Journal: bioRxiv

Article Title: Nascent extracellular matrix converts biomaterial cues into cell fate decisions

doi: 10.64898/2026.01.10.698826

Figure Lengend Snippet: a. Representative fluorescent images of nuclei and cell membrane of chondrocytes cultured in low, mid, and high modification hydrogels at day 7 (scale bars: 10 μm.) b. Quantification of divided nuclei of chondrocytes culture in low, mid and high modification hydrogels at day1, 4 and 7 (low: day 1 n = 12 regions of interest (ROIs), N = 2; day 4 n = 9 ROIs, N = 2; day 7 n = 12 ROIs, N = 3; mid: day 1 n = 8 ROIs, N = 2; day 4 n = 8 ROIs, N = 2; day 7 n = 12 ROIs, N = 3; high: day 1 n = 10 ROIs, N = 2; day 4 n = 9 ROIs, N = 2; day 7 n = 12 ROIs, N = 3) c. Quantification of divided nuclei of chondrocytes culture in low, mid and high modification hydrogels at day 7 ( low: n = 12 ROIs, N = 3; mid: n = 12 ROIs, N = 3; and high: n = 12 ROIs, N = 3). d. Representative fluorescent images of incorporation of 5-ethynyl-2’-deoxyuridine (EdU) into chondrocytes cultured in low, mid and high modification hydrogels at day 7 (scale bar: 100 μm). e. Quantification of EdU incorporation into chondrocytes cultured in low, mid and high modification hydrogels at day 7 (scale bar: 100 μm, low: n = 23 ROIs, N = 4; mid: n = 24 ROIs, N = 4; and high: n = 23 ROIs, N = 4 ). f. Quantification of chondrocyte cell aspect ratio at day 7 (low: n = 108 cells, N = 3; mid: n = 112 cells, N = 3; and high: n = 111 cells, N = 3). g. Schematic showing the downstream effects of Sox9 translocation into the nucleus, including upregulation of collagen type IIA1, aggrecan (ACAN) and Sox9, and the downregulation of collagen type IA1, versican (VCAN) and THY-1 h. Representative immunofluorescent images and quantification of Sox9 nuclear translocation calculated by the nucleus-to-cytoplasm (NC) ratio at day 7 (scale bar: 10 μm, low: n = 139 cells, N = 3; and high: n = 124 cells, N = 3). i. Ratio of COL2A1/COL1A1 and ACAN/VCAN gene expression at day 7, measured by qPCR and normalized to S18 housekeeping gene. Gene expression ratios were calculated as 2 (-(ΔCt₁ - ΔCt₂)) , where ΔCt₁ and ΔCt₂ represent Ct values of each gene normalized to S18. ( N = 4) a-i. N = number of independent experiments, error bar = standard deviation, ****p < 0.0001, **p < 0.01, *p < 0.05, ns: not significant by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies included SOX9 (NovusBio, NBP2-24659, 1:100), Collagen II (DSHB, II-II6B3, 1:100), Collagen VI (Biosynth, 70R-CR009X, 1:100), Decorin (Kerafast, ENH077-FP, 1:100), and Aggrecan (Abcam, ab3778, 1:50).

Techniques: Membrane, Cell Culture, Modification, Translocation Assay, Gene Expression, Standard Deviation

Journal: bioRxiv

Article Title: Nascent extracellular matrix converts biomaterial cues into cell fate decisions

doi: 10.64898/2026.01.10.698826

Figure Lengend Snippet: a. Timeline and schematic showing the blocking of cell adhesion to hyaluronic acid with CD44 function-perturbing antibody for 1 hour pre-embedding and for 3 consecutive days while maintaining adhesion to nECM. b. Representative fluorescent images and quantification of the incorporation of EdU in chondrocytes cultured without (Ctrl) and with CD44 inhibition (CD44i) in ‘low’ modification hydrogels at day 7. (scale bar = 100μm, low-Ctrl: n = 23 ROIs, N = 3; low-CD44: n = 27 ROIs, N = 3; high-Ctrl: n = 23 ROIs, N = 3) c. Representative fluorescent images (dashed line outlines nuclei) and quantification of Sox9 nucleus-to-cytoplasm (NC) ratio of chondrocytes cultured without (Ctrl) and with CD44 inhibition (CD44i) in low modification hydrogels at day 7. (scale bar = 10μm, low Ctrl: n = 138 cells, N = 4; low CD44i: n = 110 cells; N = 4, high-Ctrl: n =124 cells; N = 4) a-c. N = number of independent experiments, error bar = standard deviation, ****p < 0.0001, ns: not significant by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies included SOX9 (NovusBio, NBP2-24659, 1:100), Collagen II (DSHB, II-II6B3, 1:100), Collagen VI (Biosynth, 70R-CR009X, 1:100), Decorin (Kerafast, ENH077-FP, 1:100), and Aggrecan (Abcam, ab3778, 1:50).

