial cells within channels of distinct diameters 7 days after seeding. Cell cytoskeleton (F-actin) is depicted in magenta and nuclei in blue. Yellow arrows indicate the direction (arrow angle) and degree of alignment (vector length) for the corresponding region. Scale bar close-up images: 25 μm. (d, left) Degree of channel closure for the investigated channel diameters and cell types. (d, right) Relative degree of tissue alignment for the different channel diameters and cell types. Tissue alignment ranges from 0 (fully isotropic) to 1 (fully anisotropic, dashed line). Tissue across the channel and relative degree of is calculated in the central 50 % of each channel. Data displayed as average with standard deviation. N = 4 scaffolds/cell type. 1 donor/cell type. Scale bars 200 μm (unless otherwise stated).

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: In Vitro, Migration, Comparison, Plasmid Preparation, Standard Deviation

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: ADGRG1 have the capacity to serve as potential target for IVDD treatment. (A) Primary human NPCs were treated with TBHP at concentrations of 50 μM, 100 μM, 200 μM for 12 h, and the cellular ferrous ion levels were assessed by confocal microscopy using FerroOrange probe. FerroOrange fluorescence intensity was quantified, n = 3. Scale bar, 50 μm. (B) GSEA enrichment plots of gene data sets associated with oxidative damage response, ferroptosis, permeabilize mitochondria and mitochondrial respiratory chain complex assembly in TBHP‐treated primary NPCs, compared to the control. (C) Primary NPCs were treated with TBHP for 12 h and cell lysates were subjected to immunoblotting with indicated ADGRG1, IVDD and ferroptosis-related antibodies. (D) Volcano plot of differentially expressed genes in NPCs between the TBHP‐treated group and the control. |log2FC| > 2, FDR < 0.05. (E-F) Primary NPCs were incubated with TBHP for 12 h, followed by immunofluorescent staining with anti-ADGRG1 (green) and anti-4-HNE (red) antibodies and examination by confocal microscopy. The fluorescence intensity was quantified. n = 3. Scale bar, 50 μm. (G) Magnetic resonance imaging (MRI) showed the IVDD Pfirrmann grade of needle puncture IVDD rat models with 25G (MI-IVDD), 21G(MOD-IVDD) and18G (SE-IVDD), and statistical analysis was performed, n = 3. (H) Immunoblotting analysis showed the expression of IVDD markers (COL1A1) and ADGRG1 in IVDD rat models with different degrees of needle-puncture degeneration, n = 3. (I) Feature plots depict the average expression of ADGRG1 (color-scaled) across each cell cluster. (J) Immunoblotting analysis showed the expression of IVDD markers (COL1A1) and ADGRG1 in the NP tissue of clinical MI/SE degenerative IVDs. The relative ADGRG1 grayscale was quantified in (L), n = 10. (K) Representative images for colocalization analysis of ADGRG1 (green), 4-HNE (red) fluorescence in MI and SE NP tissues. n = 5. Scale bar, 50 μm. (L) Relative ADGRG1 grayscale in (J) and fluorescence intensity in (K) were quantified. All data are expressed as the mean ± SD. For panels A) and F–H), data were analyzed using one-way ANOVA with Tukey's multiple comparisons, while panel L) was assessed using a two-tailed unpaired Student's t-test. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001.

Article Snippet: After permeabilization and blocking with 10% goat serum containing 0.2% Triton X-100, the sections were incubated with primary antibodies against ADGRG1 (1:50; sc-390192, Santa Cruz Biotechnology), TOM20(11802-1-AP, Proteintech), 4-HNE(68538-1-Ig, Proteintech), 4-HNE(HY-P81208, MCE), Ferritin (Rockland 200-401-090-0100), LAMP1(65051-1-Ig, Proteintech), PAX1(sc-514352, Santa Cruz Biotechnology), FOXF1(PA5-83039, Thermo Fisher).

Techniques: Confocal Microscopy, Fluorescence, Control, Western Blot, Incubation, Staining, Magnetic Resonance Imaging, Expressing, Two Tailed Test

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: In vitro evaluation of the cargo transfer capacity and therapeutic potential of A1TP-HX-EVs. (A) Immunoblotting analysis confirmed the overexpression of GFP-tagged ADGEG1 protein in NPCs by retrovirus. (B) The live-cell workstation demonstrated the uptake of AIE-labeled A1TP-HX-EVs by NPCs in both the control group (GFP) and the ADGRG1 overexpression group (ADGRG1-GFP) within 24 h. (C) Representative images from the live-cell workstation showed the uptake of DPA-labeled HX-EVs at different concentrations by TBHP-treated NPCs within 24 h. Scale bar, 50 μm. (D) Representative images from the live-cell workstation showed the uptake of AIE-labeled A1TP-HX-EVs (DPA 10 μM) by primary NPCs within 24 h after treatment with different concentrations of TBHP for 12h. Scale bar, 50 μm. (E) AIE fluorescence intensity in (C-D) were quantified. n = 3. ∗ P < 0.05. ∗∗ P < 0.01. ns, not significant. (F) Primary NPCs were stained with FerroOrange probe and assessed by confocal microscopy. n = 3. Scale bar, 50 μm. (G) GSEA enrichment analysis of ferroptosis and oxidative damage response-related gene sets in A1TP-HX-EVs‐treated group and TBHP group NPCs. (H) Primary NPCs were induced with 100 μM TBHP for 12 h, and then treated with EVs, HX-EVs or A1TP-HX-EVs for 24 h. Cell lysates were immunoblotted with antibodies against IVDD markers and ferroptosis-related proteins and ADGRG1. (I) GSEA enrichment analysis of the mitochondrial respiratory chain complex assembly and transcriptional activation of mitochondrial biogenesis-related gene sets in A1TP-HX-EVs‐treated group and TBHP group NPCs. (J-K) Representative oxygen consumption traces of primary NPCs induced with TBHP and then treated as indicated for 24 h. Maximal respiration of NPCs were quantified. n = 3. (L) Primary NPCs were induced with TBHP, and then treated as indicated for 24 h, followed by immunofluorescent staining with anti-TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. All data are expressed as the mean ± SD. For B) and E), two‐way ANOVA with Tukey 's multiple comparison tests were used for statistical analysis. For panels F) and K-L), one‐way ANOVA with Tukey's multiple comparison tests were used for statistical analysis. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001. ns, not significant.

