Journal: Research

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

doi: 10.34133/research.1462

Figure Lengend Snippet: Microcurrent stimulation regulates intracellular Ca 2+ signaling and nuclear factor of activated T cells (NFAT) activation in cartilage spheroids. (A) Representative fluo-4 acetoxymethyl ester (Fluo-4 AM) calcium imaging snapshots of cartilage spheroids in the 0-, 50-, and 200-μA groups, captured at their respective peak-response time points (2.4, 4.3, and 12.2 s). Quantitative analysis of (B) the Ca 2+ signal area under the curve (AUC) (Δ F / F 0 · s ) and (C) the responsive cell ratio in the 0-, 50-, and 200-μA groups during the first 60 s after stimulation onset. (D) Representative immunofluorescence staining images of NFAT and 4′,6-diamidino-2-phenylindole (DAPI) in cartilage spheroids from the 0 μA, 50 μA, 50 μA + verapamil, 50 μA + BAPTA-AM, 50 μA + CsA, and 200 μA groups at day 12. BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid acetoxymethyl ester; CsA, cyclosporin A. (E) Quantitative analysis of the NFAT nuclear/cytoplasmic fluorescence ratio in each group. (F) Representative Western blot images showing the protein expression levels of NFATc1 in cartilage spheroids from the 0 μA, 50 μA, 50 μA + verapamil, 50 μA + BAPTA-AM, 50 μA + CsA, and 200 μA groups at day 12, with β-actin used as the loading control. (G) Quantitative analysis of the relative protein expression levels of NFATc1 in each group based on the Western blot results shown in (F). Data are presented as mean ± SD ( n = 5). ns, not significant; ** P < 0.01; **** P < 0.0001.

Article Snippet: Samples were then incubated overnight at 4 °C with an anti-NFATc1 primary antibody (sc-7294, Santa Cruz Biotechnology, Dallas, TX, USA, 1:200), followed by incubation with the corresponding fluorescence-conjugated secondary antibody (Thermo Fisher Scientific, 1:500) for 1 h at room temperature in the dark.

Techniques: Activation Assay, Spheroids, Imaging, Analysis, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Control

Journal: Research

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

doi: 10.34133/research.1462

Figure Lengend Snippet: In vivo validation of the effects of microcurrent stimulation on cartilage matrix formation after subcutaneous implantation. (A) Schematic illustration summarizing the beneficial and detrimental responses to microcurrent stimulation in engineered chondrocyte spheroids, highlighting coordinated Ca 2+ /calcineurin/nuclear factor of activated T cells (NFAT)-mediated anabolic signaling at 50 μA and dysregulated stress-associated responses at 200 μA. (B) Schematic illustration of subcutaneous injection of cartilage spheroid suspensions from the 0-, 50-, and 200-μA groups into nude mice. (C) Representative gross images of samples after 2 weeks of subcutaneous implantation in nude mice. (D) Representative hematoxylin and eosin (H&E), toluidine blue (TB), Safranin O, and type II collagen (COL II) immunohistochemical staining images of samples from the 0-, 50-, and 200-μA groups after 2 weeks of subcutaneous implantation in nude mice. The right panels show enlarged views of the corresponding regions in the left panels. Quantitative analysis of (E) COL II content, (F) glycosaminoglycan (GAG) content, and (G) compressive modulus of samples from the 0-, 50-, and 200-μA groups after 2 weeks of subcutaneous implantation in nude mice. Data are presented as mean ± SD ( n = 5). ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Samples were then incubated overnight at 4 °C with an anti-NFATc1 primary antibody (sc-7294, Santa Cruz Biotechnology, Dallas, TX, USA, 1:200), followed by incubation with the corresponding fluorescence-conjugated secondary antibody (Thermo Fisher Scientific, 1:500) for 1 h at room temperature in the dark.

Techniques: In Vivo, Biomarker Discovery, Spheroids, Injection, Immunohistochemical staining, Staining, Analysis