Techniques: Blocking Assay, Cell Culture, Inhibition, Modification, Standard Deviation

Journal: bioRxiv

Article Title: Nascent extracellular matrix converts biomaterial cues into cell fate decisions

doi: 10.64898/2026.01.10.698826

Figure Lengend Snippet: a. Timeline and schematic showing the blocking of cell adhesion to nECM using an integrin β1 (ITGB1) function perturbing antibody for 1 hour pre-embedding and for 7 consecutive days . b. Representative fluorescent images of the incorporation of EdU in chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in high and low modification hydrogels at day 7 (scale bar = 100 μm). c. Quantification of the incorporation of EdU in chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in high and low modification hydrogels at day 7 (low-Ctrl: n = 23 ROIs, N = 3; low-ITGB1i: n = 30 ROIs, N = 3; high-Ctrl: n = 23 ROIs, N = 3; low-ITGB1i: n = 27 ROIs, N = 3). d. Representative fluorescent images of Sox9 immunofluorescence (dashed line outlines nuclei) of chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in low and high modification hydrogels at day 7 (scale bar = 10 μm). e. Quantification of Sox9 nucleus-to-cytoplasm (NC) ratio of chondrocytes cultured without (Ctrl) and with ITGB1 inhibition (ITGB1i) in low and high modification hydrogels at day 7 (low-Ctrl: n = 138 cells, N = 4; low-ITGB1i: n = 133 cells, N = 4; high-Ctrl: n = 124 cells, N = 4; high-ITGBi: n = 138 cells, N = 4). a-e. N = number of independent experiments ****p < 0.0001, *p < 0.05, error bar = standard deviation, ns: no significant difference by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies included SOX9 (NovusBio, NBP2-24659, 1:100), Collagen II (DSHB, II-II6B3, 1:100), Collagen VI (Biosynth, 70R-CR009X, 1:100), Decorin (Kerafast, ENH077-FP, 1:100), and Aggrecan (Abcam, ab3778, 1:50).

Techniques: Blocking Assay, Cell Culture, Inhibition, Modification, Immunofluorescence, Standard Deviation

Journal: Bioactive Materials

Article Title: Bioinspired scaffold recapitulating chondrogenic ontogeny and microenvironment for functional cartilage regeneration

doi: 10.1016/j.bioactmat.2025.11.041

Figure Lengend Snippet: Biological functional analysis of tri-layer scaffold systems. A) Representative images of migrated cells stained with crystal violet at 48 h. B) Number of migrating cells in control and CE-SKP. C) Glycosaminoglycan concentrations in control and CE-SKP at 7 and 14 days. D) qPCR analysis for COL2, SOX9, and ACAN. E) Western blotting of COL2 and SOX9. F) Alizarin red staining of control and CPH. G) ALP activity of control and CPH. H) qPCR analysis for COL1, COL10, OCN, and RUNX2. I) Western blotting of COL10, COL1, and RUNX2. J) Representative images of migrated cells stained with crystal violet at 48 h. K) Number of migrating cells in control and P2G3. L) Scanning electron microscope micrographs of P2G3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: After blocking the membranes for an hour at room temperature using fast blocking solution, the membranes were incubated with primary antibodies specific for SOX9 (1:2000, 67439-1-Ig, Proteintech), COL2 (1:200, NBP1-91056, Novus Biologicals), COL10 (1:500, 26984-1-AP, Proteintech), COL1 (1:2000, 67288-1-Ig, Proteintech), RUNX2 (1:200, 20700-1-AP, Proteintech), FGF2 (1:500, 11234-1-AP, Proteintech), FGF18 (1:500, 60341-1-Ig, Proteintech) and GAPDH (1:50000, 60004-1-Ig, Proteintech) for an entire night at 4 °C.

Techniques: Functional Assay, Staining, Control, Western Blot, Activity Assay, Microscopy

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Neutral Buoyancy as a Simple Approach to Simulated Microgravity

doi: 10.1007/s13770-025-00781-2

Figure Lengend Snippet: The feasibility of the simulated microgravity in Optiprep with a neutral buoyancy regarding the stemness maintenance and trilineage differentiation of h BMSCs was evaluated. A qRT-PCR analysis and B western blotting determined the expression of OCT4, SOX2, and NANOG (stemness markers) in h BMSCs cultured at 3D-sim-μg (in Optiprep ), 3D-1 g (in CCM), and 2D-1 g (in CCM). C qRT-PCR analysis, D immunofluorescence staining, E histological analysis, and F quantitative analysis of osteogenic differentiation markers (RUNX2, OCN, and alizarin red S), adipogenic differentiation markers (PPARγ, CEBP/α, and Oil red O), and chondrogenic differentiation markers (SOX9, ACN, and Alcian blue) of h BMSCs cultured at 3D-sim-μg and 3D-1 g (n = 3; *p < 0.05 and **p < 0.01)

Article Snippet: The specific TaqMan Primers (Thermo Fisher Scientific) used were: OCT4 (Hs04260367_gH), SOX2 (Hs04234836_s1), NANOG (Hs02387400_g1), RUNX2 (Hs00231692_ml), osteocalcin (OCN; Hs00609452_gl), PPARγ (Hs01115513_m1), CEBP/α (Hs00269972_s1), SOX9 (Hs00165814_m1), aggrecan (ACN; Hs00153936_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs02786624_g1), with GAPDH serving as the housekeeping gene.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Cell Culture, Immunofluorescence, Staining