Article Snippet: After permeabilization and blocking with 10% goat serum containing 0.2% Triton X-100, the sections were incubated with primary antibodies against ADGRG1 (1:50; sc-390192, Santa Cruz Biotechnology), TOM20(11802-1-AP, Proteintech), 4-HNE(68538-1-Ig, Proteintech), 4-HNE(HY-P81208, MCE), Ferritin (Rockland 200-401-090-0100), LAMP1(65051-1-Ig, Proteintech), PAX1(sc-514352, Santa Cruz Biotechnology), FOXF1(PA5-83039, Thermo Fisher).

Techniques: In Vitro, Western Blot, Over Expression, Labeling, Control, Fluorescence, Staining, Confocal Microscopy, Activation Assay, Comparison

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Taurine is the key small molecule in A1TP-HX-EVs that activated the AMPK/NRF2 pathway to regulate nucleus pulposus cell repair. (A) The LC-MS/MS analysis was used to detect the differential active small molecule components between placental HX-EVs and EVs. (B) The SMPDB enrichment analysis identified pathways related to small molecules that are up-expressed in HX-EVs compared to EVs. The metabolic pathways marked in red are related to ferroptosis inhibition and mitochondrial function. (C) Volcano plot of small molecule in HX-EVs versus EVs. |log2FC| > 0.5, FDR <0.05. (D) The content of taurine in placental MSC (pMSC), hypoxia-induced pMSC(HX-pMSC) and their derived EVs was detected by ELISA. n = 3. (E) Primary NPCs cells were induced with TBHP, and then treated with EVs, HX-EVs, and A1TP-HX-EVs for 24 h. The cell lysates were subjected to ELISA assay to detect taurine content. (F) Two shRNA lentiviruses were designed to knock down TAUT a key enzyme in taurine uptake in pMSC. (G) The content of taurine in TAUT-sh1-pMSC and TAUT-sh2-pMSC derived EVs (KD-HX-EVs) was detected by ELISA. n = 3. (H) Primary NPCs were induced with TBHP, and then treated with A1TP-HX-EVs and A1TP-KD-HX-EVs for 24 h. Cell lysates were immunoblotted with indicated antibodies. (I) Primary NPCs were induced with TBHP, and then treated with A1TP-HX-EVs and A1TP-KD-HX-EVs for 24 h, followed by immunofluorescent staining with anti- TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. (J) A CDO1-overexpressing retrovirus was designed to overexpress CDO1 in pMSCs. (K) The content of taurine in CDO1-OE-pMSC derived EVs (OE-EVs) was detected by ELISA. n = 3. (L) Primary NPCs were induced with TBHP, and then treated with treated A1TP-EVs and A1TP-OE-EVs for 24 h. Cell lysates were immunoblotted with indicated antibodies. (M) Primary NPCs were induced with TBHP, and then treated with A1TP-EVs and A1TP-OE-EVs for 24 h, followed by immunofluorescent staining with anti-TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. (N-O) Representative oxygen consumption traces of primary NPCs induced with TBHP and then treated with A1TP-HX-EVs, A1TP-KD-HX-EVs, or A1TP-OE-EVs for 24 h. Maximal respiration of NPCs were quantified. n = 3. All data are expressed as the mean ± SD. For E), I), M) and O), one‐way ANOVA with Tukey's multiple comparison tests were used for statistical analysis. For D), G) and K), two‐tailed unpaired Student's t‐tests were used for statistical analysis. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001. ns, not significant.

Article Snippet: After permeabilization and blocking with 10% goat serum containing 0.2% Triton X-100, the sections were incubated with primary antibodies against ADGRG1 (1:50; sc-390192, Santa Cruz Biotechnology), TOM20(11802-1-AP, Proteintech), 4-HNE(68538-1-Ig, Proteintech), 4-HNE(HY-P81208, MCE), Ferritin (Rockland 200-401-090-0100), LAMP1(65051-1-Ig, Proteintech), PAX1(sc-514352, Santa Cruz Biotechnology), FOXF1(PA5-83039, Thermo Fisher).

Techniques: Liquid Chromatography with Mass Spectroscopy, Inhibition, Derivative Assay, Enzyme-linked Immunosorbent Assay, shRNA, Knockdown, Staining, Comparison, Two Tailed Test