Journal: Bioactive Materials

Article Title: Reprogramming macrophage mechanosensation via TRPV4 modulating mechano-immunotherapy controls fibrotic encapsulation of biomaterial implants

doi: 10.1016/j.bioactmat.2026.06.020

Figure Lengend Snippet: Characterization of RN-1734 effects on J774a macrophages and LPS-induced calcium flux. A) Schematic of RN-1734 mechanism: selective TRPV4 inhibition reduce intracellular calcium influx. B) Brightfield images showing macrophage morphology after RN-1734 treatment (80 μM); Scale bar = 50 μm. C) Dose-dependent effects of RN-1734 on macrophage attachment after 1 h (n = 4), D) Live/dead staining at 24 h (n = 3), E) proliferation at day 1 and day 3 measured by alamarBlue (n = 3). F) Schematic of live-cell calcium imaging under LPS stimulation. G) Representative Fura-2 F 34 0/F380 ratio traces over 6 min. H) Quantification of F340/F380 ratio changes relative to respective baseline of each cell, 3 dishes (n > 76 cells per dish) I) Representative Fura-2 images of J774a cells pre- and post-LPS stimulation; Color scale: red = high [Ca 2+ ], yellow/green = intermediate, blue/purple = low [Ca 2+ ]; Scale bar = 100 μm ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Changes in intracellular calcium were measured using a ratiometric calcium indicator dye Fura-2 AM (ThermoFisher.

Techniques: Inhibition, Staining, Imaging

Journal: Bioactive Materials

Article Title: Reprogramming macrophage mechanosensation via TRPV4 modulating mechano-immunotherapy controls fibrotic encapsulation of biomaterial implants

doi: 10.1016/j.bioactmat.2026.06.020

Figure Lengend Snippet: RN-1734-mediated modulation of TPRV4 downstream signalling following LPS stimulation. A-C) Dose-dependent ELISA quantification of A) TNF-α, B) IL-6 and C) MCP-1 cytokine secretion from macrophage cultures following 24 h LPS stimulation (n = 3), D) Representative western blots of macrophage lysates after 24 h post LPS stimulation for iNOS, ARG-1, p65, and α-tubulin. E-G) Protein quantification of western blots for iNOS, ARG-1, and p65 normalised to α-tubulin (n = 3). H) Schematic of GSK1016790A (GSK) mechanism: selective agonism of TRPV facilitating increased intracellular calcium influx. I) Representative Fura-2 imaging of macrophage cultures treated with GSK; Color scale: red = high [Ca 2+ ], yellow/green = intermediate, blue/purple = low [Ca 2+ ]; scale bar = 30 μm. J-K) ELISA quantification of TNF-α and IL-6 secretion from macrophage cultures following 24 h of GSK stimulation (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Changes in intracellular calcium were measured using a ratiometric calcium indicator dye Fura-2 AM (ThermoFisher.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Imaging

Journal: Bioactive Materials

Article Title: Reprogramming macrophage mechanosensation via TRPV4 modulating mechano-immunotherapy controls fibrotic encapsulation of biomaterial implants

doi: 10.1016/j.bioactmat.2026.06.020

Figure Lengend Snippet: Stiffness-dependent modulation of macrophage TPRV4 activity and signalling. A) Fabrication of 10% (stiff) and 5% (soft) GelMA hydrogels (scale bar: 2.5 mm) validated by compression test, B) Compression curves (inset: linear region of the curve). C) J774. a2 macrophage viability on GelMa hydrogels measured by alamarBlue and complementary (n = 3) D) brightfield imaging of macrophage morphology 24 h post-seeding; scale bar = 100 μm. E) Fura-2 (F340/380 ratio) calcium imaging traces of macrophage cultures over 60 min and F) quantification of changes from peak ratio relative to baseline (t = 0) (n > 50 per dish, 3 dishes), with G) representative culture images at t = 40mins; Color scale: red = high [Ca 2+ ], yellow/green = intermediate, blue/purple = low [Ca 2+ ]; scale bar = 100 μm. H–J) ELISA of TNF-α, IL-6, and MCP-1 after 24 h on GelMA (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Changes in intracellular calcium were measured using a ratiometric calcium indicator dye Fura-2 AM (ThermoFisher.

Techniques: Activity Assay, Imaging, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: Effect of Microcurrent Stimulation on Cartilage Regeneration in Engineered Chondrocyte Spheroids

doi: 10.34133/research.1462

Figure Lengend Snippet: Effects of microcurrent stimulation on chondrocyte proliferation in a 2-dimensional (2D) in vitro culture system. (A) Schematic illustration of chondrocytes cultured in 6-well plates under microcurrent stimulation. (B) Relative DNA content of chondrocytes in the 10-, 25-, 50-, 100-, and 200-μA groups compared with that of the control (Ctrl, 0 μA) group at day 9. (C) Relative optical density (OD) values of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 5, as determined by the CCK-8 assay. (D) Representative Live/Dead staining images of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups from days 1 to 9. (E) Quantitative analysis of cell viability in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 9. (F) Representative 5-ethynyl-2′-deoxyuridine (EdU) staining images and corresponding quantification of the EdU-positive rate of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 9. (G) Representative Ki67 staining images and corresponding quantification of the Ki67-positive rate of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 9. Data are presented as mean ± SD ( n = 5). ns, not significant; *** P < 0.001; **** P < 0.0001.

Article Snippet: Samples were washed with PBS and incubated in working solution containing 2 μM calcein-AM and 4 μM propidium iodide using a calcein-AM/propidium iodide Live/Dead staining kit (Beyotime Biotechnology, Shanghai, China) for 20 min at 37 °C in the dark.

Techniques: In Vitro, Cell Culture, Control, CCK-8 Assay, Staining, Analysis

Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: For the mPTP opening assay, cells were first incubated with MitoTracker Red (Beyotime, C1032) at 37°C for 30 min to visualize mitochondria, then stained with calcein‐AM solution (Beyotime, C2009) at 37°C for 30 min.

Techniques: Inhibition, Staining, Membrane, Permeability, Analysis, Activity Assay, Western Blot, Micro-CT